5 – 7 June 2018 Konzerthaus Freiburg · Germany 3D Cell ...

PROGR AMME AND BOOK OF ABSTR AC TS

5 – 7 June 2018 Konzerthaus Freiburg · Germany

3D Cell Culture 2018How close to ‘in vivo‘ can we get? Models, Applications & Translation

www.dechema.de/3DCC2018

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contents

committeeHansjörg Hauser Helmholtz Centre for Infection Research, Braunschweig/DJens M. Kelm Competence Centre TEDD, Wädenswil/CHUwe Marx TissUse GmbH, Berlin/DTobias May InSCREENeX GmbH, Braunschweig/DThomas Noll University of Bielefeld/DRalf Pörtner Hamburg University of Technology/DMarkus Rimann Zurich University of Applied Sciences, Wädenswil/CHHeinz Ruffner Novartis Institutes for BioMedical Research, Basel/CHKarin Tiemann DECHEMA e. V., Frankfurt Main/D

venueKonzerthaus FreiburgKonrad-Adenauer-Platz 179098 FreiburgGermanyWebsite: www.konzerthaus.freiburg.de

organiser and contactDECHEMA e.V.Theodor-Heuss-Allee 2560486 Frankfurt am MainGermany

Christopher Diaz MaceoPhone: +49 (0)69 7564-243Fax: +49 (0)69 7564-176E-mail: [email protected]

As of 17.05.2018Subject to alterations. Submission title and authors information as provided by the authors. No proof by DECHEMA.

LECTURE PROGRAMME 4

Tuesday, 5 June 2018

Wednesday, 6 June 2018

Thursday, 7 June 2018

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EXHIBITORS 11

FLOOR PLAN – POSTER SESSION / EXHIBITION 12

POSTER PROGRAMME 14

LECTURE ABSTRACTS 27

POSTER ABSTRACTS 77

LIST OF PARTICIPANTS 201

© Cover images on “3D”: Katharina Schimek, Technical University Berlin/D, Gerd Lindner, Technical University Berlin/D Background image: Sandra Laternser/Ursula Graf-Hausner, ZHAW, Wädenswil/CH

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14:45 Image-based quantification of immunotherapies effects in 3D environment K. Yan¹; L. Daszkiewicz¹; L. Price¹; ¹ OcellO B.V., Leiden/NL

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15:05 3D culture models for investigaing recruitment of stem cells to the vascular niche Y. Atlas¹; C. Gorin²; C. Chaussain²; S. Germain¹; L. Muller¹; ¹ CIRB, Collège de France, Paris/F; ² Decartes University, Dental School, Paris/F

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15:25 STATARRAYS©: microcavity arrays as a useful tool to detect single cell migration in a 4D co-culture model of human bone marrow E. Gottwald¹; S. Giselbrecht¹; R. Truckenmüller¹; V. Colditz²; C. Nies²; ¹ 300MICRONS GmbH, Karlsruhe/D; ² Karlsruhe Institute of Technology (KIT), Karlsruhe/D

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15:45 Coffee Break / Posters / Exhibition

Predictive Model Systems

Chair: T. May¹; ¹InSCREENeX GmbH, Braunschweig/D

16:15 keynote lecture3D human liver spheroid systems for analyses of liver diseases, liver function, drug metabolism and toxicity M. Ingelman-Sundberg¹; ¹ Karolinska Institutet, Stockholm/S

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17:00 Novel predictive 3D cultivation models for validating small molecules against KSHV infection T. Dubich¹; C. Lipps¹; T. May²; M. Stadler¹; T. Schulz³; D. Wirth¹; ¹ Helmholtz Centre for Infection Research, Braunschweig/D; ² InSCREENeX GmbH, Braunschweig/D; ³ Institute of Virology, Hannover Medical School, Hannover/D

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17:20 Three-dimensional tumor cell growth stimulates autophagic flux and recapitulates chemotherapy resistance C. Bingel¹; E. Koeneke¹; J. Ridinger¹; A. Bittmann¹; M. Sill²; H. Peterziel¹; J. Wrobel¹; I. Rettig¹; T. Milde¹; U. Fernekorn³; F. Weise³; A. Schober³; O. Witt¹; I. Oehme²; ¹ CCU Pediatric Oncology, German Cancer Research Center (DKFZ), Heidelberg/D; ² German Cancer Research Center (DKFZ), Heidelberg/D; ³ Dpt of Nano-Biosystem Technology, TU Ilmenau/D

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17:40 keynote lectureSynthetic Biology-Inspired Treatment Strategies of the FutureM. Fussenegger¹; ¹ ETH Zurich/CH

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18:25 Poster Session / Poster Party (18:25 – 21:00)

18:30 SECTION MEMBER ASSEMBLY (Room K3+K4 / 18:30 – 19:30) DECHEMA Working Groups Cell Culture Technology and Medical Biotechnology


09:30 Registration

10:30 Welcome Address

Effects of Microenvironment

Chair: H. Hauser¹; ¹ Helmholtz Centre for Infection Research, Braunschweig/D

10:35 keynote lectureEngineering organoid development M. Lutolf¹; ¹Ecole Polytechnique Fédérale de Lausanne/CH

11:20 Macromolecular crowding in 2D and 3D culture systems: creating of cell and stem cell specific microenvironments M. Raghunath¹; N. Kohli¹; ¹ Zurich University of Applied Sciences, ICBT, Wädenswil/CH

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11:40 Redefining cell culture environment with combinatorial biomatrices A. Thomas¹; ¹ B CUBE Center for Molecular Bioengineering, Center for Molecular and Cellular Bioengineering – TU Dresden/D

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12:00 iPSC-derived neurospheroids recapitulate development and pathological signatures of brain microenvironment A. Terrasso¹; D. Simão¹; N. Bayó-Puxan²; F. Arez¹; M. Silva¹; M. Sousa¹; S. Creysells³; P. Gomes-Alves¹; N. Raimundo⁴; E. Kremer³; P. Alves¹; C. Brito¹; ¹ iBET, Instituto de Bio-logia Experimental e Tecnológica, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidede Nova de Lisboa, Oeiras, Portugal;, Oeiras/P; ² Institute of Biomedicine of the University of Barcelona (IBUB); Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535; Université de Montpellier, Barcelona; Montpellier/E; ³ Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535; Université de Montpellier, Montpellier/F; ⁴ Universitätsmedizin Göttingen, Institut für Zellbiochemie, Göttingen/D

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12:20 Expansion of mouse pancreatic organoids in a chemically defined three-dimensional matrix N. Rischert¹; H. Wurst¹; T. Moreth²; L. Hof²; E. Stelzer²; M. Huch³; F. Pampaloni²; B. Angres¹; ¹ Cellendes GmbH, Reutlingen/D; ² Goethe-Universität Frankfurt am Main/D; ³ University of Cambridge/UK

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12:40 Lunch Break / Posters / Exhibition

Imaging and Analytics

Chair: R. Pörtner¹; ¹ Hamburg University of Technology/D

14:00 keynote lectureObserving three-dimensional biological specimens with light sheet-based fluorescence microscopy (LSFM) E. H. K. Stelzer¹; ¹ Buchmann Institute for Molecular Life Sciences – Goethe University Frankfurt am Main/D

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08:30 Registration

Advanced Models – Skin

Chair: U. Marx¹; ¹TissUse GmbH, Berlin/D

09:00 keynote lectureIn vitro skin models for clinical research and transplantation S. Gibbs¹; ¹ VU University Medical Center Amsterdam/NL

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09:45 Towards an immunocompetent skin model to study and develop materials for wound healing C. Griffoni¹; B. Sentürk¹; M. Rottmar¹; K. Maniura¹; ¹ Empa - Swiss Federal Laboratories for Materials Science and Technology, St Gallen/CH

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10:05 vascSkin-on-a-chip: combination strategies of human skin-equivalents and vasculature K. Schimek¹; A. Thomas²; T. Hasenberg³; G. Giese⁴; U. Marx³; R. Lauster⁴; G. Lindner⁴; ¹ Technische Universität Berlin, FG Medizinische Biotechnologie, Berlin/D; ² Cellbricks GmbH, Berlin/D; ³ TissUse GmbH, Berlin/D; ⁴ Technische Universität Berlin/D

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10:25 MUG-Mel2, a novel highly pigmented and well characterized NRAS mutated human melanoma cell line in 3D culture B. Rinner¹; G. Gandolfi²; K. Meditz¹; M. Frisch¹; K. Wagner¹; A. Ciarrocchi²; F. Torricelli²; R. Koivuniemi³; J. Niklander³; B. Liegl-Atzwnager¹; B. Lohberger¹; E. Heitzer¹; N. Ghaffari-Tabrizi-Wizsy¹; D. Zweytick¹; I. Zalaudek¹; ¹ Medical University of Graz, Graz/A; ² Laboratorio di Ricerca Traslazionale Arcispedale S. Maria Nuova - IRCCS, Reggio Emilia/I; ³ University of Helsinki, Helsinki/FIN

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Advanced Models - Vascularization, Muscle

Chair: H. Ruffner¹; ¹Novartis Institutes for BioMedical Research, Basel/CH

11:15 Pre-vascularized cell cultivation system to generate perfused 3D co-culture models I. Prade¹; M. Busek²; M. Wiele¹; F. Sonntag²; M. Meyer¹; ¹ FILK gGmbH, Freiberg/D; ² Fraunhofer-Institut für Werkstoff- und Strahltechnik, Dresden/D

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11:35 Generation of 3D human cardiac macrotissues with tissue-like functionality M. Valls-Margarit¹; O. Iglesias-García²; C. Di Guglielmo²; L. Sarlabous¹; R. Paoli¹; J. Comelles¹; D. Blanco-Almazán¹; S. Jiménez-Delgado²; O. Castillo-Fernández³; J. Samitier¹; R. Jané¹; E. Martínez¹; Á. Raya²; ¹ Institute for Bioengineering of Catalonia, Barcelona/E; ² Center of Regenerative Medicine in Barcelona/E; ³ Institute of Micro-electronics of Barcelona, Bellaterra/E

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11:55 keynote lectureAdvanced induced pluripotent stem cell (iPSC) screens M. Müller¹; ¹ Novartis Institutes for BioMedical Research, Basel/CH

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12:40 Lunch / Posters / Exhibition


From Models to High Throughput

Chair: J. Kelm¹; ¹ Competence Centre TEDD, Wädenswil/CH

14:00 Merging high-content and high-throughput screening: Microphysiological Organ-on-a-Chip systems featuring complex human tissues with physiological structure and function P. Loskill¹; ¹ Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Stuttgart/D

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14:20 Modification of a standardized 3D in vitro tumor-stroma model for high throughput screening of candidates of new tumor therapeutica S. Hensler¹; C. Kühlbach¹; M. Mueller¹; ¹ HFU Hochschule Furtwangen, Villingen-Schwenningen/D

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14:40 Development of a matrix-based technology platform for the high throughput analysis of 3D cell cultures M. Rimann¹; A. Picenoni¹; E. Bono¹; E. Felley-Bosco²; C. Hund³; R. Pellaux³; A. Meyer³; ¹ Zurich University of Applied Sciences, ICBT, Waedenswil/CH; ² Laboratory of Molecular Oncology, Zurich University Hospital, Zurich/CH; ³ FGen GmbH, Basel/CH

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15:00 Magnetic 3D Bioprinting for High-Throughput Compound Screening and Translational Applications G. Souza¹; G. Bartholomeusz²; ¹ The University of Texas Health Science Center, Houston/USA; ² UT MD Anderson Cancer Center, Houston/USA

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15:20 Simple and robust microfluidic platform for spheroid culturing in a high-throughput manner J. Kim¹; H. Choi¹; ¹ Daegu Gyeongbuk Institute of Science and Technology, Deagu/ROK

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15:40 Microtissues meet microfluidics – next generation microphysiological tilting system K. Renggli¹; C. Lohasz¹; S. Bürgel¹; D. Fluri²; A. Hierlemann¹; O. Frey²; ¹ ETH Zürich, Basel/CH; ² Insphero AG, Schlieren/CH

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08:30 Registration

Advanced Models – From Liver to Lung

Chair: M. Rimann¹; ¹ Zurich University of Applied Sciences, Wädenswil/CH

09:00 Metabolic cross talk between human pancreatic islet and liver spheroids in a micro-physiological system - Towards a novel human ex vivo model of Type 2 Diabetes S. Bauer¹; C. Wennberg Huldt²; K. Kanebratt²; I. Durieux¹; D. Gunne¹; S. Andersson²; L. Ewart³; W. Haynes²; I. Maschmeyer¹; A. Winter¹; C. Ämmälä²; U. Marx¹; T. Andersson²; ¹ TissUse GmbH, Berlin/D; ² AstraZeneca, Mölndal/S; ³ AstraZeneca, Cambridge/UK

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09:20 Mimicking human physiology at Transwell-based barrier models of the proximal tubulus – The ZEBRA-Chip F. Schmieder¹; D. Förster²; M. Hempel¹; J. Sradnick²; B. Hohenstein²; F. Sonntag¹; ¹ Fraunhofer Institute for Material and Beam Technology IWS, Dresden/D; ² Division of Nephrology, Department of Internal Medicine III, University Hospital Carl Gustav Carus Dresden/D

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09:40 Human and mouse intestinal organoids as model system for studying drug transport T. Zietek¹; ;E. Rath²; F. Reichart³; H. Kessler³; G. Ceyhan⁴, I. Demir⁴, H. Daniel¹;¹ Technische Universität München, Freising/D; ² TUM ZIEL Institute for Food & Health, Freising/D; ³ TUM Institute for Advanced Study, Garching/D; ⁴ Dept. of Surgery, Klinikum rechts der Isar, München, Germany

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10:00 Microstructured 3D model of small intestine epithelium: breaking the mold M. García-Díaz¹; A. G. Castaño¹; G. Altay¹; N. Torras¹; R. Martin-Venegas²; R. Ferrer¹; E. Martínez¹; ¹ Institute for Bioengineering of Catalonia, Barcelona/E; ² Universitat de Barcelona/E

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10:20 Long-term culture of rat Precision-Cut Lung Slices using Lab-on-Chip technology as an ex vivo system with prolonged viability S. Konzok¹; S. Dehmel¹; V. Neuhaus¹; J. Labisch¹; S. Grünzner²; F. Sonntag²; A. Braun¹; K. Sewald¹; ¹ Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Hannover/D; ² Fraunhofer Institute for Material and Beam Technology IWS/Dresden University of Technology, Dresden/D

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From Models to Clinical and Industrial Solutions

Chair: T. Noll¹; ¹ University of Bielefeld/D

16:30 keynote lectureThe application of microphysiological systems in drug discovery using case studies from safety and efficacy questions L. Ewart¹; ¹ AstraZeneca, Cambridge/UK

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17:15 Bringing 3D Tumor Models to the clinic – predictive value for personalized medicine K. Halfter¹; B. Mayer²; ¹ SpheroTec GmbH, Munich/D; ² Hospital of the LMU Munich/D

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17:35 Single-donor iPSC derived Multi-Organ-Chips A. Ramme¹; L. Koenig¹; D. Faust¹; A. Krebs¹; T. Hasenberg¹; E. Dehne¹; U. Marx¹; ¹ TissUse GmbH, Berlin/D

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17:55 Rethinking Drug Development – 3D Disease Models for Advanced Preclinical Drug Evaluation M. Schäfer-Korting¹; S. Hedtrich¹; V. Kral¹; G. Weindl¹; J. Plendl¹; C. Thöne-Reineke¹; B. Kleuser²; R. Preissner³; A. Pries³; A. Volkamer³; R. Lauster⁴; A. Luch⁵; G. Schönfelder⁵; M. Weber⁶; ¹ Freie Universität Berlin/D; ² Potsdam University, Potsdam/D; ³ Charité Universitätsmedizin Berlin/D; ⁴ Technische Universität Berlin/D; ⁵ Federal Institute for Risk Assessment, Berlin/D; ⁶ Zuse Institute Berlin/D

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18:15 End of Lecture Programme

19:30 CONFERENCE DINNERSchlossbergrestaurant Dattler Am Schlossberg 1 79104 Freiburg

(dinner ticket required)

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Safety and Toxicity Testing

Chair: U. Marx¹; ¹TissUse GmbH, Berlin/D

11:15 keynote lecture Advanced cell models, organs on a chip & microphysiological systems in drug safety assessment: the need, the vision – and challenges to overcome A. Roth¹; ¹ F. Hoffmann-La Roche Ltd., Basel/CH

12:00 A Novel 3D Human Liver Fibrosis Model for Anti-fibrotic Drug Discovery and Safety Testing S. Messner¹; ¹ Insphero AG, Schlieren/CH

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12:20 Bioprinted kidney model to assess nephrotoxicity M. Nosswitz¹; M. Rimann²; N. Hernando³; C. Wagner³; U. Graf-Hausner¹; M. Raghunath¹; ¹ Zurich University of Applied Sciences, ICBT, Waedenswil/CH; ² Zurich University of Applied Sciences, ICBT, Wädenswil/CH; ³ University of Zurich/CH

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12:40 Microfluidic Platform for Advanced Embryotoxicity Testing in vitro J. Boos¹; A. Michlmayr¹; K. Renggli¹; O. Frey²; A. Hierlemann¹; ¹ ETH Zürich, Basel/CH; ² Insphero AG, Schlieren/CH

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13:00 Closing Remarks

13:05 Lunch / Posters / Exhibition

14:15 End of the Conference

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3D Cell Culture 20185 - 7 June 2018, Konzerthaus Freiburg Germany

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A1 - Promega GmbH A2 - Cellon S.A.A3 - Union Biometrica, Inc.A4 - UPM-Kymmene CorporationA5 - Cenibra GmbHA6 - Greiner Bio-One GmbHA7 - PreSens Precision Sensing GmbH

B1 - ChemoMetec GmbHB2 - tebu-bio GmbHB3 - Kugelmeiers AGB4 - PeproTech GmbH

Exhibitors - 1st Floor

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C1 - MBL InternationalC2 - I&L Biosystems GmbHC3 - STEMCELL Technologies Germany GmbHC4 - Fraunhofer-Institut für Werkstoff- und Strahltechnik IWS

D1 - Competence Centre TEDD, Wädenswil/CHD2 - OcellO B.V.D3 - CellSystems® Biotechnologie Vertrieb GmbHE1 - CELLnTEC Advanced Cell Systems AGE2 - abc biopply agE3 - LOT-QuantumDesign GmbHE4 - Noviocell BV

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3D Cell Culture 20185 - 7 June 2018, Konzerthaus Freiburg Germany

Exhibition Area and Poster Session1st Floor

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Exhibitors – 1st Floor A1 Promega GmbH A2 Cellon S.A.A3 Union Biometrica, Inc.A4 UPM-Kymmene CorporationA5 Cenibra GmbHA6 Greiner Bio-One GmbHA7 PreSens Precision Sensing GmbHB1 ChemoMetec GmbH

B2 tebu-bio GmbHB3 Kugelmeiers AGB4 PeproTech GmbHC1 MBL InternationalC2 I&L Biosystems GmbHC3 STEMCELL Technologies Germany GmbHC4 Fraunhofer-Institut für Werkstoff- und

Strahltechnik IWS

D1 Competence Centre TEDDD2 OcellO B.V.D3 CellSystems® Biotechnologie

Vertrieb GmbHE1 CELLnTEC Advanced Cell Systems AGE2 abc biopply agE3 LOT-QuantumDesign GmbHE4 Noviocell BV

Exhibitors – 2nd Floor F1 Corning BVF2 ariadne-service gmbh F3 RIGENERAND Srl F4 PromoCell GmbH F5 ROKIT Inc.

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1.1. Advanced cell culture models

P1.1.01 Advanced physiologically relevant 3D models for pre-clinical screening D. Sabino¹; I. Fixe¹; A. Foucher¹; F. Carpentier¹; M. Rochet¹; I. Topin¹; E. Mennesson¹; N. Normand¹; ¹ tebu-bio, Le Perray en Yvelines/F

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P1.1.02 Evaluation of EGFR induced on-target and target-mediated adverse effects in a microfluidic 3D human lung tumour – full thickness skin co-culture model J. Hübner¹; M. Raschke²; I. Rütschle¹; S. Schnurre²; S. Gräßle¹; I. Maschmeyer¹; U. Marx¹; T. Steger-Hartmann²; ¹ TissUse GmbH, Berlin/D; ² Bayer AG, Berlin/D

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P1.1.03 A 3D High-Content Screening assay as model system for polycystic kidney disease H. Bange¹; T. Booij²; W. Leonhard³; K. Yan¹; D. Peters³; L. Price¹; ¹ OcellO B.V., Leiden/NL; ² LACDR, Leiden University, Leiden/NL; ³ Leiden University Medical Centre, Leiden/NL

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P1.1.04 Parallelized Heart-on-a-chip with integrated Force Sensing incorporating human iPS-derived cardiac microtissues C. Probst¹; O. Schneider¹; S. Fuchs¹; P. Loskill¹; ¹ Fraunhofer IGB, Stuttgart/D

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P1.1.05 Establishment of an advanced in vitro model to study nanomaterial-intestinal barrier interactions C. Hempt¹; C. Hirsch¹; M. Kucki¹; P. Wick¹; T. Buerki-Thurnherr¹; ¹ Empa - Swiss Federal Laboratories for Materials Science and Technology, St.Gallen/CH

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P1.1.06 The Ocular DynaMiTES – A dynamic microfluidic in vitro system with improved predictability of ocular drug absorption N. Beißner¹; K. Mattern²; A. Dietzel²; S. Reichl¹; ¹ TU Braunschweig/ Institut für Pharmazeutische Technologie, Braunschweig/D; ² TU Braunschweig/ Institut für Mikrotechnik, Braunschweig/D

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P1.1.07 Cell Processing in Microreactors: Real-time Monitoring of Cell Metabolism Using Sensor Particles and Surface Based, Gentle Cell Detachment K. Uhlig¹; C. Gehre²; S. Prill²; M. Stahl²; C. Duschl²; E. Schmälzlin³; L. Dähne⁴; T. Hellweg⁵; ¹ Fraunhofer-Institut für Zelltherapie und Immunologie IZI, Potsdam/D; ² Fraunhofer-Institute for Cell Therapy and Immunology, Potsdam/D; ³ Colibri Photonics GmbH, Potsdam/D; ⁴ Surflay Nanotec GmbH, Berlin/D; ⁵ Bielefeld University, Bielefeld/D

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P1.1.08 Evaluation of a Novel Cell Culture Platform with Various Barrier Forming Cells for Dynamic Cultivation S. Hinkel¹; K. Mattern²; A. Dietzel²; S. Reichl¹; C. Müller-Goymann¹; ¹ TU Braunschweig/ Institut für Pharmazeutische Technologie, Braunschweig/D; ² TU Braunschweig/ Institut für Mikrotechnik, Braunschweig/D

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P1.1.09 Ready-to-use 3D spheroid culture as a standard tool I. Prieto¹; ¹ StemTek Therapeutics, DERIO/E

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P1.1.10 Dual targeting of prognostic biomarkers in the 3D microtumor model of advanced colorectal cancer C. Ilmberger¹; O. Hoffmann¹; J. Gülden²; T. Bühl²; J. Werner²; B. Mayer²; ¹ SpheroTec GmbH, Munich/D; ² Hospital of the LMU Munich, Munich/D

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P1.1.11 Permeation Measurement for 3D Skin Culture in a Membrane Insert System H. Hsu¹; K. Schimek²; U. Marx³; R. Pörter⁴; ¹ Technische Universität Hamburg- Harburg, Hamburg/D; ² Department Medical Biotechnology of Biotechnology, Technische Universität Berlin, Berlin/D; ³ TissUse GmbH - TU Berlin, Berlin/D; ⁴ Institute of Bioprocess- and Biosystems Engineering, Hamburg University of Technology, Hamburg/D

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P1.1.12 Preservation of tumor architecture and heterogeneity in long-term cultures of patient-derived explants S. Abreu¹; S. da Mata²; F. Silva³; M. Teixeira¹; T. Franchi Mendes¹; R. Fonseca⁴; B. Filipe²; S. Morgado²; I. Francisco²; M. Mesquita²; C. Albuquerque²; J. Serpa⁵; P. Chaves²; I. Rosa²; A. Felix⁵; E. R. Boghaert⁶; V. E. Santo¹; C. Brito¹; ¹ iBET/ITQB-NOVA, Oeiras/P; ² IPOLFG, Lisboa/P; ³ CEDOC-FCM-NOVA, Lisboa/P; ⁴ IPOLFG and FMUL, Lisboa/P; ⁵ IPOLFG and CEDOC-FCM-NOVA, Lisboa/P; ⁶ AbbVie, Chicago/USA

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P1.1.13 Establishment of a murine intestinal tissue model based on immortalized primary epithelial cells C. Fey¹; T. Truschel²; M. Schweinlin¹; H. Walles³; T. May²; M. Metzger³; ¹ Department of Tissue Engineering and Regenerative Medicine (TERM), University Hospital Würzburg, Würzburg/D; ² InSCREENeX GmbH, Braunschweig/D; ³ Translational Center Würzburg “Regenerative Therapies for Oncology and Musculoskeletal Diseases” (TZKME), Würzburg branch of the Fraunhofer Institute of Silicate Research (ISC), Würzburg/D

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P1.1.14 Development of a human epidermal burn wound model V. Schneider¹; ¹ Uniklinik Würzburg, Würzburg/D

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P1.1.15 Initial screening of novel copolymer micelles for biocompatibility and effects on cell motility Y. Yordanov¹; D. Aluani¹; B. Tzankov¹; V. Tzankova¹; R. Kalinova²; I. Dimitrov³; V. Bankova⁴; M. Popova⁴; B. Trusheva⁴; K. Yoncheva¹; ¹ Faculty of Pharmacy, Medical University of Sofia, Sofia/BG; ² Institute of Polymers, Bulgarian Academy of Sciences, Sliven/BG; ³ Institute of Polymers, Bulgarian Academy of Sciences, Sofia/BG; ⁴ Institute of Organic Chemistry with Center for Phytochemistry, Bulgarian Academy of Sciences, Sofia/BG

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P1.1.16 An injectable hybrid hydrogel for tissue engineering applications R. Wittig¹; B. Baumann²; M. Lindén²; ¹ Institute for Laser Technologies in Medicine & Metrology (ILM) at Ulm University, Ulm/D; ² Institute for Inorganic Chemistry II, Ulm University, Ulm/D

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P1.1.17 A tissue engineered Full Thickness Skin Equivalent based on a non-contracting, biophysical optimised collagen type-I hydrogel P. Fey¹; C. Reuter²; T. Finger¹; M. Engstler²; H. Walles³; F. Groeber-Becker¹; ¹ Fraunhofer ISC - Translationszentrum für Regenerative Therapien TLZ-RT, Würzburg/D; ² Julius-Maximilians Universität Würzburg, Würzburg/D; ³ Universitätsklinikum Würzburg, Würzburg/D

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P1.1.18 Cell on cell – functionally immortalized smooth muscle cells as building blocks for 3D tissues A. Bleisch¹; ¹ InSCREENeX GmbH, Braunschweig/D

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P1.2.03 Establishment and initial characterization of a simple 3D organotypic wound healing model S. Hensler¹; C. Kühlbach²; J. Parente³; S. Krueger-Ziolek⁴; K. Moeller⁴; M. Mueller²; ¹ HS Furtwangen, Villingen-Schwenningen/D; ² Molecular Cell Biology Lab, Institute of Technical Medicine, HFU Furtwangen, Villingen-Schwenningen/D; ³ Institute of Technical Medicine, HFU Furtwangen University, Villingen-SChwenningen/D; ⁴ Insti-tute of Technical Medicine, HFU Furtwangen University, Villingen-Schwenningen/D

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P1.2.04 Novel 3D tumour models with stromal components to evaluate the efficacy of immunotherapy with gene-engineered ROR1-specific CAR T cells J. Kühnemundt¹; ¹ University Hospital Würzburg, Department of Tissue Engineering & Regenerative Medicine, Würzburg, Germany; Würzburg/D

115

P1.2.05 Evaluation of pharmacological responses in InflammaSkin®, a fully human full-thickness ex vivo skin model reproducing key features of psoriatic lesions P. Lovato¹; C. Jardet²; E. PAGES²; A. David²; E. Braun²; H. Norsgaard¹; P. Descargues³; ¹ LEO Pharma, Ballerup/DK; ² GENOSKIN SAS, Toulouse/F; ³ Genoskin Inc., Boston (MA)/USA

117

P1.2.06 Generation of human induced pluripotent stem cells (hiPSc)-derived hepatocyte organoids to study liver size control E. Saponara¹; ¹ Novartis Institutes of Biomedical Research, Basel/CH

119

1.3 Complex and multi-cell type models

P1.3.01 Using the Real Architecture For 3D Tissue (3D RAFT™) System as a Versatile Tool to Build in vitro Epithelial Barrier Models T. Willstaedt¹; J. Langer¹; S. Schaepermeier²; S. Buesch²; T. D’Souza¹; L. Hussain¹; J. Schroeder²; ¹ Lonza Walkersville Inc., Walkersville, MD/USA; ² Lonza Cologne GmbH, Cologne/D

120

P1.3.02 Towards a three-dimensional microfluidic in vitro model to assess efficacy & safety of immune-stimulatory antibody drugs R. Nudischer¹; C. Bertinetti-Lapatki²; C. Claus³; K. Renggli⁴; C. Lohasz⁴; O. Frey⁵; A. Hierlemann⁴; A. Roth²; ¹ F. Hoffmann-La Roche Ltd., Basel/CH; ² Roche Pharma Research and Early Development, Roche Innovation Center Basel, Basel/CH; ³ Roche Pharma Research and Early Development, Roche Innovation Center Zürich, Schlieren/CH; ⁴ ETH Zürich, D-BSSE Basel, Basel/CH; ⁵ Insphero AG, Schlieren/CH

121

P1.3.03 MSCs Isolation in 3D cell culture conditions: challenges, modeling and perspectives D. Egger¹; M. Kirsch²; T. Scheper²; A. Lavrentieva²; C. Kasper³; ¹ Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna/A; ² Insti-tute of Technical Chemistry, Leibniz University Hanover, Hannover/D; ³ Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna/D

123

P1.3.04 Retina-on-a-Chip: Merging Organoid and Organ-on-a-Chip technology for complex multi-layer tissue models J. Chuchuy¹; K. Achberger²; C. Probst¹; J. Haderspeck³; J. Rogal¹; S. Liebau²; P. Loskill¹; ¹ Fraunhofer IGB, Stuttgart/D; ² Eberhard Karls Universität Tübingen, Tübingen/D; ³ Eberhard Karls Universität Tübingen, Stuttgart/D

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P1.1.1 Evaluation of local inflammatory reactions following subcutaneous injection of a pro-inflammatory cocktail in a fully human ex vivo skin model C. Jardet¹; E. Pagès¹; E. Raude²; F. Seeliger³; L. Brandén³; E. Braun¹; M. Ingeslten³; P. Descargues⁴; ¹ GENOSKIN SAS, Toulouse/F; ² LAAS CNRS, Toulouse/F; ³ Drug Safety and Metabolism, iMED Biotech Unit, Astra Zeneca, Gothenburg/S; ⁴ Genoskin Inc., Boston (MA)/USA

104

P1.1.20 A microchip array-based 3D culture system for the in vitro differentiation of osteoblasts W. Zhang¹; P. Tomakidi²; T. Steinberg²; R. Kohal³; E. Gottwald⁴; B. Altmann¹; ¹ G.E.R.N., Department of Oral and Maxillofacial Surgery, University Medical Center Freiburg, Freiburg im Breisgau/D; ² Department of Oral Biotechnology, University Medical Center Freiburg, Freiburg im Breisgau/D; ³ Department of Prosthetic Dentistry, University Medical Center Freiburg, Freiburg im Breisgau/D; ⁴ 300MICRONS GmbH, Karlsruhe/D

106

P1.1.21 Automating 3D cell culture using a wood-derived hydrogel L. Paasonen¹; ¹ UPM-Kymmene Corporation, Helsini/FIN

107

P1.1.22 Combining pluripotent stem cell-derived models of the blood-brain barrier with Multi-Organ-Chip systems L. Koenig¹; A. Ramme¹; D. Faust¹; E. Dehne¹; U. Marx¹; ¹ TissUse GmbH, Berlin/D

108

P1.1.23 Microspheres-based scaffolds from poly(3-hydroxybutyrate) for 3D cell growth D. Chesnokova¹; I. Zharkova¹; A. Bonartsev¹; V. Voinova¹; ¹ Lomonosov Moscow State University, Faculty of Biology, Moscow/RUS

109

P1.1.24 In vitro 3D bladder cancer model using PDX-derived cells R. Amaral¹; A. Ma²; H. Zhang²; K. Swiech¹; C. Pan²; ¹ University of Sao Paulo, Ribeirao Preto/BR; ² University of California Davis, Sacramento/USA

110

1.2 Innovative disease models

P1.2.01 A tissue engineering approach to model Primary Ciliary Dyskinesia N. Lodes¹; H. Walles²; S. Hackenberg³; H. Hebestreit⁴; M. Steinke²; ¹ University Hospital Würzburg, Chair of Tissue Engineering and Regenerative Medicine, Würz-burg/D; ² University Hospital Würzburg, Chair of Tissue Engineering and Regen-erative Medicine; Fraunhofer Institute for Silicate Research, Translational Center Regenerative Therapies, Würzburg/D; ³ University Hospital Würzburg, Department of Otorhinolaryngology, Plastic, Aesthetic and Reconstructive Head and Neck, Würz-burg/D; ⁴ University Hospital Würzburg, Department of Paediatrics, Würzburg/D

111

P1.2.02 Characterisation of Bordetella pertussis virulence mechanisms using engineered human airway tissue models D. Kessie¹; ¹ Julius-Maximilians Universität Würzburg, Würzburg/D

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P1.3.15 Neuronal differentiation of human iPSCs in 3DProSeed hydrogel well plate and establishment of glia co-cultures S. de Leeuw¹; V. Milleret²; B. Simona³; R. Urbanet²; M. Ehrbar²; C. Tackenberg¹; ¹ Institute for Regenerative Medicine, University of Zürich, Schlieren/CH; ² Depart-ment of Obstetrics, University hospital Zürich, Zürich/CH; ³ Ectica Technologies AG, Zürich/CH

136

P1.3.16 Contractile work contributes to maturation of energy metabolism in hiPSC-derived cardiomyocytes B. Ulmer¹; A. Stoehr²; M. Schulze¹; S. Patel³; M. Gucek³; I. Mannhardt¹; S. Funcke¹; E. Murphy³; T. Eschenhagen¹; A. Hansen¹; ¹ UKE, Hamburg/D; ² Karolinska Institutet, Huddinge/S; ³ National Heart Lung and Blood Institute, Bethesda/USA

137

P1.3.17 Development of microvascular structures inside porous fibrin coated polydioxanon and PLLA/PLGA scaffolds S. Heene¹; S. Thoms¹; R. Jonczyk¹; T. Scheper¹; C. Blume¹; ¹ Leibniz Universität Hannover, Hannover/D

138

1.4 Predictivity and validation

P1.4.01 Patient-derived 3D tumor cultures for clinical diagnostics and pre-clinical drug development. S. Basten¹; B. Herpers¹; K. Yan¹; T. Giesemann²; J. Schueler²; W. Vader³; L. Price⁴; ¹ OcellO B.V., Leiden/NL; ² Charles River, Freiburg/D; ³ Vitroscan B.V., Leiden/NL; ⁴ OcellO B.V., Leiden/D

139

P1.4.02 Detailed Cell-Material Interactions in 3D Cell Culture Systems R. Harjumäki¹; R. Nugroho²; J. Valle-Delgado²; Y. Lou¹; M. Yliperttula¹; M. Österberg²; ¹ University of Helsinki, Helsinki/FIN; ² Aalto University, Espoo/FIN

141

P1.4.03 Towards controlling the mobility of flowing cells in a hanging-drop network for microphysiological systems N. Rousset¹; M. de Geus¹; A. Kaestli¹; K. Renggli¹; A. Hierlemann¹; ¹ ETH Zürich, Basel/CH

142

2.1 Translation of models to solutions

P2.1.01 Three-dimensional in vitro co-culture model for nanoparticle-mediated transfection V. Sokolova¹; N. Bialas¹; L. Rojas¹; M. Epple¹;; ¹ Inorganic Chemistry, University of Duisburg-Essen, Essen/D

144

P2.1.02 Microphysiological system based on human liver microtissues for intrinsic clearance prediction F. Hürlimann¹; S. Mannino²; C. Lohasz³; K. Renggli³; A. Hierlemann³; L. Suter-Dick²; O. Frey¹; ¹ InSphero AG, Schlieren/CH; ² University of Applied Sciences and Arts Northwestern Switzerland, Muttenz/CH; ³ ETH Zürich, D-BSSE, Basel/CH

145

P1.3.05 WAT-on-a-Chip: Microphysiological systems integrating white adipose tissue J. Rogal¹; C. Binder²; E. Rubiu²; C. Probst²; K. Schenke-Layland³; P. Loskill¹; ¹ Fraun-hofer Institute for Interfacial Engineering and Biotechnology IGB & Eberhard Karls University Tübingen, Stuttgart/D; ² Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Stuttgart/D; ³ Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB & Eberhard Karls University Tübingen, Tübingen/D

125

P1.3.06 High content screening of intestinal organoid cultures to visualize and quantify immune responses M. Madej¹; B. Herpers¹; L. Salinaro¹; K. Yan¹; L. Daszkiewicz¹; L. Price¹;; ¹ OcellO B.V., Leiden/NL

126

P1.3.07 3D co-cultivation of beta cells and mesenchymal stromal/stem cells for diabetes therapy F. Petry¹; P. Czermak¹; D. Salzig¹; ¹ Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen, Gießen/D

127

P1.3.08 Modeling tumor microenvironment to address the dynamics of tumor, stromal and immune cell interactions S. Rebelo¹; C. Brito²; D. Simão³; ¹ iBET/ITQBAX-UNL, Oeiras/P; ² iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidede Nova de Lisboa, Oeiras, Portugal, Oeiras/P; ³ iBET, Oeiras/P

128

P1.3.09 Development of a 3D spheroid SK-MEL-28 tumor model and its characterisation J. Klicks¹; R. Rudolf¹; M. Hafner¹; ¹ Hochschule Mannheim, Mannheim/D

129

P1.3.10 Trace Amines and Fatty Acids are Essential Endogenous Signaling Factors for β-Cell Activity and Insulin Secretion S. Hauke¹; C. Schultz²; ¹ European Molecular Biology Laboratory (EMBL), Heidelberg/D; ² Oregon Health and Science University (OHSU), Portland, OR/USA

130

P1.3.11 In vitro vascularization of a human bone marrow model. K. Keskin¹; S. Sieber¹; U. Marx²; R. Lauster¹; M. Rosowski¹; ¹ Technische Universität Berlin, FG Medizinische Biotechnologie, Berlin/D; ² TissUse GmbH, Berlin/D

132

P1.3.12 Development and characterization of PDX-derived 3D tumor microtissues as platform for screening targeted molecular therapeutics F. Chiovaro¹; N. Buschmann²; I. Agarkova²; A. Maier³; S. Messner²; J. Schueler³; P. Guye²; ¹ InSphero AG, Schlieren/CH; ² Insphero AG, Schlieren/CH; ³ Charles River, Freiburg im Breisgau/D

133

P1.3.13 Imitation of the long-lived plasma cell survival niche of the human bone marrow in vitro Z. Uyar¹; S. Sieber¹; U. Marx²; R. Lauster¹; M. Rosowski¹; ¹ Technische Universität Berlin/D

134

P1.3.14 Development of a Cardiac Organoid Culture System with hiPSC-derived Cardiomyocytes M. Schulze¹; B. Ulmer¹ ; M. Lemoine¹ ; A. Fischer¹ ; T. Eschenhagen¹; ¹ University Medical Center Hamburg-Eppendorf/D

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P3.2.04 Real-Time Assay for Apoptosis using Complementation of Annexin V Luciferase Subunits T. Riss¹; K. Kupcho¹; J. Shultz¹; J. Hartnett¹; R. Hurst¹; W. Zhou²; R. Akiyoshi³; A. Niles¹; ¹ Promega Corporation, Madison/USA; ² Promega Biosciences, San Louis Obispo/USA; ³ Olympus Corporation, Tokyo/J

157

P3.2.05 Benefits of Real-Time Measurements of Cell Health in 2D or 3D Using a Plate Reader T. Riss¹; ¹ Promega Corporation, Madison/USA

158

P3.2.06 Volume Regulation of HaCaT Spheroids in Response to Hypotonic Stimuli E. von Molitor¹; ¹ Hochschule Mannheim, Mannheim/D

159

P3.2.07 Calcium signals in taste-bud like 3D cultures T. Cesetti¹; E. von Molitor¹; R. Rudolf¹; M. Hafner¹; P. Scholz²; K. Riedel²; ¹ Hochschule Mannheim, Mannheim/D; ² BRAIN AG, Zwingenberg/D

160

3.3 New devices for 3D cell culture

P3.3.01 Scaffold-Free Aggregate Cultivation of Mesenchymal Stem Cells in a Stirred Tank Bioreactor C. Kasper¹; D. Egger²; I. Schwedhelm³; J. Hansmann³; ¹ Boku, Vienna/A; ² DBT - University of Natural Resources and Life Sciences (BOKU), Vienna/A; ³ Translational Center, University Hospital Wuerzburg, Würzburg/D

161

P3.3.02 Guiding 3D cell migration in deformed synthetic hydrogel micro-structures M. Dietrich¹; H. Le Roy²; D. Brückner³; H. Engelke⁴; R. Zantl⁵; J. Rädler⁶; C. Broedersz³; ¹ Faculty of Physics and Center for NanoScience, Ludwig-Maximilians-University and ibidi GmbH, Munich/D; ² École Normale supérieure Paris-Saclay, Cachan/F; ³ Arnold-Sommerfeld Center for Theoretical Physics and Center for NanoScience, Ludwig-Maximilians-University, Munich/D; ⁴ Department of Chemistry and Center for NanoScience, Ludwig-Maximilians-University, Munich/D; ⁵ ibidi GmbH, Martins-ried/D; ⁶ Faculty of Physics and Center for NanoScience, Ludwig-Maximilians- University, Munich/D

162

P3.3.03 Scaffold-Free Aggregate Cultivation of Mesenchymal Stem Cells in a Stirred Tank Bioreactor C. Kasper¹; ¹ University of Natural Resources and Life Sciences, Vienna, Vienna/A

164

P3.3.04 Development, Characterization and Application of a Parallelizable Perfusion Bioreactor for 3D Cell Culture D. Egger¹; M. Fischer¹; A. Clementi¹; J. Hansmann²; C. Kasper³; ¹ University of Natural Resources and Life Sciences, Vienna, Vienna/A; ² University Hospital Würzburg/D; ³ University of Natural Resources and Life Sciences, Vienna, Vienna/D

165

P3.3.05 A modular perfusion microbioreactor system for oxygen level control and optimization for bone tissue engineering J. Schmid¹; M. Schieker²; R. Huber¹; ¹ University of Applied Sciences Munich, Munich/D; ² Ludwig-Maximilians University Munich (LMU), Munich/D

166

2.2 Clinical applications

P2.2.01 Silencing GALNT1 or GALNT2 suppresses malignant phenotypes of pancreatic cancer cells T. Yeh¹; M. Huang¹; ¹ National Taiwan University College of Medicine, TAIPEI/RC

147

P2.2.02 Production of clinical grade temporary epidermal substitute obtained from hESC derived keratinocytes for the treatment of sickle cell leg ulcers: a challenge for regenerative medicine S. Domingues¹; Y. Masson¹; A. Poulet¹; M. Saidani¹; J. Polentes¹; G. Lemaitre¹; M. Peschanski¹; C. Baldeschi¹; ¹ ISTEM/CECS, Corbeil-Essonnes/F

148

P2.2.03 Establishment of a Novel Functional in Vitro Assay to Investigate the Angiogenic Potential of Colonic Adenocarcinomas S. Bring Truelsen¹; G. Hagel¹; N. Mousavi²; H. Harling²; K. Qvortrup²; O. Thastrup¹; J. Thastrup¹; ¹ 2cureX A/S, Birkerød/DK; ² University of Copenhagen, Copenhagen/DK

149

3.1 Innovative, advanced analytics

P3.1.01 Imaging oxygen gradients in cell aggregates and in spheroids R. Meier¹; R. Meier¹; ¹ PreSens Precision Sensing GmbH, Regensburg/D

151

P3.1.02 Application of video analysis for the evaluation of cardiac contractility in different in vitro model systems including freshly isolated adult rat cardiomyocytes and human iPSC-derived cardiomyocytes in 2D- and 3D-culture P. Beauchamp¹; S. Adrian²; S. Longnus²; T. Suter²; C. Zuppinger³; ¹ Bern University, Bern/CH; ² Bern University Hospital, Bern/CH; ³ University Hospital Bern, Bern/CH

152

3.2 Specific assay development

P3.2.01 Microfluidics: a powerful tool to recreate in vivo environment C. Vergne¹; B. Rouffet²; S. Renard³; M. Verhulsel²; ¹ Fluigent, Villejuif/FP; ² Fluigent, Villejuif/F; ³ Fluigent GmbH, Jena/D

153

P3.2.02 An assay to characterize the impact of cigarette smoke exposure on mucociliary clearance in-vitro. S. Frentzel¹; L. Ortega Torres¹; S. Majeed¹; P. Leroy¹; F. Zanetti¹; M. van der Toorn¹; M. Peitsch¹; J. Hoeng¹; ¹ Philip Morris Products S.A., Neuchatel/CH

154

P3.2.03 Minimalistic hydrogel matrices to direct early neural progenitors from pluripotent stem cells in 3D culture A. Meinhardt¹; A. Ranga²; E. Tanaka³; M. Lutolf⁴; C. Werner⁵; ¹ Leibniz Institute of Polymer Research Dresden, Max Bergmann Center of Biomaterials Dresden/D; ² KU Leuven, Leuven/B; ³ Research Institute of Molecular Pathology, Vienna/A; ⁴ Ecole Polytechnique Fédérale de Lausanne, Lausanne/CH; ⁵ Leibniz Institute of Polymer Research Dresden, Max Bergmann Center of Biomaterials Dresden, and Center for Regenerative Therapies Dresden, TU Dresden, Dresden/D

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P3.3.14 Comparison of 2D and 3D cultures of primary hepatocytes on hepatocellular functions and hepatotoxicity H. Dinter¹; A. Ullrich²; D. Runge²; ¹ Hochschule Biberach/D; ² Primacyt Cell Culture Technology GmbH, Schwerin/D

179

P3.3.15 Funnel-Guided Positioning of Multi-cellular Microtissues to Build Macrotissues K. Manning¹; A. Thomson²; J. Morgan²; ¹ Brown University, Providence, RI/USA; ² Brown University, Providence/USA

180

P3.3.16 A novel 3D microwell array for analysis of adhesion independent micro-tumours A. Thomsen¹; C. Aldrian²; Y. Thomann³; A. Grosu²; P. Bronsert⁴; M. Leu⁵; P. Lund⁶; ¹ University Medical Center Freiburg, Freiburg/D; ² Medical Center – University of Freiburg, Freiburg/D; ³ Freiburg Material Research Center and Institute for Macro-molecular Chemistry, Freiburg/D; ⁴ Institute for Surgical Pathology, Medical Center – University of Freiburg, Freiburg/D; ⁵ abc biopply ag, Solothurn/CH; ⁶ Department of Radiation Oncology, Ortenau-Klinikum, Offenburg/D

181

P3.3.17 Integration of 3d printed hollow hydrogel fiber with microfluidic system to develop a perfusable nephron model. A. Akkineni¹; D. Förster²; J. Sardnick²; F. Schmieder³; F. Sonntag³; M. Gelinsky¹; A. Lode¹; ¹ Centre for Translational Bone, Joint and Soft Tissue Research, TU Dresden/D; ² University Hospital Carl Gustav Carus, TU Dresden, Dresden/D; ³ Fraunhofer Institute for Material and Beam Technology IWS, Dresden/D

183

3.5 High-throughput and automatisation

P3.5.01 Impedance analysis of viability of Schistosoma mansoni larvae for drug screening application M. Modena¹; K. Chawla¹; F. Lombardo²; S. Burgel¹; G. Panic²; J. Keiser²; A. Hierlemann¹; ¹ ETH Zürich, Basel/CH; ² University of Basel/CH

185

P3.5.02 Magnetic 3D Bioprinting for High-Throughput and Automated Hepatotoxicity Testing G. Souza¹; B. Larson²; ¹ The University of Texas Health Science Center, Houston/USA; ² Biotek Instruments, Inc., Winooski/USA

187

P3.5.03 Cytotoxicity Evaluation of Nanoparticles using Automatic 3D Cell Culture System M. Heo¹; ¹ Korea Research Institute of Standards and Science, Yuseong-gu, Daejeon/ROK

189

P3.5.04 Automated large-scale production and deposition of spheroids K. Tröndle¹; ¹ University of Freiburg, Technical Faculty, Freiburg/D

190

P3.3.06 Gelatin-based hydrogels for 3D cell culture: stability at physiological temperatures by UV-crosslinking or nanoparticles K. Kruppa¹; A. Lavrentieva²; T. Scheper¹; I. Pepelanova³; ¹ Institute of Technical Chemistry, Leibniz University Hanover/D; ² Institute of Technical Chemistry/Leibniz University Hanover/D; ³ Institute of Technical Chemistry, Hannover/D

167

P3.3.07 A tubing-free, microfluidic tilting platform for the realization of in vivo-like drug exposure scenarios for three-dimensional microtissues C. Lohasz¹; O. Frey²; K. Renggli¹; A. Hierlemann¹; ¹ ETH Zürich, Basel/CH; ² Insphero AG, Schlieren/CH

168

P3.3.08 Organ-on-a-Disc – Enabling technology for the parallelization and automation of microphysiological systems S. Schneider¹; O. Schneider¹; F. Erdemann¹; C. Probst¹; P. Loskill¹; ¹ Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Stuttgart/D

170

P3.3.09 Porous, ultralight 3D tubular scaffolds from short electrospun nanofibers M. Merk¹; C. Adlhart¹; ¹ ZHAW Zürcher Hochschule für Angewandte Wissenschaften, Wädenswil/CH

171

P3.3.10 Enhanced cardiomyocyte maturation in a microfluidic system as a potential platform for pharmacological screening T. Kolanowski¹; M. Busek²; S. Grünzner³; F. Sonntag²; K. Guan¹; ¹ TU Dresden, Faculty of Medicine Carl Gustav Carus, Institute of Pharmacology and Toxicology, Dresden/D; ² Fraunhofer Institute of Material and Beam Technology IWS, Dres-den/D; ³ Fraunhofer Institute of Material and Beam Technology IWS; TU Dresden, Faculty of Manufacturing Technology, Dresden/D

173

P3.3.11 Autonomous Plug&Play Multi-Organ-Chips with Integrated Pumping and Sensing F. Sonntag¹; C. Probst²; S. Grünzner³; M. Busek⁴; P. Loskill⁵; ¹ Fraunhofer-Institut für Werkstoff- und Strahltechnik IWS, Dresden/D; ² Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Stuttgart/D; ³ Fraunhofer Institute for Material and Beam Technology IWS / Dresden University of Technology, Dresden/D; ⁴ Fraun-hofer Institute for Material and Beam Technology IWS, Dresden/D; ⁵ Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB / Eberhard Karls University Tübingen, Stuttgart/D

175

P3.3.12 vasQchip: A blood vessel scaffold for the reconstruction and 3D bioprinting of 3D-tissues in vitro U. Schepers¹; ¹ Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen/D

177

P3.3.13 A non-invasive microscopy platform for the online monitoring of human induced pluripotent stem cell aggregation in suspension cultures in small-scale stirred tank bioreactors I. Schwedhelm¹; D. Egger²; P. Wiedemann³; T. Schwarz⁴; H. Walles¹; J. Hansmann⁴; ¹ University Hospital Würzburg/D; ² University of Natural Resources and Life Scienc-es, Vienna/A; ³ Mannheim University of Applied Sciences, Mannheim/D; ⁴ Fraunhofer Institute for Silicate Research ISC, Würzburg/D

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3.6 3D printing

P3.6.01 Characterization of GelMa and alginate hydrogels for bioprinting: printability, polymerization and biocompatibility L. Raddatz¹; C. Schmitz¹; P. Gellermann¹; M. Kirsch¹; D. Geier²; S. Beutel¹; T. Becker²; T. Scheper³; I. Pepelanova¹; A. Lavrentieva¹; ¹ Institute of Technical Chemistry, Leibniz University Hanover, Hannover/D; ² Institute of Brewing and Beverage Technology, Forschungszentrum Weihenstephan, Technical University Munich/D; ³ Institute of Technical Chemistry/Leibniz University Hanover, Hannover/D

192

P3.6.03 Engineering bio-mimetic vasculature with photolithographic fabrication techniques A. Thomas¹; K. Schimek¹; G. Giese²; A. Kreuder¹; T. Grix¹; L. Kloke³; ¹ Technische Universität Berlin/D; ² Freie Universität Berlin/D; ³ Cellbricks GmbH, Berlin/D

193

P3.6.04 Characterisation of bioprinted mandibular osteoblasts for engineering an in vitro jaw bone model A. Amler¹; A. Thomas¹; T. Grix¹; R. Lauster¹; L. Kloke²; ¹ TU Berlin, Berlin/D; ² Cellbricks GmbH, Berlin/D

194

P3.6.05 3D Bioprinting of hydrogels for viral Infection and transduction with viral gene vectors T. Hiller¹; ¹ TU Berlin, Berlin/D

195

P3.6.06 3D-printed drug delivery systems for cell therapy:A new approach for the treatment of Diabetes Mellitus A. Pössl¹; P. Schlupp¹; T. Schmidts¹; F. Runkel¹; ¹ Technische Hochschule Mittelhessen, Gießen/D

196

P3.6.07 Imaging of O2 concentration and spatial distribution in 3D bioprinted hydrogel scaffolds using O2 sensing nanoparticles A. Akkineni¹; A. Lode¹; E. Trampe²; K. Koren²; F. Krujatz¹; M. Kühl²; M. Gelinsky¹; ¹ Technische Universität Dresden, Dresden/D; ² University of Copenhagen/DK

197

P3.6.08 Modelling of a microfluidic device to study tumor cell extravasation C. Kühlbach¹; R. Glunz¹; M. Mueller¹; F. Baganz²; V. Hass¹; ¹ HFU Hochschule Furt-wangen University, Villingen-Schwenningen/D; ² UCL University College London, London/UK

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Lecture Abstracts

27

Redefining cell culture environment with combinatorial biomatrices Alvin Kuriakose Thomas, Richard Wetzel, Dejan Husman, Nadine Schmieder-Galfe,

Robert Wieduwild, Yixin Zhang

Technische Universität Dresden, Center for Molecular and Cellular Bioengineering

B CUBE Center for Molecular Bioengineering, Arnoldstraße 18, 01307 Dresden,

Germany

The natural extracellular environment (ECM) is a complex interplay of extracellular

matrix components such as glycosaminoglycans (GAGs), proteoglycans, soluble factors

and cell-cell interactions. The ECM architecture is largely under-represented, in the

present-day cell culture models, used for biological and pharmacological research. A

biologically relevant environment for specific in vitro cell culture of choice, should

incorporate the essential inputs in a tailored manner in order to recapitulate the in vivo

milieu. In addition, developmental stages or pathological states of the cells depend on

specialized environments. The in vitro ECM mimetic developments today, is currently

limited to approaches which results in universal or “one-serves-all” solutions e.g.:

chemically treated plastic ware, protein coatings or basement membrane extracts.

We have developed a platform technology based on a biocompatible non-covalent

modular hydrogel system that can serve the demand for being chemically defined and

customizable. It incorporates GAGs and peptides enabling the versatile presentation of

essential cues from a biomimetic matrix. Our technology enables a screening approach

to identify the relevant composition for the cell type of choice.

We describe here, a number of screening setups to identify optimal conditions for the

maintenance of stemness or directed differentiation of stem cells. The biomatrix

presenting chondroitin sulphate along with a defined dose of an adhesion ligand; RGD,

promoted similar proliferation of human Mesenchymal Stromal Cells (MSCs) but

improved the preservation of naive stemness markers in comparison to currently used

plastic or fibronectin coated surface. Interestingly, Neuronal Precursor Cells (NPCs)

favoured chondroitin sulfate for expansion. However, neuronal differentiation was

Macromolecular crowding in 2D and 3D culture systems: creating of cell and stem cell specific microenvironments

Nicole Kohli, Zurich University of Applied Sciences, Wädenswil, Switzerland;

Michael Raghunath, Zurich University of Applied Sciences, Wädenswil, Switzerland

Cell culture studies are performed in standard medium, an aqueous salt solution with

varying addition of serum. This highly diluted condition does not mimic the natural

crowded environment of cells within tissues, where molecular interactions are

modulated by excluded volume effects. In order to remedy these limitations,

scientists mimic these crowded conditions by adding inert high-mass molecules. This

approach is called macromolecular crowding (MMC) [1]. We have shown that MMC

substantially enhances extracellular matrix (ECM) deposition and remodelling in

monolayer cell cultures. This gave rise to the Scar-in-a-Jar system allowing to study

the full depositional cascade of collagen and to screen antifibrotic drugs [2]. The

enhanced deposition of tissue-specific ECM under MMC has been demonstrated in a

variety of human and animal cells. Empowering stem cells to make their own

microenvironments via MMC leads to an accelerated differentiation of stem cells, for

example in adipogenesis. The improved microarchitecture of a collagen IV cocoon

around freshly differentiated adipocytes even unleashes dormant brown

differentiation potential in human bone marrow-derived stem cells [3]. While MMC

obviously generates ultraflat 3D systems, the question arises whether MMC also

would be efficacious in thicker systems, those, that are traditionally addressed as 3D.

We show here, that human mesenchymal stem cells (MSC) cultured in collagen

derived hydrogels and differentiated into white adipocytes using a classical induction

cocktail also generate fully surrounding collagen IV cocoon under MMC. Additionally,

enhanced deposition with a finer meshwork was observed for fibronectin. This

demonstrates efficacy of MMC also in hydrogel systems. Finally, MMC was shown in

bilayered skin constructs to accelerate the formation of the dermo-epidermal junction,

as evidenced by collagen VII deposition [4]. Thus, MMC has become a valuable tool

in 3D tissue engineering and cell culture systems.

[1] Chen CZC et al. Adv Drug Deliv Rev, 63(4-5):277-290, 2011

[2] Chen CZC et al. Br J Pharmacol, 158(5):1196-209, 2009

[3] Lee HCM et al. Sci Rep, 21173, 2016

[4] Benny P et al. Tissue Engineering A, 21(1-2):183-92, 2015.

28 29

iPSC-derived neurospheroids recapitulate development and pathological signatures of brain microenvironment

Ana Paula Terrasso1,2, Daniel Simão1,2, Neus Bayó-Puxan3,4,5, Francisca Arez1,2,

Marta M Silva1,2, Marcos F Sousa1,2, Sophie Creysells4,5, Patrícia Gomes-Alves1,2,

Nuno Raimundo6, Eric J Kremer4,5, Paula M Alves1,2 Catarina Brito1,2 1IBET, Instituto de Biologia Experimental e Tecnológica, Portugal; 2Instituto de

Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa,

Portugal; 3IBUB, Spain; 4Institut de Génétique Moléculaire de Montpellier, CNRS

UMR 5535, France; 5Université de Montpellier, France; 6Universitätsmedizin

Göttingen, Institut für Zellbiochemie, Germany

Brain microenvironment plays an important role in neurodevelopment and

pathology, where extracellular matrix (ECM) and soluble factors modulate multiple

cellular processes. Neural cell culture typically relies on the use of heterologous

matrices that poorly resemble the brain ECM or reflect its pathological features.

We have previously demonstrated that perfusion stirred-tank bioreactor-based 3D

differentiation of human neural stem cells (NSC) - pSTR-neurospheroids, sustains

the concomitant differentiation of the three neural cell lineages (neurons,

astrocytes and oligodendrocytes) and the establishment of physiologically relevant

cell-cell interactions 1,2.

Here, we hypothesized that if the pSTR-neurospheroid strategy would also allow

the deposition of native neural ECM components and diffusion of secreted factors,

it would be possible to: (i) mimic the cellular and microenvironment remodeling

occurring during neural differentiation without the confounding effects of

exogenous matrices; (ii) recapitulate the pathological phenotypes of diseases in

which alteration of homotypic and heterotypic cell-cell interactions and ECM

components are relevant. To demonstrate the first point, we analyzed pSTR-neurospheroid differentiation by

quantitative transcriptome (NGS) and proteome (SWATH-MS). Data showed that

neurogenic developmental pathways were recapitulated, with significant changes

at cell membrane and ECM composition, diverging from the 2D differentiation

profile. A significant enrichment in structural proteoglycans typical of brain ECM,

along with downregulation of basement membrane constituents was observed.

observed on a heparan sulphate containing matrix, an abundant GAG in the natural

environment, but poorly interrogated in biomaterial research. We further expanded our

ECM mimetic library to include growth factor mimetic peptides and thereby, identified a

unique composition for expansion of induced pluripotent stem cells (iPS) and iPS

derived neural precursor cells (iNPCs). We have proven, that iNPCs could be tuned to

maintain their stemness for up to 30 days of culture while preserving their neuronal

differentiation capacity. Hence, screening employing combinatorial biomatrices led us to

identify specific environments for different cell types and development stages.

Furthermore, our technology enables 3D culture models. Based on the combinatorial

screening, MSCs and fibroblast were co-cultured in a sandwich assay of differently

composed layers. Additionally, MSCs cultured on our material induced tubular network

formation of Human Umbilical Vein Endothelial cells. With these proof-of-principles we

show a successful transfer from 2D screening to 3D models. We have also been able to

extend our technology to be further engineered for 3D printing, or in vivo applications.

Our matrices are biologically relevant, modular, chemically defined and scalable. Our

platform technology could enable the researcher to perform high-throughput screens to

identify the appropriate environment for the defined cell based assays.

References:

1. In Vivo Examination of an Injectable Hydrogel System Crosslinked by Peptide–Oligosaccharide

Interaction in Immunocompetent Nude Mice. Advanced Functional Materials, 2017. DOI:

10.1002/adfm.201605189

2. Noncovalent hydrogel beads as microcarriers for cell culture. Angewandte Chemie Int. Ed., 2015.

DOI: 10.1002/anie.201411400

3. A Repertoire of Peptide Tags for Controlled Drug Release from Injectable Non-covalent Hydrogel.

Biomacromolecules, 2014. DOI: 10.1021/bm500186a

4. Minimal Peptide Motif for Non-covalent Peptide-Heparin Hydrogels. J. Am. Chem. Soc., 2013.

DOI: 10.1021/ja312022u

30 31

Expansion of mouse pancreatic organoids in a chemically defined three-dimensional matrix

Nadine Rischert1, Helmut Wurst1, Till Moreth2, Lotta Hof2, Ernst H.K. Stelzer2,

Meritxell Huch3, Francesco Pampaloni2, Brigitte Angres1 1Cellendes GmbH, Reutlingen, Germany, 2Goethe-Universität Frankfurt am Main,

Frankfurt am Main, Germany, 3University of Cambridge and Wellcome Trust -

Medical Research Council Stem Cell Institute, Cambridge, UK.

Organoids obtained from adult progenitor cells are emerging tissue models for drug

discovery screening since they reflect the native tissue physiology better than non-

organoid cultures1. Organoid cultures are also being developed for the cellular

therapy of diseases such as diabetes type 1 as an alternative to tissue

transplantations to overcome the limited availability of donated organs (project

LSFM4LIFE, www.lsfm4life.eu). Drug screening as well as therapeutic approaches

require an upscale of production of organoids in order to be able to provide the large

amounts of material needed. To this aim, an efficient expansion of organoids with

pharmaceutical-grade cell culture reagents is essential. In previous studies,

organoids have been grown in a 3D matrix derived from murine basement membrane

extract (e.g. Matrigel® or BME 2). Batch-to-batch variations and the animal origin

result in limited reproducibility of assay results and in organoids unsuitable for

therapeutic purposes due to pharmaceutical safety requirements. Therefore,

chemically defined 3D matrices are needed to obtain reproducible culture conditions

and thus reliable experimental outcomes, as well as compliance with pharmaceutical

regulatory rules.

Here, we present the development of a chemically defined hydrogel for the growth

and expansion of mouse pancreatic organoids. A soft hydrogel made of polyvinyl

alcohol and polyethylene glycol that contains ligands for cell adhesion and induction

of cell polarity allowed a successful and efficient propagation of the organoids for

many passages. Hyaluronic acid as a component of the hydrogel matrix further

improved the the growth efficiency and allowed more than a 6000-fold increase in cell

mass. The analysis of expression of progenitor markers like Sox9 and Lgr5 provide

first insights into the maintenance of the progenitor status of these cultures during

expansion. The expansion of pancreatic organoids in chemically defined matrices is

Moreover, higher expression of synaptic and ion transport machinery in pSTR-

neurospheroids suggest higher neuronal maturation than in 2D.

Having shown recapitulation of neural microenvironmental dynamics in pSTR-

neurospheroids, we used Mucopolysaccharidosis VII (MPSVII) as a disease case

study. MPS VII is a lysosomal storage disease caused by deficient �-

glucuronidase (�-gluc) activity, which leads to accumulation of

glycosaminoglycans (GAGs) in many tissues, including the brain. In pSTR-

neurospheroids generated from hiPSC of a MPS VII patient, the main molecular

disease hallmarks were recapitulated, such as accumulation of GAGs. Notably,

MPS VII neurospheroids showed reduced neuronal activity and a disturbance in

network functionality, with alterations both in connectivity and synchronization, not

observed in 2D cultures. These data provide insight into the interplay between

reduced �-gluc activity, GAG accumulation, alterations in the neural network, and

its impact on MPS VII-associated cognitive defects.

Overall we demonstrate that neural cellular and extracellular developmental and

pathological features are recapitulated in healthy and diseased pSTR-

neurospheroids, respectively. These can be valuable in vitro models to address

molecular defects associated with neurological disorders that affect neural

microenvironment homeostasis. Moreover, the 3D neuronal connectivity assay

developed is a new tool with potential to assess other lysosomal storage diseases

and neurodegenerative diseases that have variable phenotypes.

References 1. Simão, D. et al. Perfusion Stirred-Tank Bioreactors for 3D Differentiation of

Human Neural Stem Cells. Methods Mol. Biol. (2016).

doi:10.1007/7651_2016_333

2. Simão, D. et al. Modeling human neural functionality in vitro: three-

dimensional culture for dopaminergic differentiation. Tissue Eng. Part A 21, 654–

668 (2015).

Acknowledgments SFRH/BD/78308/2011, SFRH/BD/52202/2013 and SFRH/BD/52473/2014 PhD

fellowships from FCT, Portugal and iNOVA4Health-UID/Multi/04462/2013,

supported by FCT/ MEC, through national funds and co-funded by FEDER under

the PT2020 Partnership Agreement.

32 33

Observing three-dimensional biological specimens with light sheet-based fluorescence microscopy (LSFM)

Ernst H.K. Stelzer

Physical Biology (IZN, FB 15, CEF-MC II, BMLS) Goethe Universität, Frankfurt am Main, Germany

[email protected], +49 (69) 798 42547, x42545

A major objective of the Physical Biology Group is to perform experiments in the life sciences under close-to-natural conditions, i.e. to rely on three-dimensional biological specimens such as cysts, organoids, spheroids, blastoids, tissue sections and small model organisms. The scientific projects relate to developmental biology, including embryogenesis and tissue formation, as well as to cell biology, e.g. taking account of the role of specific pathways. Methods for specimen preparation and mounting are regularly adapted.

This talk concentrates on the development of new microscopes and image processing pipelines that are capable of handling millions of large-scale images. Applications from our research and our collaborations relate to three-dimensional cell biology in general and spheroids as well as organoids in particular.

The optical sectioning capability is fundamental for dynamic three-dimensional imaging. One of the very few instruments, which can claim this property is light sheet-based fluorescence mi-croscopy (LSFM).

In general, fluorescence microscopy provides a high contrast, since only specifically labelled cellular components are observed while all other structures remain “dark”. However, funda-mental issues are: 1) Excitation light degrades endogenous organic compounds and bleaches fluorophores. 2) A specimen provides only a finite number of fluorophores, which limits the number of collectable emitted photons. 3) Organisms are adapted to a solar flux of 1.4 kW/m2. Thus, irradiance should not exceed a few mW/mm2 or nW/µm2 in live imaging assays.

LSFM makes a sincere effort to address these challenges by decoupling the excitation and emission light pathways. The significance of the illumination-based optical sectioning property is that the viability and the fluorescence signal of a living specimen are retained while millions of images are recorded for days or even weeks.

Particular benefits of LSFM are: (i) good axial resolution, (ii) imaging along multiple directions, (iii) deeper tissue penetration due to the low numerical aperture of the illumination objective lens, (iv) high signal-to-noise ratio, (v) unrestricted compatibility with fluorescent dyes and proteins, (vi) reduced fluorophore bleaching and (vii) photo-toxicity at almost any scale, (viii) millions of pixels recorded in parallel and (ix) excellent specimen viability. www.researcherid.com/rid/A-7648-2011orscholar.google.com/citations?user=EV5RvqkAAAAJ

a major step forward for the preparation of pharmaceutically-grade material. The next

goal is translating these findings into a defined matrix for the growth of human

pancreatic organoids to provide a cell-based therapeutic approach for the treatment

of diabetes type 1.

1 Huch, Meritxell, et al. "The hope and the hype of organoid research." Development 144.6 (2017): 938-941. The LSFM4LIFE project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 668350

34 35

periphery of tumoroids derived from primary gastric tumor cells, with few T cells

penetrating into the tumoroid interior. This was reflected by shorter T cell infiltration

distance readouts, much lower infiltrated T cell count but no significant reduction in

tumoroid volume measurement, suggesting a mechanism by which these tumours

may be resistant to killing by T cells. Where invasive tumoroids were used, such as

MDA-MB-231, T cells predominantly ‘attacked’ invasive protrusions rather than

infiltrating the main body of the tumoroids. Quantitatively, this was reflected by

reduced tumoroid protrusion length and low T cell counts in the tumoroid and no

significant reduction in tumoroid volume measurement. The relative spatial and

geometric measurements provided by OMiner enabled the differentiation of various

tumour infiltration and killing patterns in response to different immunotherapeutic

treatments.

CONCLUSSIONS

A 3D environment allows the different cell types to engage in a more realistic setting

than when cells are grow in a monolayer. Using image-based analysis, various

immune-tumor interactions can be visualised and quantified. This represents a new,

highly powerful tool for cancer immunotherapy drug developers to select the most

promising treatments and understand better the spatial cellular context not detected

by alternative techniques.

Raw image overlay Tumoroids segmentation mask T cells segmentation mask Segmentation mask Overlay

Fig 1. MCF7 cell line was pregrown in 3D environment to form tumoroids. T cells

were stained with fluorescent dye and added together with immuno-modulatory

compounds. Infiltration into tumoroids and T cell-mediated killing was captured using

3D stack imaging and next quantified with the OMiner platform immuno-analysis

pipeline

Image-based quantification of immunotherapies effects in 3D environment

Kuan Yan, Lidia Daszkiewicz, Leo Price, OcellO B.V. Leiden, The Netherlands;

BACKGROUND

Delivering on the promises of cancer immunotherapy is hampered by a lack of in vitro

testing platforms that enable the early selection of promising treatments. Compared

to conventional 2D culture, 3D cultures can more faithfully reproduce the organization

of a tissue, recapitulating complex cell-cell interactions. However, readouts are

typically limited to biochemical measurements of viability or cytokine release and the

rich phenotypic information describing critical interactions between immune cells and

tumor is squandered. We therefore extended the OMiner software platform to enable

the 3D analysis of interactions between tumor and immune cells in a 3D co-culture

system.

METHODS

Tumor cells were cultured in a hydrogel to form 3D tumoroids. T cells labeled with a

fluorescent dye were added together with immuno-modulatory compounds and the

behavior of the immune cells and subsequent tumoroid killing was quantified with 3D

imaging and phenotypic analysis with the OMiner platform. Individual T cells and

tumoroids were identified from the 3D image stack using automated image

processing and machine learning algorithms. The phenotype of T cells and tumoroids

was determined, as well as their spatial relationship to describe the localization and

infiltration of T cells with respect to each tumoroid.

RESULTS

Automated 3D image and data analysis with OMiner enabled discrimination of

immune-tumor cell interactions depending on activation status of T cells. Counting of

T cells within each tumoroid region and measurement of the distance of T cell

penetration into the tumoroid provided a measurement of T cell infiltration into

tumoroids derived from MCF7 breast cancer cells. Measurement of tumoroid size

provided a readout of tumoroid killing. In contrast, T cells formed a ring around the

36 37

STATARRAYS©: microcavity arrays as a useful tool to detect single cell migration in a 4D co-culture model of human bone marrow

Eric Gottwald, Stefan Giselbrecht, Roman Truckenmüller, Vera Colditz*, Cordula

Nies*, 300MICRONS GmbH, Karlsruhe, Germany, * Karlsruhe Institute of

Technology, Karlsruhe, Germany

Introduction: Hematopoietic stem cells (HSC) reside in specialized environments of

the bone marrow, the so-called niches. Although there is general agreement about

the niche concept, the location(s), the cellular composition, and the size are still a

matter of debate. This discussion implies that there are some effects which cannot be

covered by some models whereas others can. One of the effects that can directly be

linked to differences in cellular behavior is the position of hematopoietic stem cells in

co-culture models. In 2010, Jing et al. [1] could show in a 2D-model of human HSC

from peripheral blood in co-culture with mesenchymal stromal cells (MSC), isolated

from bone marrow aspirates of healthy donors, that HSCs in this model formed three

subpopulations, one of which was only loosely adhering to the MSC feeder layer,

another one which was more tightly attached to the surface (phase bright cells) of the

MSC and finally one that displayed migration underneath the MSC layer (phase dim

cells) once they adhered to the MSC membrane. In subsequent experiments it was

shown that the third subpopulation displayed typical stem cell phenotype and

behavior as judged by FACS analysis and fluorescence microscopy. We, therefore,

tried to setup a 3D model of the human hematopoietic bone marrow niche to analyze

whether HSC in co-culture with MSCs display a more uniform behavior in 3D

environment with regards to stem cell maintenance.

Methods: To achieve this, we used the so-called STATARRAYS©, a new polymer

film-based 3D cell culture platform in 96 well format that comprises 169 microcavities

arranged in a hexagonal array. The microcavity arrays are manufactured by

microthermoforming and are typically made of tissue culture polystyrene or

polycarbonate. We cultivated the STATARRAYS© for up to 14 days and analyzed the

behavior of the HSC in 3D over time (4D) by taking confocal z-stacks of a subset of

microcavities inside one well. For cell tracking, the HSC have been labeled with

CellTracker Green-CMFDA and for immunofluorescence were stained for CD34

3D culture models for investigaing recruitment of stem cells to the vascular niche

Yoann Atlas1, Caroline Gorin2, Catherine Chaussain2, Stéphane Germain1, Laurent Muller1

1. CIRB, Collège de France, Paris, France 2. Dental School, Descartes University, Paris, France

Angiogenesis is a key event of organogenesis, both as a means for delivering

oxygen and nutrients, and through the paracrine interactions of endothelial cells with

perivascular cells, mesenchymal stem cells as well as pericytes. A mature vascular

component consisting in endothelial capillaries covered with perivascular cells should

thus be included in any 3D cell co-culture model aiming at getting closer to in vivo

situations. Indeed, stem cells and pericytes share molecular markers and

perivascular distribution. We have developed assays for investigation of capillary

network formation in hydrogels that allowed characterization of endothelial progenitor

cells (Ferratge et al, StemCellRes 2017) and of angiogenic potential of stem cells.

Indeed, we have shown that mesenchymal stem cells from the dental pulp (DPSC)

promote capillary formation through the secretion of the potent angiogenic factors

VEGF and HGF under the control of FGF-2. We thus proposed that pre-conditioning

DPSC with FGF2 prior to embedding in 3D hydrogels together with endothelial cells

improves vascularization (Gorin et al, Stem Cell Trans Med 2016). More recently, we

have investigated the recruitment of DPSC and the maturation of the vascular wall

through generation of basement membrane, since these two factors are required for

generation of functional blood vessels and support stemness.

Using lightsheet microscopy for rapid and largescale (mm3) analysis of 3D hydrogels,

we could measure the engagement of endothelial cells in capillary networks and

characterize culture conditions that control perivascular

recruitment of DPSC on endothelial capillaries, allowing some

DPSC to migrate and spread along capillaries, acquiring

typical perivascular cell morphology (purple cell on the green

capillary). The signaling pathways involved are currently

under investigation, with special focus on the PDGF-BB

pathway. Since DPSC express high levels of pericyte markers, we have established

morphological parameters in order to quantify recruited stem cells in the vascular

niche. Finally, we have also analyzed the participation of recruited DPSC to the

generation of basement membraneand shown that stem cells deposit type IV

collagen in the vascular niche, whereas isolated DPSC do not.

38 39

3D human liver spheroid systems for analyses of liver diseases, liver function, drug metabolism and toxicity

Magnus Ingelman-Sundberg, Delilah Hendriks, Tracey Hurrell, Inger Johansson, Sabine Vorrink, Åsa Nordling, Mikael Kozyra, Volker Lauschke,

Karolinska Institutet, Department of Physiology and Pharmacology, Stockholm, Sweden

Hepatic in vitro systems should be able to provide a cellular phenotype similar to the

situation in vivo in man. In recent years several 3D models mimicking appropriate

liver functions have been presented. Using a novel 3D PHH spheroid ULA plate

model based on chemically defined media, we observed that drug metabolism was

preserved for several weeks of cultivation and that transcriptomic, proteomic and

metabolomic analyses revealed a similar phenotype as in the corresponding livers in

vivo or in freshly isolated hepatocytes. In addition using this 3D spheroid systems we

have been able to mimic chronic drug toxicity, different liver diseases like NAFLD,

NASH and fibrosis and found the system suitable for evaluation of mechanisms

behind and for identification of novel drug candidates.

References: Bell CC, et al., Comparison of Hepatic 2D Sandwich Cultures and 3D Spheroids for Long-term

Toxicity Applications: A Multicenter Study. Toxicol Sci. 2018 Apr 1;162(2):655-666.

Vorrink S, et al., Prediction of drug-induced hepatotoxicity using long-term stable primary hepatic 3D spheroid

cultures in chemically defined conditions. Toxicol Sci. 2018 Mar 24. doi: 10.1093/toxsci/kfy058.

Vorrink SU, et al., Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term

3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics. FASEB J.

2017 Jun;31(6):2696-2708.

Bell CC et al., Transcriptional, Functional, and Mechanistic Comparisons of Stem Cell-Derived Hepatocytes,

HepaRG Cells, and Three-Dimensional Human Hepatocyte Spheroids as Predictive In Vitro Systems for

Drug-Induced Liver Injury. Drug Metab Dispos. 2017 Apr;45(4):419-429.

Lauschke VM, et al, Novel 3D Culture Systems for Studies of Human Liver Function and Assessments of the

Hepatotoxicity of Drugs and Drug Candidates. Chem Res Toxicol. 2016 Dec 19;29(12):1936-1955.

Hendriks DF, et al., Hepatic 3D spheroid models for the detection and study of compounds with

cholestatic liability. Sci Rep. 2016 Oct 19;6:35434.

Bell CC et al., Characterization of primary human hepatocyte spheroids as a model system for drug-induced

liver injury, liver function and disease. Sci Rep. 2016 May 4;6:25187.

(HSC) and CD105 (MSC). We then analyzed the number and position of the entire

HSC population of a subset of microcavities on a single cell level.

Results: Although the two stem cell populations were mixed prior to incubation, after

inoculation we could observe a segregation of the HSCs into two subpopulations.

The first one residing near the top of the microcavities and the second one residing at

the bottom (fig. 1).

HSC stained with

CellTracker Green and

immunofluorescence stai-

ning (CD34) after 14 days

in STATARRAY© culture.

This effect was even more pronounced after longer cultivation periods. Moreover, the

cells at the top of the microcavities lost their CD34-expression over time whereas the

“bottom population” kept its expression and even seemed to proliferate as the total

number of HSCs inside a given microcavity increased.

Discussion: We could show that STATARRAYS© are useful 3D systems for static

cell culture. Moreover, we have shown that 4D experiments (3D over time) can easily

be automated since the microcavity arrays are self-referencing. HSC in co-culture

with MSC segregated in two distinct subpopulations, one of which lost their stem cell

character and one of which maintained the stem cell character over the entire

cultivation period of 14 days. Moreover, the total number of HSCs in this

subpopulation increased over time by 62% indicating the usefulness for stem cell

expansion as well.

40 41

Three-dimensional tumor cell growth stimulates autophagic flux

and recapitulates chemotherapy resistance Corinna Bingel1,§, Emily Koeneke1,§, Johannes Ridinger1,§, Annika Bittmann1, Martin

Sill2, Heike Peterziel1, Jagoda K. Wrobel1, Inga Rettig1,3, Till Milde1,4,6, Uta

Fernekorn5, Frank Weise5, Andreas Schober5, Olaf Witt1,6 and Ina Oehme1* 1Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ), INF 280, D-69120 Heidelberg,

Germany and German Consortium for Translational Cancer Research (DKTK) 2Division of Biostatistics, German Cancer Research Center, Heidelberg, Germany 3current address: Roche Diagnostics GmbH, Mannheim, Germany 4Center for Individualized Pediatric Oncology (ZIPO) and Brain Tumors, Department of Pediatric Oncology, Hematology

and Immunology, University Hospital Heidelberg, Germany 5Department of Nano-Biosystem Technology, Technische Universität Ilmenau, Germany 6Translational Program, Hopp Children’s Cancer Center at NCT Heidelberg (KiTZ)

§ These authors contributed equally to this work.

Current preclinical models in tumor biology are limited in their ability to recapitulate

relevant (patho-) physiological processes, including autophagy. Three-dimensional

(3D) growth cultures have frequently been proposed to overcome the lack of

correlation between two-dimensional (2D) monolayer cell cultures and human tumors

in preclinical drug testing. Besides 3D growth, it is also advantageous to simulate

shear stress, compound flux and removal of metabolites, e.g. via bioreactor systems,

through which culture medium is constantly pumped at a flow rate reflecting

physiological conditions. Here, we show that both static 3D growth and 3D growth

within a bioreactor system modulate key hallmarks of cancer cells, including

proliferation and cell death as well as macroautophagy, a recycling pathway often

activated by highly proliferative tumors to cope with metabolic stress. The autophagy-

related gene expression profiles of 2D-grown cells are substantially different from

those of 3D-grown cells and tumor tissue. Autophagy-controlling transcription factors,

such as TFEB and FOXO3, are upregulated in tumors, and 3D-grown cells have

increased expression compared with cells grown in 2D conditions. Combining

cytotoxic treatment with compounds affecting late autophagic flux, such as

chloroquine, renders the 3D-grown cells more susceptible to therapy. Altogether, the

model presented here is a valuable tool to study drug response of tumor cells, as it

mimics tumor (patho-)physiology more closely, including the upregulation of tumor

relevant pathways such as autophagy.

Novel predictive 3D cultivation models for validating small molecules against KSHV infection

Tatyana Dubich1, Christoph Lipps1, Tobias May2, Marc Stadler1, Thomas Schulz3,

Dagmar Wirth1 1Helmholtz Centre for Infection Research, Braunschweig/Germany, 2InSCREENeX

GmbH, Braunschweig/Germany, 3Institute of Virology, Hannover Medical School,

Hannover/Germany

Kaposi’s sarcoma associated herpes virus (KSHV) infections can result in formation

of systemic tumor of endothelial origin, primary affecting immunosuppressed

patients. Up to date there is no targeted therapy and no vaccine available. To

overcome the lack of reliable in vitro and in vivo models for the investigation of the

viral pathogenesis and screening of potential antiviral compounds we developed a

cell culture system based on a growth controlled endothelial human cell line that

preserves properties of primary endothelial cells and gives rise to functional vessels

when transplanted to mice. Virus infection in 2D is associated with massive

alterations of cellular transcriptome reflecting endothelial to mesenchymal transition

(EndMT) typical for KS lesions in patients. 3D spheroids formed from infected cells

exhibit highly invasive growth, mimicking the tumorigenic properties of cells that are

observed in vivo. Upon engraftment in mice infected cells form lesions sharing

morphologic properties and cellular markers with Kaposi’s sarcoma. Notably, while

rapid viral loss is observed in standard 2D cell culture conditions, 3D culture and also

in vivo environment supports maintenance of virus. This underlines the importance of

3D culture conditions to closely mimic virus-induced tumorigenesis.

The systems were evaluated for identification and validation of novel antiviral

compounds. Making use of the 2D cell culture system we identified compounds

which reduce viral load in latently infected cells. Several compounds inhibiting

invasiveness in the 3D culture model could be identified. Of note, the 3D conditions,

but not the 2D evaluation correlated with significant reduction of tumor formation

when evaluated in the humanized mouse model, thus underlying the predictive power

of the 3D cell culture system for compound validation.

Taken together, the human growth-controlled endothelial cells support the

establishment of potent in vitro and in vivo models that allow investigation of the

pathogenesis of KSHV infection and validation of novel antiviral compounds.

42 43

In vitro skin models for clinical research and transplantation

Susan Gibbs, VU University medical Center and Academic Center for Dentistry, Amsterdam, The Netherlands

Human healthy and disease skin models are required for safety testing of all substances coming into contact with the skin and for efficacy as well as safety testing of novel drugs. Furthermore skin substitutes are being developed to apply directly to chronic wounds and deep burns to promote optimal healing with reduced scar formation. Skin tissue engineering has come a long way since the days of Bell et al who cultured the first bi-layered skin equivalent consisting of a reconstructed epidermis of a fibroblast populated collagen hydrogel. Complexity has been increased to introduce e.g. pigment forming melanocytes, endothelial cells and immune cells. Furthermore disease models representing e.g. fibrosis, melanoma and allergy have been developed. Current challenges now lie with introducing the skin appendages e.g. hair since the hair shaft is an important penetration route for substances applied to the skin. Regarding clinical applications, transplantation of viable hair follicles within the skin substitute might result in hair restoration which would be the first step towards reintroducing temperature regulation into the damaged skin area as well as having a substantial psychological impact for the patients. In this lecture, developments towards a next generation of skin models will be presented along with advancements in skin-on-chip.

Synthetic Biology-Inspired Treatment Strategies of the Future

Prof. Dr. Martin Fussenegger ETH Zurich, Department of Biosystems Science and Engineering, Basel, Switzerland

Since Paracelsus’ (1493-1541) definition that the dose makes the drug, the basic treatment strategies have largely remained unchanged. Following diagnosis of a disease the doctor prescribes specific doses of small-molecule drugs or protein pharmaceuticals which interfere with disease-associated molecular targets. However, this treatment concept lacks any diagnostic feedback, prophylactic impact and dynamic dosage regimen. We have pioneered the concept of metabolic prostheses which, akin to mechanical prosthesis replacing defective body parts, interface with host metabolism to detect and correct metabolic disorders. Metabolic prostheses consist of designer cells containing synthetic sensor-effector gene networks which detect critical levels of disease metabolites, processes pathological input with Boolean logic and fine-tune in-situ production and release of protein therapeutics in a seamless, self-sufficient and closed-loop manner. When implanted inside insulated, immunoprotective and autovascularizing microcontainers the metabolic prostheses connect to the bloodstream, constantly monitor the levels of disease-associated metabolites and trigger an immediate therapeutic response to prevent, attenuate or correct the disease. With their unique characteristic to dynamically link diagnosis to dose-specific in-situ production and delivery of protein pharmaceuticals, metabolic protheses will enable new treatment strategies in the future. To highlight the impact of synthetic biology on future biomedical applications, we will present our latest generation of remote-controlled gene switches, biosensor circuits and metabolic prostheses tailored to diagnose, prevent and cure high-prevalence medical conditions including diabetes, cancer and pain.

contact:Prof. Dr. Martin Fussenegger ETH Zurich, Department of Biosystems Science and Engineering,Basel, Switzerland Tel: +41 61 387 31 60 E-Mail: [email protected]

44 45

vascSkin-on-a-chip: combination strategies of human skin-equivalents and vasculature.

K. Schimek, TU Berlin, Berlin/Germany; A. Thomas, TU Berlin, Berlin/Germany; T.

Hasenberg, TissUse GmbH, Berlin/Germany; G. Giese, TU Berlin, Berlin/Germany;

A.K. Lorenz, TissUse GmbH, Berlin/Germany; U. Marx, TissUse GmbH,

Berlin/Germany; R. Lauser, TU Berlin, Berlin/Germany; G. Lindner, TU Berlin,

Berlin/Germany

The skin is the outermost barrier of the human body. Its various different cell types

are involved in the protection of the inner environment from the various outer

environmental stimuli. Drugs and cosmetics applied to the skin have to be screened

for toxicity and efficacy. In the last few decades, human skin equivalents composed

of one or two different cell types were improved substantially and are these days

widely used for animal free tests of these substances. However, limitations to

accurately predict the human outcomes still exist due to the simplicity of these

models. In particular, the implementation of a perfused vasculature would pave the

way to mimic structures and physiological responses of human native skin in vitro

more closely. Recent advances in microfluidic systems offer great potential in this

context. The two-organ-chip, a variant of TissUse multi-organ-chip platform,

comprises a miniaturized circulatory system with an integrated micropump. This

provides pulsatile circulation of microliter-volume of medium to the tissues in a similar

way to blood. Skin equivalents can be implemented either within 96well cell culture

inserts or in direct contact with the medium flow. In this study, perfusable vascular

channels have been successively integrated into a skin equivalent using

photopatterning technique. Following attachment of the endothelial cells to the

channel walls, the vascularized skin equivalents have been cultured with continuous

pulsatile flow conditions inside our two-organ-chip for 7 to 14 days. Histological

endpoint analysis showed a regular dermal/epidermal architecture of the skin

equivalents and consistency and vitality after long-term co-culture. The endothelial

cells covered all walls forming a viable fluid tight layer. Further, a physiological-like

elongation and orientation with the direction of flow could be demonstrated. These

results suggests the MOC a useful system for engineering a vascularized skin

construct promoting long-term culture and in vitro evaluation of topically and

systemically applied drugs and cosmetics.

Towards an immunocompetent skin model to study and develop materials for wound healing

Chiara Griffoni, Berna Sentürk, Markus Rottmar, Katharina Maniura-Weber

Empa Swiss Federal Laboratories for Materials Science and Technology, St. Gallen,

Switzerland

The increasing occurrence of chronic skin wounds has been pushing forward both

the development of novel therapeutic options and skin tissue engineering research in

general. One of the major issues in wound care is the absence of models enabling to

assess new treatments designed to improve healing. Additionally existing skin in vitro

models do not reflect the complexity of the physiological environment in which

chronic wounds occur, as they generally lack an immune component. An

immunocompetent wound healing model that closely mimics the human skin could

thus greatly improve the evaluation of novel wound treatments. As a first step, a

suitable co-culture medium for fibroblasts, keratinocytes and monocyte-derived

macrophages was identified by analyzing cell viability, morphology, proliferation,

differentiation and polarization. In parallel, in vitro skin encompassing a collagen-

based dermis seeded with primary fibroblasts and a stratified epithelium obtained

after air-lift culture of primary keratinocytes was established. Full thickness wounds

were created with a biopsy punch and healing was assessed in terms of wound

closure rate, while cytokine secretion was measured to monitor inflammatory

responses. A fibrin hydrogel was used as a wound filling material, leading to a

decrease of the wound area over time and no significant change in interleukin

expression. When encapsulating primary macrophages into a collagen gel, they were

found to be viable in the hydrogels over 7 days of culture, maintaining the ability to

elicit inflammation through cytokine secretion when stimulated, thus demonstrating

the feasibility to include the macrophages into the skin model in a next step. For

future applications, the described platform could be used for evaluating novel wound

healing treatments at a pre-clinical stage and also to understand the molecular

mechanisms of skin disease processes where immune cells have a central role.

46 47

Pre-vascularized cell cultivation system to generate perfused 3D co-culture models

I. Prade1; M. Busek2; M. Wiele1; F. Sonntag2; M. Meyer1, 1FILK, Freiberg/Germany; 2Fraunhofer IWS, Dresden/Germany

Introduction Microfluidic cultivation platforms significantly improve the supply of cells in vitro. A

key challenge however is the sufficient distribution of oxygen and nutrients within a

3D tissue construct. We present a novel approach for the cultivation of a 3D cell

culture model using a pre-vascularized collagen matrix connected to a perfused

bioreactor system.

Results The matrix consists of a chemically cross-linked fibrous collagen material and

contains perfusable vascular-like collagen hollow fibers. The fibers are coupled with a

microfluidic circulation system and continuously permeated with nutrients and

oxygen. This system was used to grow a co-culture for several days with human

primary endothelial cells seeded within the hollow tubes. Confocal microscopy

analyses revealed the absence of necrotic areas. To characterize the distribution of

oxygen in the cell culture module, fluorescence sensors and immune fluorescence

staining against hypoxia signaling markers were performed. Additionally, cell type

specific marker expression was investigated in histological sections and in Western

Blot.

Conclusion The results demonstrate that our system is able to support the growth of 3D cell

culture models by providing a biocompatible 3D matrix and a microfluidic circulation.

The integrated hollow fibers allow the supply of nutrients and oxygen immediately

after cell seeding. The resulting tissue construct is biodegradable and vascularized.

Abstract NRAS mutation in melanoma has been associated with aggressive tumor biology and poor

prognosis. Although targeted therapy has been tested for NRAS mutated melanoma, response

rates still appear much weaker, than in BRAF mutated melanoma. While plenty of cell lines

exist, however, only few melanogenic cell lines retain their in vivo characteristics. In this

work we present an intensively pigmented and well-characterized cell line derived from a

highly aggressive NRAS mutated cutaneous melanoma, named MUG-Mel2. We present the

clinical course, unique morphology, angiogenic properties, growth characteristics using in

vivo experiments and 3D cell culture, and results of the exome gene sequencing of an

intensively pigmented melanogenic cell line MUG-Mel2, derived from a cutaneous metastasis

of an aggressive NRAS pQ61K mutated melanoma. Amongst several genetic alterations,

mutations in GRIN2A, CREBP, PIK3C2G, ATM, and ATR were present. These mutations,

known to reinforce DNA repair problems in melanoma, might serve as potential treatment

targets. In vitro and in vivo imaging achieved an enormous contribution to the detailed

characterization of the new established cell line MUG-Mel2. The aggressive and fast growing

behavior in animal models and the obtained phenotype in 3D culture reveal a perfect model

for research in the field of NRAS mutated melanoma.

MUG-Mel2, a novel highly pigmented and well characterized NRAS mutated human melanoma cell line in 3D culture

B. Rinner¹; G. Gandolfi²; K. Meditz¹; M. Frisch¹; K. Wagner¹; A. Ciarrocchi²; F. Torricelli²;

R. Koivuniemi³; J. Niklander³; B. Liegl-Atzwnager¹; B. Lohberger¹; E. Heitzer¹; N. Ghaffari-Tabrizi-Wizsy¹; D. Zweytick¹; I. Zalaudek¹

¹ Medical University of Graz, Graz/A; ² Laboratorio di Ricerca Traslazionale Arcispedale S. Maria Nuova - IRCCS, Reggio Emilia/I

³ University of Helsinki, Helsinki/FIN

48 49

structural organization. Tissue-like functionality of electrostimulated constructs is

demonstrated by their electrocardiogram (ECG)-like signals, which account for

improved synchronization and electrical signal propagation, and their ability to predict

drug-induced cardiotoxicity. Overall, this technology allows for the production of a 3D

model of human cardiac tissue with an increased complexity, improved physiological

relevance and ability to predict drug-induced cardiotoxicity.

Generation of 3D human cardiac macrotissues with tissue-like functionality

Maria Valls-Margarit*, Institute for Bioengineering of Catalonia (IBEC), Barcelona;

Olalla Iglesias-García*, Center of Regenerative Medicine in Barcelona (CMRB),

Barcelona; Claudia Di Guglielmo, Center of Regenerative Medicine in Barcelona

(CMRB); Leonardo Sarlabous, Institute for Bioengineering of Catalonia (IBEC),

Barcelona; Roberto Paoli, Institute for Bioengineering of Catalonia (IBEC),

Barcelona; Jordi Comelles, Institute for Bioengineering of Catalonia (IBEC),

Barcelona; Dolores Blanco-Almazán, Institute for Bioengineering of Catalonia (IBEC),

Barcelona; Senda Jiménez-Delgado, Center of Regenerative Medicine in Barcelona

(CMRB); Oscar Castillo-Fernández, Institute of Microelectronics of Barcelona, IMB-

CNM (CSIC), Bellaterra. Josep Samitier, Institute for Bioengineering of Catalonia

(IBEC), Barcelona; Raimon Jané, Institute for Bioengineering of Catalonia (IBEC),

Barcelona; Elena Martínez#, Institute for Bioengineering of Catalonia (IBEC),

Barcelona; Ángel Raya#, Center of Regenerative Medicine in Barcelona (CMRB),

Barcelona.

* These authors contributed equally to this work. # Share senior co-authorship.

Sub topic - Advanced cell culture models: Complex and multi-cell type models

In vitro 3D models of human cardiac tissue hold great promise in disease modelling,

drug screening and toxicity testing, and regenerative medicine applications. Human

cardiac tissue constructs developed thus far recapitulate some of the complexity and

electromechanical functionality of the native myocardium through the generation of

microengineered models. However, the production of human macroscale tissues

displaying in vivo-like complexity and, therefore, tissue-like functionality, is still an

unmet challenge. Here, we report the design and fabrication of a platform for the

production of human cardiac macrotissues from human induced pluripotent stem

cells (hiPSC). The methodology developed is size scalable in nature, and allows on-

line non-destructive measurements by using a novel parallelized perfusion bioreactor

with electrostimulation capabilities. Constructs cultured in this platform develop into

macroscopically contractile structures displaying in vivo-like electrophysiological

properties, functional response to drugs and cardiomyocytes with a high level of

50 51

Merging high-content and high-throughput screening: Microphysiological Organ-on-a-Chip systems featuring complex

human tissues with physiological structure and function Peter Loskill

Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Nobelstraße

12, 70569 Stuttgart, Germany;

Research Institute for Women’s Health, Eberhard Karls University Tübingen,

Silcherstr. 7/1, 72076 Tübingen, Germany

Drug discovery and development to date has relied on animal models, which are

useful, but fail to resemble human physiology. The discovery of human induced

pluripotent stem (hiPS) cells has led to the emergence of a new paradigm of drug

screening using human patient- and disease-specific organ/tissue-models. One

promising approach to generate these models is by combining the hiPS

technology with microfluidic devices tailored to create microphysiological

environments and recapitulate 3D tissue structure and function. Such

microphysiological organ-on-a-chip systems (OoCs) combine human genetic

background, in vivo-like tissue structure, physiological functionality, and

“vasculature-like” perfusion.

Using microfabrication techniques, we have developed multiple OoCs that

incorporate complex human 3D tissues and keep them viable and functional over

multiple weeks, including a “Retina-on-a-chip”, a “Heart-on-a-chip” and a

“White adipose tissue(WAT)-on-a-chip”. The OoCs generally consist of three

functional components: organ-specific tissue chambers mimicking in vivo

structure and microenvironment of the respective tissues; “vasculature-like”

media channels enabling a precise and computationally predictable delivery of

soluble compounds (nutrients, drugs, hormones); “endothelial-like” barriers

protecting the tissues from shear forces while allowing diffusive transport. The small

scale and accessibility for in situ analysis makes our OoCs amenable for both

massive parallelization and integration into a high-content-screening approach.

The adoption of OoCs in industrial and non-specialized laboratories requires

enabling technology that is user-friendly and compatible with automated workflows.

We have developed technologies for automated 3D tissue generation as well

as flexible plug&play connection of individual OoCs into multi-organ-chips. These

technologies paired with the versatility of our OoCs pave the way for applications in

drug development, personalized medicine, toxicity screening, and mechanistic

research.

Advanced induced pluripotent stem cell (iPSC) screens Matthias Müller, Novartis Institutes for BioMedical Research, Basel/CH

Motor neuron (MN) diseases are progressive disorders resulting from degeneration

of neuromuscular junctions (NMJs), which form the connection between MNs and

muscle fibers. There is currently no effective treatment to promote NMJ regeneration

in such disease. Studying only MNs or muscle cannot give a comprehensive and

complete view of the neuromuscular diseases. To increase our understanding and

find a cure for such conditions, easily controllable and monitorable cell culture

models would allow a better dissection of certain molecular and cellular events that

cannot be teased apart. Unfortunately, there is currently no confirmed robust human

in vitro model for neuromuscular diseases available. Through the use of iPS

technology we were able to generate and miniaturize human neuromuscular

processes in a well. When iPS derived neurons were co-cultured with iPS derived

myotubes contraction could be detected which demonstrate the physiological

relevance of the assay system. The use of a G-Camp reporter to monitor the Ca

changes and indirectly the contraction as well as full automatization of the whole cell

culture process allows high well to well reproducibility and ultimately enhanced

compound screening capability. Indeed positive and negative regulators could be

detected which validates the developed co-culture system. Use of this fully human

model will provide more accurate prediction of drug effects in humans. This defined

human-based NMJ system is a first step in creating functional in vitro systems.

Currently we are developing the next generation co-culture models. The goal is to

enhance the system by adding astrocytes and/or microglia and to generate 3D

cultures which will provide a more physiological organization.

52 53

Development of a matrix-based technology platform for the high throughput analysis of 3D cell cultures

M. Rimann1, A. Picenoni1, E. Bono1, E. Felley-Bosco2, C. Hund3, R. Pellaux3, A.

Meyer3

1Institute of Chemistry and Biotechnology (ICBT), Zurich University of Applied

Sciences, Waedenswil, Switzerland; 2Laboratory of Molecular Oncology, Zurich

University Hospital, Zurich, Switzerland; 3FGen GmbH, Basel, Switzerland

The screening of large cell libraries is an important process in pharmaceutical

discovery and R&D, e.g. to define drug targets or develop effective medicines.

Crucial for proper cell behavior is the stiffness of the cells’ microenvironment. The

goal of this project is the implementation of a screening platform based on 3D

cultivation of primary human cancer cells encapsulated in alginate-based hydrogels

providing different E-moduli. To this end, hydrogel compositions exhibiting different

physical and biological properties will be designed, tested and finally utilized in the

automated nanoliter-reactor (NLR) cultivation system that enables high throughput

analysis of 3D cell cultures.

In the present study, we are focusing on human mesothelioma cells. Mesothelioma is

the direct cause of asbestos exposure. We successfully encapsulated mesothelioma

cell line ZL55 into alginate beads (bead diameter, 1 – 1.6 mm). Three types of

alginates with E-moduli of 1.7 ± 0.1 kPa up to 10.3 ± 1.7 kPa were tested, showing a

cell viability of 50 – 70% the day after the encapsulation. Cells were proliferating and

formed spheroids (spheroid diameter, 40 – 55 µm) up to 14 days of culture, with an

oval morphology that characterized some cell spheroids at day 14.

Further analyses are currently running to correlate the material stiffness to

cell/spheroid proliferation and behavior in order to simulate the in vivo tumor

microenvironment. Afterwards, the process will be adapted to the NLR-technology to

produce smaller, monodisperse capsules (bead diameter 200 and 500 µm) in a high

throughput manner with cells obtained from patients suffering of mesothelioma. The

final goal will be the generation of clonal patient derived cell lines which then can find

application for personalized drugs screening as wells as cancer cell models.

Funding: CTI, Project Numbers: 18364.2 PFLS-LS

Modification of a standardized 3D in vitro tumor-stroma model for high throughput screening of candidates of new tumor therapeutica Sabine Hensler,1 Claudia Kühlbach,1 Margareta M. Müller1

1 Hochschule Furtwangen University, Germany

During the last decades essential progress has been made in understanding the role of

stromal reactions that support tumor growth and progression. Our lab has shown that tumor

progression in the HaCaT model of human skin carcinogenesis in vivo is dependent upon the

persistent recruitment of neutrophils and macrophages into the tumor microenvironment.

Tumor cells activate inflammatory as well as fibroblastic cells via the secretion of specific

growth factors like IL-6, G-CSF, GM-CSF and PDGF and induce the expression of

progression promoting cytokines like HGF, MCP-1 and VEGF and proteases (e.g. MMP-9

and MMP-13) (Linde et al 2012, Lederle et al, 2006, Lederle et al 2011). To better

understand the cellular interactions necessary for this crosstalk and specifically to analyze

the dynamic interaction between tumor associated fibroblasts and the inflammatory

compartment that seems to play a major role in establishing a tumor promoting environment

we extended our 3D organotypic in vitro model for carcinomas (Gutschalk et al. 2013) to

analyze the interaction of macrophages, neutrophils and fibroblasts in the tumor

microenvironment of epithelial tumors. Additionally we successfully incorporated HUVEC

endothelial cells into the system and could demonstrate an in vivo like differentiation towards

a tumor supporting phenotype of all stromal cells. Therefore, our new model provides an in

vitro tissue context to analyze tumor stroma interactions and represents an excellent

possibility for a targeted interference in the respective interaction processes as well as for the

analysis of angiogenic processes in the tumor microenvironment. As such, the model is

highly suitable for pharmaceutical screening of novel therapeutics, since it allows the

analysis of drugs in a 3D tissue like context that highly resembles the in vivo environment.

For the use in high throughput screening for new cancer therapeutic candidates, we adapted

the 3D tumor stroma model to the use of a chemically inert hydrogel matrix and are

miniaturizing it to a 96-well format. Easy visualization of tumor cells in the model is achieved

by GFP transfection of tumor cells (currently lung and breast carcinoma and glioblastoma).

54 55

cardiomyocyte spheroids, and CYP induction/inhibition in hepatocyte spheroids.

These endpoints are easy to image and analyze, and while they do not depend solely

on fluorescent imaging for results, their label-free and non-lytic nature allow for

multiplexing with other endpoints. Our results demonstrate that magnetic 3D

bioprinting is singularly built to engineer assays that can model cellular and tissue

function in vitro for high-throughput and high-content screening.

Furthermore, significant efforts have been undertaken in the field of cancer to

develop clinically relevant pre-clinical models to permit the translation of research

advances into agents that can improve patient outcomes. Patient-derived xenograft

(PDX) models in which patient tumor tissue implanted into immune-deficient mice

provide a more accurate reflection of human tumor biological characteristics than

immortalized cancer cell lines. These models provide possibilities for better

preclinical testing of new therapies for the treatment and better outcomes of cancer.

A significant drawback associated with the use of PDX systems is that their

generation requires immense resources, are costly and time-consuming. Also, they

have limited use as platforms for high throughput drug screens. Utilizing the

technology of magnetic 3D bioprinting we developed a PDX-derived ex-vivo tumor

tissue platform for use in high throughput drug screens. The PDX-derived ex-vivo

tumor tissue generated through magnetic 3D bioprinting mirrors the original PDX

tumor in both tissue architecture and genetic signature. We confirm the predictive

value of our screening template by demonstrating a similar response outcome

between our model and a PDX mouse system generated from the same original

tumor tissue and treated with the same panel of drugs. Utilizing an Inflammatory

Breast Cancer PDX system as our model system, we screened our ex-vivo tumor

tissue template with the anti-cancer drug library and identified lead candidates that

had a stronger anti-tumor activity compared to the standard of care agents used to

treat IBC patients in the clinics. Incorporating our ex-vivo tumor tissue high

throughput screening platform as an early step in a PDX system will permit the

identification of new and effective tumor-specific therapies in a time, cost and

resource efficient manner.

Magnetic 3D Bioprinting for High-Throughput Compound Screening and Translational Applications

Glauco R. Souza1,2 and Geoffrey Bartholomeusz3 1University of Texas Health Science Center at Houston, Texas 77030

2Nano3D Biosciences, Inc., Houston, Texas 77030 3University of Texas MD Anderson Cancer Center, Houston, Texas 77045

High-Throughput Screening and Automation

The limited relevance of non-human tissues and the need to reduce the dependence

on animal-intensive tests define an unmet need for in vitro assays with fewer ethical

challenges and the potential for needed translational capabilities. Thus, there is a

demand for an in vitro assay that is predictive of in vivo drug response, uses human

cells, and is adaptable to high-throughput screening. To meet such need, biomedical

research has gravitated towards 3D cell culture as scientists seek cellular models

and assays that represent in vivo tissue more accurately than traditional 2D

monolayers. Challenges in 3D cell culture both technical challenges information and

handling, but also analytical challenges, as the density of 3D cell cultures can make

imaging difficult. Towards that end, we discuss a recently developed platform for 3D

cell culture, magnetic 3D bioprinting, that addresses the technical issues of 3D cell

culture. The principle behind magnetic 3D bioprinting is the magnetization of cells

using a biocompatible nanoparticle assembly (NanoShuttle), which can then be

rapidly aggregated into spheroids using mild magnetic forces. Magnetization occurs

at the cellular level by the binding of nanoparticles to cell membranes, and as a

result, spheroids can be scaled down to small sizes (<1,000 cells) for high-throughput

formats (384- and 1536-well). The magnetization of the spheroid also allows for the

spheroids to be held in place with magnetic forces during liquid transfer, thereby

improving sample retention. With this system, we can more easily create

representative models in vitro for research. To overcome the analytical challenges

that all 3D cell culture platforms encounter, particularly light and reagent penetration

in 3D cell cultures, we designed and created unique assays that allow for image-

based endpoints that escape these limitations and others with the use of magnetic

fields. These include wound healing in 3D rings, contraction in spheroids; beating in

56 57

Microtissues meet microfluidics – next generation microphysiological tilting system

Kasper Renggli1, Christian Lohasz1, Sebastian Bürgel1, David Fluri2,

Andreas Hierlemann1, Olivier Frey2

1ETH Zurich, Dept. of Biosystems Science and Engineering, Basel, Switzerland 2InSphero AG, Schlieren, Switzerland

The better in vitro models reflect function and structure of their in vivo counter parts,

the more predictive cell-based assays will become. The combination of 3D tissue

engineering approaches with microfluidics technology to realize microphysiological

systems comprising of several cell/organ models (body-on-a-chip formats) addresses

this issue [1].

We developed a tubing-free and simple-to-use microfluidic platform that enables

culturing and interconnection of different types of 3D microtissues under

physiological flow conditions (Figure 1). Open media reservoirs are located at both

ends of each channel, and perfusion flow is generated through tilting the device back

and forth on an automated system inside an incubator. This design offers multiple

attractive features: (i) simple microtissue loading and harvesting through standing-

drop ports; these ports enable direct access to the culture compartments from the top

throughout the experiment and provide sufficient oxygenation via the air-liquid

interface; (ii) optimized accommodation of the microtissues in dedicated culture

compartments that enable physiological flow conditions and shear-forces without

tissue adhesion and functional loss; (iii) tubing-free medium transfer and circulation

by hydrostatic pressure build-up upon platform tilting; (iv) the injection moulded

polystyrene chips are biocompatible, transparent for optical microscopy and stable

for long-term cultures without problems of protein adsorption and small molecule

absorption that have been observed with PDMS; (v) optical accessibility from

underneath renders the device compatible with automated read-out and analysis

instruments, so that tissue integrity and morphology can be controlled during

experiments; (vi) simplicity in handling and operation, which results in high

robustness and reproducibility of experimental data; and (vii) scalability is achieved

by operating multiple devices in parallel on an automated tilting platform in a

standard well-plate-compatible format.

Simple and robust microfluidic platform for spheroid culturing in a high-throughput manner

Jin-Young Kim and Hongsoo Choi

DGIST-ETH Microrobotics Research Center, Daegu Gyeongbuk Institute of Science and Technology, Daegu, South Korea

In line of the increased interest in in-vitro three-dimensional cell culture methods for the last years, multi-cellular spheroids have become a widely used 3D microtissue model. It is because their scaffold-free cellular structure mimics better in-vivo environments with respect to oxygen and nutrient gradients as well as with respect to the presence of a natural extracellular matrix (ECM). In addition, microfluidic platforms have been considered as a promising tool for biological applications, because they represent physiological conditions more closely with regard to liquid-to-cell ratios and fluid residence times. In this study, a microfluidic platform for long-term culturing of multiple spherical

microtissues (MTs) in a high-throughput manner has been developed. The device was fabricated in a conventional 96-well format and consists of 6 separate MT compartments, inter-connected through perfusion channels. After spheroids formed externally using the U-bottom well plate was transferred into the microfluidic device, they were cultured under continuous perfusion. The tilting tower system has been developed in a conventional 96-well plate rack format that provide gravity-driven flow between the two open reservoirs of the device by automated tiling in a high-throughput way without additional pumps. It makes the system operation in a conventional incubator simple and robust. The tilting tower system offers independent access to each device which is compatible for robot arms of an automated plate loader. In addition, each floor of the tilting tower system can be easily attached and detached so the height of the system can be modified in a straightforward way, depending on various experiment conditions such as space in an incubator and the number of devices to run. Furthermore, the two open medium reservoirs make sampling easy during long-term MT culturing and ensure sufficient gas exchange. Spheroids have been cultured over 8 days in the device, while growth has been

monitored using an optical microscope. Albumin secretion has been quantified using an enzyme-linked immunosorbent assay (ELISA). Higher growth rates of human hepatic carcinoma (HepG2) MTs were observed when cultured under continuous perfusion in the microdevice in comparison to static cultures in a conventional well plate. Furthermore, multiple tumor MTs (HCT116) were exposed to various concentration of 5-fluorouracil for the system characterization. The platform concept allows for experiments in a highly parallelized fashion through

arraying of many perfusion channels in parallel and adding more compartments. Currently the tilting system consists of 10 floors which can culture up to 600 MTs but it can be easily increased by addition the tilting floors. Further, multi-tissue experiments are made available when loading different organotypic spheroids or tumor spheroids into interconnected compartments. This will not only allow for further improving the culturing environment, but also for investigation of inter-organ effects using multiple spheroids. Finally, it also has a potential for commercialization and automation with existing techniques since it was developed in a conventional 96 well plate and rack format.

58 59

The application of microphysiological systems in drug discovery using case studies from safety and efficacy questions

Lorna Ewart, IMED Biotech Unit, Drug Safety and Metabolism, AstraZeneca, Cambridge, UK

Microphysiological systems, which encompass the Organ-Chip models, are designed to closely mimic the cellular microenvironment. As such, these models are poised to transform drug discovery and development by providing greater insight into biological mechanisms that drive disease. Within AstraZeneca, we believe that these models offer tremendous value and are committed to collaborative endeavors with the engineers and biologists who are at the heart of model development. This presentation will showcase our collaborations and will bring to life how microphysiological systems can impact our industry. Specifically the presentation will address the needs for characterization and validation of these models as well as some of the existing challenges precluding broader adoption within the industry.

[1] U. Marx, et al., Biology-inspired microphysiological system approaches to solve the prediction

dilemma of substance testing, ALTEX, 33, 272–321, 2016.

Figure 1: Design and operation of the microfluidic tilting platform for microtissue spheroids. (a) Layout

of the device. Each channel harbors ten spheroid compartments. (b) Close-up side view and (c) top

view of a spheroid compartment, depicting the standing drop formed on top of the compartment and

barrier structures protecting the spheroid from shear stress. (d) Assessment of the velocity of liquid

around the microtissue compartment for a flow rate of 10 �L min–1 using computational fluid dynamics

simulations (e) Tilting of the platform induces gravity-driven flow and microtissue perfusion in the

channels.

60 61

Single-donor iPSC derived Multi-Organ-Chips Anja Ramme1*, Leopold Koenig1, Daniel Faust1, Anna Krebs1, Tobias Hasenberg1,

Eva Dehne1 and Uwe Marx1 1TissUse GmbH, Oudenarder Str. 16, 13347 Berlin, Germany

*presenting author: [email protected]

TissUse Multi-Organ-Chip (MOC) platform contributes to the ongoing development of systemic substance testing in vitro. Current in vitro and animal tests for drug development are failing to emulate the systemic organ complexity of the human body and, therefore, often do not accurately predict drug toxicity.

We have developed a universal MOC platform, the size of a standard microscopic slide, for long-term culture of human iPSC derived, primary- or cell line-based 3D organ equivalents. These organoids are interconnected through a microfluidic system. An integrated on-chip micropump provides physiological pulsatile fluid flow at a microliter scale thus supporting improved nutrition and oxygen supply. Moreover, these minute amounts of enriched cultivation medium enable crosstalk between the organoids. The transparent MOC´s support life tissue imaging, as well as the integration of commonly used Transwell® inserts. We cultured iPSC derived human organoids in the multi-organ-chips for up to two weeks. Data on iPSC derived intestinal organoids, hepatocyte spheroids, and endothelial cells will be presented.

Rationale and approaches to combine multiple autologous iPSC derived 3D organ equivalents into functional multi-organ arrangements at long-term homeostasis are discussed. These further developments will lead to personalized donor specific Multi-Organ-Chips, ready to be used for example, for individualized drug response assays.

BRINGING 3D TUMOR MODELS TO THE CLINIC – PREDICTIVE VALUE FOR

PERSONALIZED MEDICINE

Halfter K, SpheroTec GmbH, Munich/Germany; Mayer B, Hospital of the LMU,

Munich/Germany

Current decision-guiding algorithms in cancer drug treatment are based on decades

of research and the result of numerous clinical trials. For the majority of patients, this

data, as summarized in guidelines, is successfully applied for the systemic

management of the disease. For a number of patients however, treatment

stratification according to clinically based risk criteria will not be sufficient. This

includes patients with recurrent or metastatic disease, geriatric patients, as well as

patients with chemoresistant tumors. Persisting medical conditions, drug intolerance

or other comorbidities may also interfere with guideline-based treatment selection.

The most effective treatment options are ideally identified prior to the start of clinical

drug therapy. This review will discuss the implementation of three-dimensional (3D)

cell culture models as a preclinical testing paradigm for the efficacy of clinical cancer

treatment. Patient tumor-derived cells in 3D cultures duplicate the individual tumor

microenvironment with a minimum of confounding factors. Prior to clinical

implementation of such personalized tumor models, a high quality of methodological

and clinical validation is essential and should follow similar recommendations as

other biomarkers. A non-systematic literature search was performed to demonstrate

the small number of studies that have been conducted in this area of research using

a prospective trial design. This may explain the current reluctance of many

physicians and insurance providers in implementing this type of assay into the clinical

diagnostic routine. Achieving valid and reproducible results with a high level of

evidence is central in improving the benefit of preclinical 3D tumor models.

62 63

From start on, BB3R trained the next generation of scientists by introducing the first

graduate school worldwide on 3R education. The established graduate program aims

to qualify PhD students for a subsequent career in the field of 3R-related life science

or science administration.

References: 1 Arrowsmith et al., 2011; 2 Zoschke et al., 2016; 3 Hönzke et al., 2016 a;4 Alnasif et al., 2014; 5 Hönzke et al., 2016 b, 6 Maschmeyer, 2015; 7 Zoschke et al., 2015

Rethinking Drug Development - 3D Disease Models for Advanced Preclinical Drug Evaluation

Monika Schäfer-Korting, Sarah Hedtrich, Vivian Kral, Günther Weindl, Johanna

Plendl, Christa Thöne-Reineke, Freie Universität Berlin, Germany; Burkhard Kleuser,

Potsdam University, Germany; Robert Preissner, Axel Pries, Andrea Volkamer,

Charité Universitätsmedizin Berlin, Germany; Roland Lauster, Technische Universität

Berlin, Germany; Andreas Luch, Gilbert Schönfelder, Federal Institute for Risk

Assessment, Germany; Marcus Weber, Zuse Institute Berlin, Germany

Among the terminated projects in drug development, three major reasons stand out:

insufficient efficacy (51%), non-profitable market (29%), and unacceptable adverse

effects (19%)1. Major reasons for non-reliable preclinical drug evaluation include

species differences.

Established in 2014 by BMBF funding, the Berlin-Brandenburg research platform

BB3R pools research activities of Freie Universität Berlin, Charité Universitätsmedizin

Berlin, Federal Institute of Risk Assessment, Technische Universität Berlin, Zuse

Institute Berlin and University of Potsdam to replace, reduce, and refine animal tests.

Here, we present latest results out of this consortium. First, we emulated non-

melanoma skin cancer by co-culturing tumor cells with normal skin models. This 3D

cell culture system allowed us not only to assess the effects of ingenol mebutate, but

also provided insights into the cancer-associated changes of the skin barrier

function2. Second, exposure to pro-inflammatory cytokines mimics selected features

of atopic dermatitis and gave insights into the disease pathway3. These organotypic

models currently serve to investigate novel drug delivery systems like nanocarriers4,5.

However, organotypic (skin) disease models lack the connection to other organotypic

models or the systemic circulation. The human-on-a-chip currently combining up to

four organs allows the study of substance exposure and elimination6. Organotypic

disease models and/or human-on-a-chip approaches help eliminating poorly

performing drug candidates at early stages of pre-clinical evaluation, thus reducing

the number of animal tests7. Yet, to the best of our knowledge in drug development,

animal experiments will not be overcome in full. Thus, the refinement of animal

experiments, e.g. by strain specific dosing of analgetics and narcotics, is subject of

the BB3R consortium, too.

64 65

with adequate insulin response and islet functionality only maintained in co-cultures.

Further development of this model using tools inducing impaired glucose regulation

will provide a unique in vitro system emulating human type 2 diabetes mellitus.

Metabolic cross talk between human pancreatic islet and liver spheroids in a microphysiological system - Towards a novel human

ex vivo model of Type 2 Diabetes

Sophie Bauer, TissUse GmbH, Berlin, Germany; Charlotte Wennberg Huldt,

AstraZeneca, Mölndal, Sweden; Kajsa Kanebratt, AstraZeneca, Mölndal, Sweden;

Isabell Durieux, TissUse GmbH, Berlin, Germany; Daniela Gunne, TissUse GmbH,

Berlin, Germany; Shalini Andersson, AstraZeneca, Mölndal, Sweden; Lorna Ewart,

Astra Zeneca, Cambridge, UK; William G. Haynes, AstraZeneca, Mölndal, Sweden;

Ilka Maschmeyer, TissUse GmbH, Berlin, Germany; Annika Winter, TissUse GmbH,

Berlin, Germany; Carina Ämmälä, AstraZeneca, Mölndal, Sweden; Uwe Marx,

TissUse GmbH, Berlin, Germany; Tommy B. Andersson, AstraZeneca, Mölndal,

Sweden.

T2DM is a multi-organ disease. Disease phenotype and response to treatments are

dependent on both metabolically functional organs and a relevant interplay between

them. Here we present a model of organ cross-talk between human pancreatic islet

microtissues and human liver spheroids, two key organs involved in glucose

homeostasis. In our setup both tissues were cultured in spatially separated cavities,

which were interconnected by a microfluidic channel. An integrated on-chip

micropump circulated medium through the microfluidic channel system enabling

transport of substances. Functional coupling of liver and pancreatic islets was

evaluated by measuring the insulin concentration in media released from the islet

microtissues in response to a glucose load similar to an in vivo glucose tolerance

test. Insulin increased glucose uptake by the liver spheroids accelerating the rate of

glucose disappearance from the media, while the liver alone did not consume

glucose as efficiently. As glucose levels fell, the accumulation of insulin in the media

decreased and by 48 h, both glucose and insulin had reached steady state levels,

supporting a homeostatic feedback loop between islet microtissues and liver

spheroids. Exposed to high glucose over time, islet microtissues cultured in the

absence of liver spheroids had reduced ability to release insulin, indicating that the

prolonged hyperglycaemia impaired islet function. The results, thus, indicate that the

glucose regulation in the two-organ combination reflects human glucose homeostasis

66 67

correct position of the insert within the microfluidic circuit. The cellular barrier was

formed by seeding endothelial cells (BEOC) at the bottom of the 0,4 µm Transwell

membrane and renal proximal tubular cells (RPTEC/TERT1) at the top of it. By using

different modulations of the integrated pneumatic micro pump different physiological

and pathophysiological conditions like the ischemic phase of acute kidney injury or

the hypertonic stress of chronic kidney disease could be applied.

Figure 1: Schematic view of the ZEBRA-Chip. A – 24-well Transwell insert; B – microfluidic system with closed loop circuit and integrated micro pump; C – holder system for reversible integration of the Transwell; D – pneumatic connectors to actuate the system by an external control unit; E – Lid to close the Transwell holder and insert during cultivation

References [1] Yin J, Wang J. Renal drug transporters and their significance in drug-drug interactions. Acta

pharmaceutica Sinica. B 2016; 6: 363–373.

[2] Wilmer MJ, Ng CP, Lanz HL, Vulto P, Suter-Dick L, Masereeuw R. Kidney-on-a-Chip Technology

for Drug-Induced Nephrotoxicity Screening. Trends in biotechnology 2016; 34: 156–170.

[3] Thuenauer R, Rodriguez-Boulan E, Römer W. Microfluidic approaches for epithelial cell layer

culture and characterisation. The Analyst 2014; 139: 3206–3218.

[4] King SM, Higgins JW, Nino CR, et al. 3D Proximal Tubule Tissues Recapitulate Key Aspects of

Renal Physiology to Enable Nephrotoxicity Testing. Frontiers in physiology 2017; 8: 123.

[5] Wei J, Song J, Jiang S, et al. Role of intratubular pressure during the ischemic phase in acute

kidney injury. American journal of physiology. Renal physiology 2017; 312: F1158-F1165.

A

B

C

D

E

Mimicking human physiology at Transwell based barrier models of the proximal tubules – The ZEBRA-Chip

Florian Schmieder1, Deborah Förster2, Melanie Hempel1, Jan Sradnick2, Bernd

Hohenstein2, Frank Sonntag1 1 Fraunhofer Institute for Material and Beam Technology IWS, Dresden, Germany

2 Division of Nephrology, Department of Internal Medicine III, University Hospital Carl

Gustav Carus Dresden

The kidney is the central organ for the elimination of toxic species from the human

body. Thus permanent renal dysfunction, as it occurs in chronic kidney disease,

leads to hemodialysis and early death. Due to the fact that the majority of molecular

transporters, that play an important role in secretion and reabsorption of metabolites,

are located in the proximal tubule, most toxic effects arise in that part of the kidney

[1]. Mimicking the physiology of the proximal tubule in vitro seems an adequate way

to study these effects. Moreover it could lead to artificial cell based kidney models for

studying kidney diseases and drug effects in vitro.

In vitro kidney models While the development of cellular models of the tubular barrier has been proven by

many researchers in the past [2,3] it remains hard to recapitulate these experiments

due to the limited flexibility of the setup and the complex cultivation conditions of the

microfluidic approaches. In contrast Transwell based cellular models of the proximal

tubules offer the beneficial opportunity of using a variety of human cell types, easy

culturing conditions and the use of automated generation of tissue constructs [4].

Nevertheless physiological conditions, regarding pressure and shear stress of the

blood capillary and the inner tubular lumen of the proximal tubules, need to be

applied also in Transwell based cellular models to reconfigure the in vivo situation of

healthy or diseased kidney [5].

The ZEBRA-Chip (Zellbsiertes artifizielles Nierensystem) To overcome the limitations of standard Transwell based cellular kidney models we

developed a microphysiological system – the ZEBRA-Chip. It consists of a closed

loop microfluidic circuit with an integrated pneumatically driven micro pump that

constantly circulates the cell culture media at the blood site of the tubular barrier. The

Transwell insert could be reversibly integrated into a holder system that ensures the 68 69

Human and mouse intestinal organoids as model system for studying drug transport

T. Zietek, Technical University of Munich, Freising, Germany; E. Rath, TUM ZIEL

Institute for Food & Health, Freising, Germany; F. Reichart, TUM Institute for Advanced Study, Garching, Germany; H. Kessler, TUM Institute for Advanced Study,

Garching, Germany; G. Ceyhan, Dept. of Surgery, Klinikum rechts der Isar, München,

Germany; I. Demir, Dept. of Surgery, Klinikum rechts der Isar, München, Germany;

H. Daniel, Technical University of Munich, Freising, Germany

Research on intestinal transport processes is of high interest for studies on nutrient

absorption as well as for drug uptake studies. We previously showed that 3-

dimensional intestinal organoids are a useful tool for assessing intestinal nutrient

transport in vitro and that compounds up to 4 kDa in size are able to enter the

organoid lumen. Now we could demonstrate that 3D mouse and human intestinal

organoids are a valuable in vitro model for the screening of pharmacological

compounds.

Mouse intestinal organoids for studying absorption of anti-cancer drugs Organoids generated from wild type and transporter knockout mice were used for

studying PEPT1-mediated absorption of the antibiotic cefadroxil and bioavailability of

novel peptide drugs / prodrugs. These novel compounds are promising candidates

for cancer therapy and tumor characterization specifically targeting the integrin

subtype αVβ3. We could demonstrate that some of these drugs are transported by

the peptide transporter PEPT1. Furthermore, we tested absorption of these compounds in human intestinal organoid cultures derived from healthy human

subjects.

70 71

Long-term culture of rat Precision-Cut Lung Slices using Lab-on-Chip technology as an ex vivo system with prolonged viability

S. Konzok1, S. Dehmel1, V. Neuhaus1, J. Labisch1, S. Grünzner2,3, F. Sonntag3, A.

Braun1, K. Sewald1 1 Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Hannover,

Germany 2 Technische Universität Dresden, Dresden, Germany 3 Fraunhofer Institute for Material and Beam Technology IWS, Dresden, Germany

In order to assess chronic inflammatory mechanisms as well as repeated dose

toxicity, development and optimization of appropriate test systems for long-term

culture remains crucial. Precision-Cut Lung Slices (PCLS) have been reported to be

a potent ex vivo system, containing an intact microanatomy and functional local

immunology. Lab-on-Chip (LOC) systems represent a cutting edge technology to

augment in vitro cell tests, allowing new functional read-outs for a prolonged period

of time through the use of dynamically perfused media. In the present study, PCLS

technique and LOC technology were combined in order to prolong the viability of ex

vivo rat lung tissue.

PCLS from rat lung tissue were cultured for up to 13 days under perfused conditions

using a closed circulation LOC system. Long-term viability, structural integrity and

immune cell responsivity was assessed through LDH assays and LIVE/DEAD

stainings, H/E stainings and LPS stimulation with subsequent measurement of pro-

inflammatory cytokines, respectively.

Both LDH assays and LIVE/DEAD stainings reveal an improved viability of rat PCLS

in the dynamically perfused LOC over a period of 13 days. H/E stainings further

showed an improved preservation of the microanatomy in comparison to classical

submersed cultivation over a period of 13 days. Immune response of the organotypic

tissue within the LOC system during the first 5 days remained similar to the

submerse control culture.

Cultivation under dynamically perfused conditions using the LOC technology greatly

prolongs the viability of rat PCLS and helps preserve an intact microanatomy. Albeit

no changes in the immune response were seen, the increased viability through

perfused conditions may be a first step towards repeated dose toxicity studies in this

organotypic model.

Microstructured 3D model of small intestine epithelium: breaking the mold

María García-Díaz1, Albert G. Castaño1, Gizem Altay1, Núria Torras1, Raquel Martin-

Venegas2, Rut Ferrer2, Elena Martínez1

1Institute for Bioengineering of Catalonia (IBEC), Barcelona, Spain 2Faculty of Pharmacy, Universitat de Barcelona (UB), Barcelona, Spain

Most of the standard in vitro cell culture models of the intestinal mucosa lack key

features of the human small intestine such as the organized 3D structure. Recent

advances in tissue engineering have enabled the development of 3D models that

better recapitulate the small intestine architecture, mostly based on laborious

sequential micromolding. However, despite their great potential, cell culture systems

that faithfully biomimic the tissue topographies are not routinely implemented. The

main limitation is the difficulty in microfabricating soft materials with complex 3D

geometries, high aspect ratio and curvature using efficient and simple methods. The

aim of this work is to engineer a hydrogel-based in vitro model of the intestinal

epithelium that account for the 3D villi-like microstructure using a simple and cost-

effective photolithography process. We demonstrate that finger-like microstructures

of anatomic and predictable dimensions can be easily fabricated by

photopolymerization using poly(ethylene) glycol diacrylate (PEGDA). In order to

provide bioactive functionalization, we co-polymerize PEGDA with acrylic acid. This

system allows for the fine-tuning of the ligand density through the amount of

carboxylic acid groups, while preserving the soft mechanical properties of the

scaffold. This fabrication process can be further tuned for the fabrication of hydrogels

directly onto permeable membranes, enabling their assembly into cell culture inserts.

Caco-2 cells seeded on the synthetic 3D villi-like scaffolds formed monolayers of

differentiated enterocytes with proper barrier properties. Through these results, this

microfabrication technology raises up as a promising tool to faithfully replicate

microscale three-dimensional topographies at the tissue level in soft hydrogels that

does not require molding and demolding steps.

72 73

Bioprinted kidney model to assess nephrotoxicity M. Nosswitz1, M. Rimann1, N. Hernando2, C. Wagner2, U. Graf-Hausner1, M.

Raghunath1 1Institute of Chemistry and Biotechnology (ICBT), Zurich University of Applied

Sciences, Waedenswil, Switzerland, 2Institute of Physiology, University of Zurich,

Zurich, Switzerland

Nephrotoxic side effects of newly developed drugs are widespread. A reliable

assessment of drug-induced nephrotoxicity in vitro can close an important gap in the

drug development chain. Narrowing down a pool of potential drug candidates prior to

clinical trials saves money and time, and helps reducing animal experimentation,

often resulting in wrong conclusions and predictions.

The goal of the study was to rebuild and dynamically cultivate the proximal tubule

(PT; first part of the nephron after the glomerulus) in an in-house developed PDMS

perfusion chip. Either bioprinting or handcrafting were used to create tubular

structures inside a gelatin-based hydrogel. For the bioprinting approach, a gelatin

scaffold was printed combined with a sacrificial ink that was removed to build hollow

structures in the crosslinked scaffold. Handcrafted tubes were fabricated with thin

wires that were removed after scaffold crosslinking. The inner tubular walls were then

seeded with proximal tubule epithelial cells (RPTEC/tert1) followed by cultivation

under dynamic conditions after a monolayer has established.

Physiologically relevant marker expression and transporter gene expression of

RPTEC/tert1 cells grown on plastic in 2D were compared to those grown in the 3D

perfusion system. Establishment of a tight ZO-1-positive monolayer, dome formation

and induction of specific genes (phosphate transporters, glucose transporters) in the

3D flow system indicates the formation of a functionally active renal epithelium.

Here we present a human proximal tubule model established in a porous gelatin

hydrogel grown under dynamic flow conditions. The goal is developing an assay to

assess albumin (BSA) uptake in real-time to monitor epithelial integrity/functionality

after drug exposure.

Standardization, refinement and upscaling of such a PT system can lead to an

important contribution to improve future drug development processes.

Funding: SNF No.: 20PC21_161566 / 1

A Novel 3D Human Liver Fibrosis Model for Anti-fibrotic Drug Discovery and Safety Testing

Radina Kostadinova1, Monika Kijanska1, Natalia Zapiorkowska-Blumer1, Aparna

Neelakandhan1, Sabrina Steiert1, Patrick Guye1, Simon Messner1

1InSphero AG, Wagistrasse 27A, 8952 Schlieren

Liver fibrosis is the excessive accumulation of extracellular matrix proteins such as

collagen, which can lead to cirrhosis, liver failure and transplantation. Anti-fibrotic

therapies aim at inhibiting the induction of hepatic stellate cells (HSC), preventing the

deposition of extracellular matrix proteins (ECM). Beside the HSC, presence of other

liver cell types is crucial to recapitulate liver disease states. Here we describe a novel

3D model that contains all relevant cell types associated with liver fibrosis (hepatocytes,

Kupffer cells (KC), liver endothelial cells (LEC) and HSC) as a highly suitable tool for

liver fibrosis research. To this end, the previously described 3D Human Liver Microtissue

model was engineered to incorporate additionally HSC from primary origin. The resulting

3D InSight™ Human Liver Fibrosis Model has been characterized under basal and

induced liver fibrosis conditions on a morphological and phenotypic level by

immunostaining techniques and gene expression analysis. Using cell-type specific

markers and IHC we demonstrated the presence of hepatocytes, HSC, KC and LEC

during the cultivation and treatment period. TGF-b1 treatment for 7 days of the 3D liver

model induced activation of HSCs as detected by up to 8-fold increased expression of a-

smooth muscle actin (a-SMA). The treatment of the microtissues with TGF-b1 also

promoted the gene expression of the ECM proteins collagen type I, III, IV and VI and

pro-fibrotic markers such as platelet-derived growth factor beta (PDGFb) and lysyl

oxidase (Lox). In addition, IF staining showed an increased deposition of Collagen type

IV upon TGF-b1 treatment. Inhibition of fibrosis induction with anti-fibrotic drugs targeting

ALK5, thymidine kinases or FXR agonists effectively blocked fibrosis development. At

the same time, monitoring of albumin secretion, cellular viability and cytotoxicity allowed

assessment of toxic side effects of anti-fibrotic compounds. In summary, we

demonstrated that TGF-b1 treatment induces liver fibrosis in vitro and that this model

system allows studying efficacy and toxicity of anti-fibrotic drugs. The 3D Human Liver

Fibrosis model is thus a novel, biologically relevant in vitro model of liver fibrosis,

suitable for high-throughput efficacy and safety screening of anti-fibrotic drugs.

74 75

Microfluidic Platform for Advanced Embryotoxicity Testing in vitro

Julia A. Boos1, Astrid Michlmayr1, Kasper Renggli1, Olivier Frey2 and Andreas Hierlemann1

1ETH Zürich, Department of Biosystems Science and Engineering, Basel, Switzerland 1InSphero AG, Schlieren, Switzerland

We present a microfluidic platform, which enables the seamless integration of a metabolic

activation system into the embryonic stem cell test (EST). The EST will be entirely executed in

a microfluidic platform, in which metabolic competence will be included by using a multi-organ

configuration. Primary human liver microtissues (hLiMTs) – the primary organ for

biotransformation – will be cultured together with embryoid bodies (EBs) in a microfluidic

hanging-drop network (HDN) [1]. Hanging drops constitute an ideal environment for the

formation and cultivation of EBs and, at the same time, help to maintain the metabolic activity

of the hLiMTs over prolonged time. The individual drops are fluidically interconnected by

microchannels. By gently tilting the device, gravity-driven flow allows for perfusion and

constant interaction between the microtissues. The test substance is metabolized by the liver

and is immediately transported to the embryonic tissue in the neighboring compartments. In

this way, not only the direct toxicity of a test compound but also that of highly reactive or

transiently stable metabolites can be assessed.

The entire assay will be realized on a single tubing-free platform (Figure 1). For EB formation,

the chip is operated in a “hanging-drop configuration”. For the readout, the chip is flipped

upside down to provide a surface for EB outgrowth. In this “standing-drop configuration”, the

absence of beating cardiomyocytes within the outgrowth serves as an indicator for impaired

embryonic development. Preliminary results showed highly uniform EBs that successfully

differentiated and developed contracting cardiomyocytes. Further, functional hLiMTs were

cultivated on-chip and remained viable over 10 days.

Figure 1. Concept of the microfluidic EST assay with metabolic competence.

[1] Frey. O. et al, Reconfigurable microfluidic hanging drop network for multi-tissue interaction

and analysis, Nature Communications, 5, 4250 (2014)

76 77

Poster Abstracts

Advanced physiologically relevant 3D models for pre-clinical screening

Dora Sabino, Isabelle Fixe, Alexandra Foucher, Flavien Carpentier, Maxime Rochet,

Isabelle Topin, Eric Mennesson, Nadia Normand

tebu-bio, 39 rue de Houdan, 78612, Le Perray en Yvelines, France

[email protected]

Human in vitro cellular models have been developed to answer screening

needs in early stages of drug development, toxicity assessment, drug metabolism,

induction or inhibition studies. Several retrospective studies highlight the need for

further development of organotypic models as a high number of new chemical

entities have been withdrawn from the market due to low PK/PD dynamics profiles,

unexpected or severe adverse drug reactions. All the aforementioned issues impact

on cost and drug development time. Therefore, pre-clinical human cellular models

are crucially needed to evaluate the efficacity and safety of NCEs before they enter

clinical trials.

Despite the extensive work done so far, emulating all functions and responses

of an organ remains a challenging task, as the experimental microenvironment

impacts on function and assay readout ability. Ideally, a 3D in vitro system would be

able to better predict toxicity in physiological environment, biotransformation, drug-

drug interaction, NCE adverse effects, and long-term toxicity in preclinical stages.

Recent, seminal studies demonstrate the added scientific relevance of 3D cellular

cultures in providing impactful answers to these questions.

In this work, we describe, an experimental setup that addresses 3D human

cellular co-cultures. Importantly, we put emphasis in the early detection of

inflammatory responses in the after chemical insult or pathology, taking advantage of

live imaging, flow and molecular data adapted to the STING pathway and others.

With these results, we want to contribute and expand the 3D model in vitro toxicity

prediction field, and provide appropriate and amenable experimental procedures that

can be adapted, reproduced and extended to different molecular, mechanistic and

cellular questions.

Sub topic - Advanced cell culture models Advanced models for substance testing

78 79

A 3D High-Content Screening assay as model system for polycystic kidney disease

H. Bange1,2, T.H. Booij1, W. N. Leonhard3, K. Yan2, D.J.M. Peters3, L.S. Price1,2.

1Division of Toxicology, LACDR, Leiden University, the Netherlands, 2OcellO B.V., Leiden, the Netherlands, 3Department of

Human Genetics, LUMC, the Netherlands

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in

either the Pkd1 or Pkd2 gene. The most important characteristic of this disease is the

formation of cysts in the kidney, which reduces renal function and will lead to end

stage renal disease. Although the cause of the disease is known, it is still not fully

known why these mutations lead to cyst formation. Since cysts cannot form in

conventional in vitro 2D cell culture, current research on ADPKD relies heavily on the

use of animal models. The lack of proper in vitro models makes the study of this

disease all the more challenging.

To address this, we developed a 3D high-content in vitro screening assay usable for

mechanistic studies as well as target and drug discovery in ADPKD. This system

uses kidney collecting duct Pkd1 KO cells, which spontaneously form small cysts

when cultured in a 3D hydrogel. cAMP inducer Forskolin is added to stimulate the

cyst swelling in the presence of the test compounds. To examine the effect of the

compounds on the swelling, cysts are fixed, stained and imaged. The 3D image

stacks are analyzed with OminerTM image analysis software which can measure

diverse phenotypic characteristics, including cyst size, nucleus shape and thickness

of cyst wall. This also enables us to identify compounds that are effective and do not

influence cell viability, and discard compounds which have undesired therapeutic

profiles.

To validate this platform, we screened a library of previously characterized kinase

inhibitors, identifying previously implicated targets in ADPKD such as mTOR, CDK

and HER2. We also identified Syk and Chk as novel targets. Furthermore, by

examining specific as well as dual inhibitors of mTOR and PI3K we observed

unexpected roles of these targets in cyst swelling.

We then screened a collection of 2320 natural products and bioactive compounds.

Hit compounds were validated first in vitro, and then in a knockout mouse model of

PKD. One of these compounds proved effective in reducing cyst progression and

renal function in a dose-dependent manner.

Evaluation of EGFR induced on-target and target-mediated adverse effects in a microfluidic 3D human lung tumour – full thickness skin

co-culture model

Juliane Hübner1, Marian Raschke², Isabel Rütschle1, Susanne Schnurre²,

Sarah Gräßle1, Ilka Maschmeyer1, Uwe Marx1 and Thomas Steger-Hartmann²

1TissUse GmbH, Oudenarder Str. 16, 13347 Berlin, Germany

²Bayer AG, Investigational Toxicology, 13353 Berlin, Germany

Microphysiological systems are increasingly contributing to the preclinical prediction

of mode of action and adverse outcome pathways of new chemical and biological

entities. The recent advent of robust human multi-organ-chip systems enables the

establishment of co cultures of human drug target tissues with healthy organ

equivalents prone to off-target effects of the respective drug.

Here we established a chip-based 5-day co-culture composed of human

mucoepidermoid carcinoma H292 cell-based lung tumour spheroids and human skin

equivalents. We investigated the impact of repeated Cetuximab exposure on the

systemic behaviour of the co-culture and on individual tissue responses. We

compared on-target antibody effects with response data for Afatinib – a small

molecule benchmark drug. Finally, we investigated target-mediated adverse effects

on healthy skin equivalents.

This co-culture has the potential to provide a platform for evaluation of the

therapeutic window of drug candidates.

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Parallelized Heart-on-a-chip with integrated Force Sensing incorporating human iPS-derived cardiac microtissues

Christopher Probst, Oliver Schneider, Stefanie Fuchs, Peter Loskill

Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Nobelstraße 12,

70569 Stuttgart, Germany;

The development of pharmaceutical compounds still relies mainly on animal models,

which are limited regarding the transferability to the human organism. Advances in stem

cell biology, namely the advent of induced pluripotent stem cells (iPS), have opened up

new possibilities for pharmaceutical research and disease modeling. In recent years,

due to the increasing interdisciplinarity of research, the combination of microfabrication,

tissue engineering and (stem) cell biology has led to a new paradigm in 3D cell culture,

the emergence of microphysiological systems (MPS), also referred to as organ-on-a-

chip systems. Cardiovascular diseases are still one of the major causes of mortality

globally and better in-vitro models are necessary to develop new treatment therapies.

Hence, we developed a highly parallelized and multi-layered microphysiological Heart-

on-a-chip system capable of integrating a large number of human iPS-derived cardiac

microtissues. The MPS consists of the following functional components: anisotropic

cardiac tissue chambers, vasculature-like media channels, a semipermeable membrane

acting as an “endothelial-like” barrier, and integrated force sensors. In situ force

measurement allows for a direct assessment of contractility development and maturation

of the cardiac microtissues. The MPS is able to keep the 3D cardiac microtissues viable

and functional with physiological beat rates for multiple weeks. By combining optical flow

motion tracking and integrated fluorescently-tagged elastic polymer-layers, we were able

to characterize beating motion and forces simultaneously using simple microscopy

techniques. By assessing the physiological response to cohorts of drug compounds from

different categories, the functionality of the cardiac microtissue was validated. The

developed MPS is extremely versatile and can be used for drug toxicity screening and

therapeutic applications.

In conclusion, we developed a novel 3D model system to study ADPKD. Using this

assay, we identified potential new targets and gained new insight into to the role of

existing targets. Moreover, we found a new molecule active in reducing cyst size,

which was validated in vivo. These results show the applicability of this model for

mechanistic studies, target discovery and compound testing for ADPKD

82 83

The Ocular DynaMiTES – A dynamic microfluidic in vitro system with improved predictability of ocular drug absorption

N. Beißner 1,3, K. Mattern 2,3, A. Dietzel 2,3, S. Reichl 1,3 1 Institut für Pharmazeutische Technologie, Technische Universität Braunschweig, Mendelssohnstr. 1,

38106 Braunschweig, Germany; 2 Institut für Mikrotechnik, Technische Universität Braunschweig, Alte

Salzdahlumer Str. 203,38124 Braunschweig, Germany; 3 Zentrum für Pharmaverfahrenstechnik,

Technische Universität Braunschweig, Franz-Liszt-Str. 35 A, 38106 Braunschweig, Germany

Common preclinical test systems are often questioned concerning their reliability in

predicting the best drug candidates for later stages of the development process [1].

Therefore, improved in vitro models are needed. These should allow emulation of the

dynamic human physiology to finally get a more reliable prediction of the effect in

humans. For this purpose our interdisciplinary team of engineers and pharmacists

developed a Dynamic Micro Tissue Engineering System (DynaMiTES;

see Figure 1) [2]. This novel microfluidic device can be loaded with well-known cell

culture insert systems after a slight adaption of the cell cultivation. Furthermore, it

allows application of shear stress, simulation of concentration gradients and

continuous monitoring of cellular barrier properties. With this the DynaMiTES

provides highly controlled dynamic experimental conditions for its application in drug

absorption tests.

As a first in vitro model a prevalidated human hemicornea (HC) construct [3,4] was

adapted to the DynaMiTES [5]. In the present study this Ocular DynaMiTES was

utilized under the envisaged dynamic conditions. To investigate the benefit of

dynamic conditions, they were compared to static conditions concerning sodium

fluorescein permeation with and without the addition of different concentrations of

Figure 1: Experimental setup for dynamic absorption tests utilizing the DynaMiTES. The DynaMiTES is

connected to a syringe pump to simulate dynamic in vivo-like shear stress and variable concentrations [5]

Establishment of an advanced in vitro model to study nanomaterial-intestinal barrier interactions

Claudia Hempt, Cordula Hirsch, Melanie Kucki, Peter Wick, Tina Buerki-Thurnherr

Empa, Swiss Federal Laboratories for Materials Science and Technology,

Lerchenfeldstrasse 5, 9014 St. Gallen, Switzerland

In recent years food industry has embraced the benefits of nanotechnology. A variety

of engineered nanomaterials (ENM) are used as food additives to provide for exam-

ple new tastes, anti-microbial properties or to improve the nutritional value. On the

other hand nanostructured silica particles are mainly used as anti-caking agents that

do not generate novel food characteristics but rather serve as processing aids.

However, the impact and translocation of ENMs through the gut epithelium is poorly

investigated and understood.

Therefore we aim to establish an advanced in vitro intestinal barrier model that

closely mimics the in vivo situation and allows mechanistic studies on the potential

interaction of ENMs with mucus, the epithelial cell surface and immune cells. This

model will thus include enterocytes (Caco-2), goblets cells (HT-29-MTX), B-

lymphocytes (Raji) and M-cells.

The individual monocultures have been successfully established and display cell

type-specific morphology, formed intact barriers and expressed cell-type specific

antigens. First results on the interaction and uptake of food-relevant ENMs in the

enterocyte monoculture showed that these particles did not affect barrier integrity,

cell viability or induce the formation of reactive oxygen species up to a concentration

of 50 µg/ml.

The next steps will be the establishment of the co- and triple cultures and subsequent

studies on the impact and translocation of food grade nanomaterials in these more

physiological models.

84 85

Cell Processing in Microreactors: Real-time Monitoring of Cell Metabolism Using Sensor Particles and Surface Based, Gentle Cell

Detachment Katja Uhlig; Christian Gehre, Sebastian Prill; Maike Stahl; Claus Duschl, Fraunhofer-

Institute for Cell Therapy and Immunology, Potsdam, Germany; Elmar Schmälzlin,

Colibri Photonics GmbH, Potsdam, Germany; Lars Dähne, Surflay Nanotec GmbH,

Berlin, Germany; Thomas Hellweg, Bielefeld University, Bielefeld, Germany

The establishment of valuable cell models and their exploitation for addressing

important issues in a number of disciplines such as tissue engineering, drug

development or toxicology, is a complex task that requires control of a multitude of

processes. Recently, we have introduced a novel perfusion microbioreactor for the

cultivation of hepatocytes. Through the integration of mircobeads that contain

oxygen-sensitive chromophores it is possible to measure the O2-concentration in

real-time in the medium in the immediate vicinity of the cells. By adjusting the flow of

the medium and the number of cells our microsystem allows the monitoring of their

metabolic activity over several weeks. Based on this approach we were able to

assess the toxicity of a number of hepatotoxic compounds. In particular, due to the

access to the dynamics of the metabolic activity of the cells on time scales from

minutes to weeks we were able to identify a hitherto unknown toxic effect on the

mitochondrial respiration of the widely used pain killer paracetamol. Recent work

includes the integration of a second class of microsensor beads that allows the

monitoring of the pH in the cell clusters and the establishment of more relevant cells

models. Results will be presented based on proliferation competent human

hepatocytes based on the upcyte technology and primary mouse hepatocytes.

Finally, we will address an issue that concerns the noninvasive pre- and

postprocessing of adherent cells. Detachment of cells from their cultivation substrate

in 2D arrangement can now be executed without invasive enzyme cocktails by

employing thermoresponsive polymer coatings. Our approach is straight forward to

apply and considerably less expensive than products already available on the

market. The polymer can easily be patterned on µm-scales using spotting or µ-

contact printing. We exploit this feature of the coatings for the development of cell

assays and for cocultures with highly defined geometric relations.

benzalkonium chloride, a common ocular preservative. The results showed improved

correlation of the dynamic results with published human in vivo data in contrast to

static conditions and human in vivo data. This underlines the particular value of our

DynaMiTES and its potential to improve the common preclinical test practice.

References:

[1] Marx, U., Walles, H., Hoffmann, S., Lindner, G., Horland, R., Sonntag, F., Klotzbach, U., Sakharov,

D., Tonevitsky, A., Lauster, R., 2012. ’Human-on-a-chip’ developments: A translational cuttingedge

alternative to systemic safety assessment and efficiency evaluation of substances in laboratory

animals and man?, Alternatives to Laboratory Animals, Vol. 40, pp. 235–257

[2] Mattern, K., Beißner, N., Reichl, S., Dietzel, A., 2017. DynaMiTES – A dynamic cell culture platform

for in vitro drug testing PART 1 – Engineering of microfluidic system and technical simulations,

European Journal of Pharmaceutics and Biopharmaceutics, Article in press

[3] Hahne, M., Reichl, S., 2011. Development of a serum-free human cornea construct for in vitro drug

absorption studies: The influence of varying cultivation parameters on barrier characteristics,

International Journal of Pharmaceutics, Vol. 416, pp. 268-279

[4] Hahne, M., Zorn-Kruppa, M., Guzman, G., Brandner, J. M., Haltner-Ukomado, E., Wätzig, H.,

Reichl, S., 2012. Prevalidation of a human cornea construct as an alternative to animal corneas for in

vitro drug absorption studies, Journal of Pharmaceutical Sciences, Vol. 101, pp. 2976–2988

[5] Beißner, N., Mattern, K., Dietzel, A., Reichl, S., 2017. DynaMiTES – A dynamic cell culture platform

for in vitro drug testing PART 2 – Ocular DynaMiTES for drug absorption studies of the anterior eye,


86 87

It was shown, that the platform can be easily integrated in existing work flows and

protocols. Furthermore, a short-term cultivation of 3 hours as well as elongated

cultivation of 24 hours had no negative impact on cell viability and cell morphology.

This study reveals that the application as dynamic barrier model is feasible.

Subsequent studies will focus on long-term dynamic cultivation as well as on

dynamic permeability studies.

References:

[1] Mattern, K., Beißner, N., Reichl, S., Dietzel, A., 2017. DynaMiTES – A dynamic cell culture platform

for in vitro drug testing PART 1 – Engineering of microfluidic system and technical simulations,


[2] Beißner, N., Mattern, K., Dietzel, A., Reichl, S., 2017. DynaMiTES – A dynamic cell culture platform

for in vitro drug testing PART 2 – Ocular DynaMiTES for drug absorption studies of the anterior eye,


Evaluation of a Novel Cell Culture Platform with Various Barrier-Forming Cells for Dynamic Cultivation

S. Hinkel 1,3, K. Mattern 2,3, A. Dietzel 2,3, S. Reichl 1,3, C. C. Müller-Goymann 1,3 1 Institut für Pharmazeutische Technologie, Technische Universität Braunschweig, Mendelssohnstr. 1,

38106 Braunschweig, Germany; 2 Institut für Mikrotechnik, Technische Universität Braunschweig, Alte

Salzdahlumer Str. 203, 38124 Braunschweig, Germany; 3 Zentrum für Pharmaverfahrenstechnik,

Technische Universität Braunschweig, Franz-Liszt-Str. 35 A, 38106 Braunschweig, Germany

In vitro models of barrier-forming cells are commonly used to study the permeability

of new substances in the early phase of drug development. It is essential to imitate

cell situations preferably close to in vivo conditions to reduce drug failures and thus

animal research, time and costs. For this purpose, the novel cell culture platform

DynaMiTES (Dynamic Micro Tissue Engineering System) is based on the idea of

combining advantages of static cell culture insert systems with those of microfluidic

devices for imitating flow conditions and shear stress. DynaMiTES enables dynamic

permeability studies as well as dynamic cultivation of various cell types. Furthermore,

it combines online TEER-measurements, versatility in use and comparability with

conventional insert systems for example Corning Transwell® or Greiner ThinCertTM.

The DynaMiTES was initially developed as a dynamic ocular cell culture platform for

improved in vitro drug absorption studies across the cornea [1,2]. Due to its universal

usability and the possibility to integrate standard cell culture inserts, it can easily be

transferred to other in vitro dynamic tissue models. For the evaluation of dynamic

cultivation of cell monolayers in DynaMiTES, the common cell line MDCK-I (Madin-

Darby canine kidney-I), which shows particularly pronounced barrier properties, as

well as the endothelial cell lines hCMEC/D3 (human cerebral microvascular

endothelial cell line) and cEND (murine cerebral endothelial cell line) were used to

study the influence of DynaMiTES cultivation on the cell status.

Peristaltic

DynaMiTES

Figure 1: Schematic view of the assembled DynaMiTES connected to a peristaltic pump for dynamic cultivation (A) and plan view of the disassembled DynaMiTES, from left: bottom, middle and top layer (B)

A B

88 89

Dual targeting of prognostic biomarkers in the 3D microtumor model of advanced colorectal cancer

Ilmberger C, SpheroTec GmbH, Munich/Germany; Hoffmann O, SpheroTec GmbH,

Munich/Germany; Gülden J, Hospital of the LMU, Munich/Germany; Bühl T, Hospital

of the LMU, Munich/Germany, Werner J, Hospital of the LMU, Munich/Germany;

Mayer B, Hospital of the LMU, Munich/Germany.

Introduction:

Advanced colorectal cancer (CRC) represents an inter- and intraheterogenous

disease. Molecular complexity is suggested a main factor why only subgroups of

patients benefit from targeted therapy directed against a single molecule. This is

further strengthened by the finding that single agent treatment often fails in clinical

trials. We hypothesize that the simultaneous inhibition of different signaling pathways

involved in tumor progression will improve prognosis of CRC patients.

Methods:

Immunohistochemical co-expression profiling of the drugable targets CD44v6, MUC-

1 and Hsp90 was performed in 89 primary CRC referenced by the corresponding

mucosa. Prognostic impact of the dual expression pattern on 10-year overall survival

was tested in multivariate analysis. Prognostic relevant biomarkers were inhibited

with targeted therapy using the 3D microtumor model generated from CRC cell lines

and patient derived tumor tissue.

Results:

Co-expression of CD44v6/MUC-1, identified in 24.72% of the primary CRC correlated

with the detection of lymphatic invasion (p=0.048) and a positive nodal status

(p=0.012). Co-expression of CD44v6/Hsp90 was found in 20.22% of the primaries

mainly diagnosed G3/G4 (p=0.026). Simultaneous expression of MUC-1 and Hsp90

was a rare event (8.99%), but these tumors were of advanced size (>4.7 cm,

p=0.022) and revealed lymphatic invasion (p=0.035). Most interestingly, marker

Ready-to-use 3D spheroid culture as a standard tool to screen for drugs targeting cancer.

I Prieto, O Leis, J Gumuzio, E Ruiz and AG Martin,

StemTek Therapeutics, Biscay, Spain

Current attrition rate in drug development for novel cancer treatments is

unacceptably high, with only a 5.1% probability of any given therapeutic candidate to

succesfully complete clinical trials. Hit selection relays on conventional 2D monolayer

based cell culture systems to select potential candidates with therapeutic activity.

However, 2D cell culture has proved to be a very poor predictor of in vivo efficacy,

compromising the chances that a drug candidate will ever reach the patient’s bed.

Biology is not flat. Tissues, organs and tumors grow in 3D in their natural

environment, therefore it is imperative to recreate this 3D architecture to better

understand how a potential compound will interact with tumor cells and thus select

those with better chances of success.

There is a growing body of preclinical evidence showing that 3D cancer spheroids

respond to candidate drugs much like in vivo experiments and that this effect highly

correlates with clinical trial results. This assay was originally developed to culture

stem cells from many tissues as well as from tumors. There are multiple reports

showing that spheroid derived cells are enriched in tumor initiating or cancer stem

cells, derived from cell lines and fresh tumors as well. These spheroids represent the

initial stages in tumor formation, providing increased biological relevance for finding

novel anti-cancer compounds.

Here we describe the development of a ready-to-use 3D tumor spheroid model assay

to profile compound activity against cancer cells. Furthermore, a case of compounds

preventing hypoxia-inducible transcription factors (HIFs) activity is presented. The

use of 3D tumor spheroid systems represent the strategy of choice for fast,

reproducible, efficient and high throughput screening of anti-cancer compounds.

90 91

Permeation Measurement for 3D Skin Culture in a Membrane Insert System

Hao-Hsiang Hsu1, Katharina Schimek2, Uwe Marx3, Ralf Pörtner1

1Institute of Bioprocess- and Biosystems Engineering, Hamburg University of

Technology, Hamburg, Germany; 2Technische Universität Berlin, Department Medical Biotechnology of Biotechnology,

Berlin, Germany 3TissUse GmbH, Berlin, Germany

Background and novelty

Permeation and diffusion are important factors for skin model development and

characterization. Standardized methods for the determination of these parameters on

skin are the use of Franz diffusion cells, tape stripping and imaging methods, just like

fluorescence recovery after photobleaching (FRAP), fourier-transform-infrared

(FTIR), two-photon fluorescence correlation spectroscopy in combination with

fluorescence correlation spectroscopy (FCS) or coherence tomography. These

methods require either large samples or cost-intensive equipment. Another

alternative is to measure the permeation directly in a membrane insert system. This

system is used for skin model cultivation in order to allow a two-phase culture. The

advantages of this system are the small size and the easy handling, which reduces

costs and facilitates parallelization of experiments. But parameters like system size

(96 or 12 well) and membrane constellations (material, pore size) can influence the

permeation of this system. Therefore, a method was developed to determine

permeation and diffusion coefficient within these systems (Hsu et al. 2017). Here the

sensibility of this method and influencing factors are evaluated.

Experimental approach

Several permeation experiments were executed in transwells®-systems to determine

the permeation of fluorescein sodium salt through 2 % agarose gel and a 3D cell

combinations, but not individual biomarkers, were associated with poor prognosis

(CD44v6/Hsp90: p=0.031 and MUC-1/Hsp90: p=0.034). Targeting co-expressed

biomarkers in the 3D microtumor model resulted in a strong reduction of cell viability

(CD44v6/Hsp90 treatment: 66.5% and MUC-1/Hsp90: 52.1%) and was superior to

single agent treatment. Efficacy of dual inhibition strategy could be further improved

with 5-FU chemotherapy.

Conclusion:

Co-expression profiling of biomarkers from different signaling pathways is superior to

single marker analysis indicating poor prognosis. Dual inhibition of co-expressed

biomarkers represents a promising treatment approach in advanced CRC.

92 93

Preservation of tumor architecture and heterogeneity in long-term cultures of patient-derived explants

Sofia Abreu1,2, Sara da Mata3, Fernanda Silva4, Marta Teixeira1,2, Teresa Franchi

Mendes1,2, Ricardo Fonseca3,5, Bruno Filipe3, Sónia Morgado3, Inês Francisco3,

Marta Mesquita3, Cristina Albuquerque3, Jacinta Serpa3,4, Paula Chaves3, Isadora

Rosa3, Ana Felix3,4, Erwin R. Boghaert6, Vítor E. Santo1,2,Catarina Brito1,2 1IBET, Instituto de Biologia Experimental e Tecnológica, Portugal; 2Instituto de

Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa,

Portugal; 3IPOLFG, Portugal; 4CEDOC-FCM-NOVA, Portugal; 5FMUL, Portugal; 6Abbvie, USA

The tumor microenvironment plays an important role on tumor drug sensitivity;

thus the incorporation of microenvironment features on cancer models is expected

to improve their predictive power. Patient-Derived Explants (PDE) have been

proposed as potential models; however there are typically short term cultures. Our

goal was to improve culture longevity and vitality to evaluate efficacy of repeated

drug treatments, taking advantage of dynamic culture systems. Fresh ovarian and

colorectal cancer (OC and CRC, respectively) samples were dissociated into PDE

and cultured in agitation. Cell viability and proliferation were assessed by an array

of readouts, e.g. immunohistochemistry, rezasurin reduction capacity and

morphometric measurements. To this date, 29 tumors were successfully cultured

as PDE, retaining the original tumor architecture and main cellular components:

epithelial cells, fibroblasts and immune cells for at least 28 days (OC) or 7 days

(CRC). Samples included all main malignant ovarian carcinoma types (low and

high grade). For CRC, epithelial neoplastic cells were preserved, in some cases

up to 3 months, and stromal components were progressively lost along culture.

The status of the original tumors was preserved for the majority of cases in terms

of driver mutations of CRC and of microsatellite stability. OC-PDE cultures were

exposed to cyclic chemotherapy treatment. After two cycles, PDE showed low

cellularity and viability compared to untreated controls, reflecting the action of the

compounds. Altogether, we established PDE dynamic cultures in which tumor

architecture and heterogeneity is preserved, replicating the original tumor features.

Moreover, we demonstrated the feasibility of performing ex vivo drug efficacy

studies employing cyclic drug exposure regimens.

This work was partially supported by FCT (iNOVA4Health – UID/Multi/04462/2013, PD/BD/105768/2014 and

SFRH/BD/52208/2013).

model. Different sizes and membranes of the system were used to investigate there

influences on this method. Furthermore 3D cell models were used to test the

sensitivity. A simulation was executed in order to see the concentration distribution in

the experiment.

Results and discussion

The results of this investigations shows that the permeation is faster in 12 well

systems in comparison to 96 well systems. With the help of the simulations with

COMSOL Multiphysics the concentration distribution on the bottom of the systems

can be shown, which could be one reason for the difference. Furthermore it was

found that the pore area of the membrane influences the permeation, not the pore

size and the material. Experiments with different 3D cell models show that the

system is sensitive to differentiated tissues with and without an enclosure cell layer

just like the skin. By consideration of the influence factors of this system, it is a

promising tool for the applications on skin model in order to measure the permeation.

Referenz

Hsu H, Kracht JK, Harder LE, Rudnik K, Lindner G, Schimek K, Marx U, Pörtner R

(2017) A Method for Determination and Simulation of Permeability and Diffusion in a

3D Tissue Model in a Membrane Insert System for Multi-well Plates. Jove (in press)

https://www.jove.com/video/56412/a-method-for-determination-simulation-

permeability-diffusion-3d

94 95

microscopy depicted essential functional structures of an intact epithelium such as

microvilli and tight-junctions. Further physiological relevant properties were indicated

by an increasing barrier integrity and functional validation studies of the transport

activity for reference substances. In addition, the immortalized cells allowed simplified

culture conditions and handling in comparison to primary cells cultured in matrigel.

Outlook. The development of an in vitro test system based on murine immortalized

primary intestinal epithelial cells provides a promising tool for a more predictive and

standardized preclinical screening. Data generated by these models will close a gap

between in vivo and in vitro, which might help to improve the interpretation of in vivo

data from mice. In a next step we will generate immortalized cells from human small

intestinal epithelial cells.

Establishment of a murine intestinal tissue model based on immortalized primary epithelial cells

Christina Fey1, Theresa Truschel2, Matthias Schweinlin1,

Heike Walles1,3, Tobias May2, Marco Metzger1,3

1�Department of Tissue Engineering and Regenerative Medicine (TERM), University

Hospital Würzburg, Röntgenring 11, 97070 Würzburg, Germany

2 InSCREENeX GmbH, Inhoffenstraße 7, 38124 Braunschweig, Germany

3 Translational Center Würzburg “Regenerative Therapies for Oncology and

Musculoskeletal Diseases” (TZKME), Würzburg branch of the Fraunhofer Institute of

Silicate Research (ISC), Röntgenring 11, 97070 Würzburg, Germany

Introduction. The main functions of the small intestine are the absorption of

essential nutrients, water and vitamins and to build up a barrier to protect us from

pathogens as well as toxic xenobiotics. This intestinal barrier is formed by tight-

junctions sealing the paracellular gap and a mucus layer covering the epithelium. In

order to investigate the uptake of drugs or infection mechanisms, suitable in vitro

models are required. To match these specifications we generated immortalized cell

clones of murine small intestinal epithelial cells together with our collaboration partner

InSCREENeX. These cells show properties comparable to the native gut and

enables a much easier handling in comparison to primary intestinal cells.

Methods. Intestinal crypts were isolated from murine small intestinal tissue samples

and expanded as enteroids over four weeks. To generate immortalized cells our

collaboration partner InSCREENeX transfected the cells lentiviral. The selected cell

clones were characterized by qPCR, histological stainings, electron microscopy and

transport assays. Epithelial barrier integrity was evaluated by TEER measurement.

Results. The characterization of the immortalized epithelial cell clones revealed

stable cell growth over a long time period and a characteristic epithelial cell

morphology. Gene and protein expression pattern of several intestinal markers

indicated a mixed cell population of gut-specific epithelial cell types. Electron 96 97

While untreated models showed homogenous staining in MTT tests, burnt areas in

wound models showed no viability that was surrounded by a ring of vital

keratinocytes at the wound edges. Burning of the models led to an increased

production and secretion of LDH, while glucose consumption was not altered.

Impedance spectroscopy showed only little influence of burning, since the stratum

corneum, which is mainly responsible for skin barrier characteristics, was not

damaged by the burning process. Histological staining showed excessive damage of

basal and suprabasal cells in the burn area, and first ingrowing cells from the wound

edges six days after injurie.

Conclusion By burning RHE models with a heated metal rod a reproducible in vitro test model for

burn wounds could be created. The models showed symptoms of injury in

histological examination, as well as in molecular and physical measurements.

Acknowledgements This work was funded by the Bavarian Research Foundation project number AZ-

1210-16 – Freeze-drying of human therapeutical cells.

Development of a human epidermal burn wound model Verena Schneider, University Hospital Würzburg Chair of Tissue Engineering and

Regenerative Medicine, Würzburg; Ives Bernardelli de Mattos, QRSkin GmbH,

University Hospital Würzburg Chair of Tissue Engineering and Regenerative

Medicine, Würzburg; Martin Funk, QRSkin GmbH, Würzburg; Heike Walles,


Medicine, Fraunhofer ISC, Würzburg; Florian Groeber-Becker, University Hospital

Würzburg Chair of Tissue Engineering and Regenerative Medicine, Fraunhofer ISC,

Würzburg

Introduction Burn injuries are a leading cause for morbidity and cause high rates of mortality in

low- and middle-income countries, with an estimated 180.000 deaths per year

(WHO). There are numerous products on the market for the treatment of severe burn

wounds, which still show severe disadvantages e.g. in aspects of efficacy. The

standard procedure to test the efficacy of a new product are animal and two-

dimensional (2D) cell culture models. While both show only limited comparability to

the in vivo situation, the latter one also involves painful treatments for the animals. A

three-dimensional (3D) test system based on human primary cells could be an

improvement for the preclinical testing of new applications and wound dressings.

Therefore, a human burn wound model which is based on a reconstructed human

epidermis was developed that holds the potential to replace, or at least reduce,

animal based test procedures.

Experimental methods Reconstructed human epidermis (RHE) models were generated, using primary

juvenile keratinocytes cultured at the air liquid interface according to the opensource

reconstructed human epidermis protocol initially published by Pumay et al and

refined by Groeber et al. . Burn injuries were introduced by contact with an 83°C

heated metal rod for 5 seconds. Models were examined 24hours, 48 hours and 6

days after injuring. The properties of models were characterized by viability testing,

electrical impedance measurement, secretion of ß-lactate and lactate dehydrogenase

(LDH), glucose consumption; and histological examination.

Results and Discussion

Development of a human epidermal burn wound model Verena Schneider, University Hospital Würzburg Chair of Tissue Engineering and

Regenerative Medicine, Würzburg; Ives Bernardelli de Mattos, QRSkin GmbH,


Medicine, Würzburg; Martin Funk, QRSkin GmbH, Würzburg; Heike Walles,


Medicine, Fraunhofer ISC, Würzburg; Florian Groeber-Becker, University Hospital

Würzburg Chair of Tissue Engineering and Regenerative Medicine, Fraunhofer ISC,

Würzburg

Introduction Burn injuries are a leading cause for morbidity and cause high rates of mortality in

low- and middle-income countries, with an estimated 180.000 deaths per year

(WHO). There are numerous products on the market for the treatment of severe burn

wounds, which still show severe disadvantages e.g. in aspects of efficacy. The

standard procedure to test the efficacy of a new product are animal and two-

dimensional (2D) cell culture models. While both show only limited comparability to

the in vivo situation, the latter one also involves painful treatments for the animals. A

three-dimensional (3D) test system based on human primary cells could be an

improvement for the preclinical testing of new applications and wound dressings.

Therefore, a human burn wound model which is based on a reconstructed human

epidermis was developed that holds the potential to replace, or at least reduce,

animal based test procedures.

Experimental methods Reconstructed human epidermis (RHE) models were generated, using primary

juvenile keratinocytes cultured at the air liquid interface according to the opensource

reconstructed human epidermis protocol initially published by Pumay et al and

refined by Groeber et al. . Burn injuries were introduced by contact with an 83°C

heated metal rod for 5 seconds. Models were examined 24hours, 48 hours and 6

days after injuring. The properties of models were characterized by viability testing,

electrical impedance measurement, secretion of ß-lactate and lactate dehydrogenase

(LDH), glucose consumption; and histological examination.

Results and Discussion

98 99

An injectable hybrid hydrogel for tissue engineering applications Rainer Wittig, Institute for Laser Technologies in Medicine & Metrology (ILM) at Ulm

University, Ulm, Germany

Bernhard Baumann, Institute for Inorganic Chemistry II, Ulm University, Ulm,

Germany

Mika Lindén, Institute for Inorganic Chemistry II, Ulm University, Ulm, Germany

The generation of an instructive biomaterial that mimics the natural environment and

thus promotes proliferation and selective differentiation of precursor / stem cells is a

great challenge for tissue engineering. To address this demand, we investigated the

RADA16-I self-assembling peptide in combination with nanosized mesoporous silica

drug carriers for injectable bone engineering. RADA16-I is soluble in water, but in

response to the addition of salts it rapidly forms a hydrogel consisting of a three-

dimensional nanofiber network with structural similarities to collagen. Suspensions of

MC3T3-E1 pre-osteoblasts containing RADA16-I peptide and mesoporous silica

nanocarriers differing in size, shape, and surface functionalities were combined with

physiological media, and the resulting hybrid hydrogels were analyzed with regard to

their cell content and distribution, their biocompatibility and nanocarrier internalization

efficiency. Three-dimensional hybrid hydrogels allowed for long-term culture and

differentiation of pre-osteoblasts. Nanocarrier size, shape and surface chemistry

critically influenced biocompatibility and uptake characteristics, which could be further

correlated with matrix-specific parameters such as RADA16-I adsorption and

resulting changes in surface charge. In this proof of concept study, we verified the

biocompatibility of our approach and identified relevant parameters for the

interactions of silica nanocarriers and cells within peptidic matrices. Future

investigations will focus on the temporal control of nanocarrier-mediated drug

delivery, aiming at the optimization of precursor cell differentiation for advanced

tissue engineering in vivo.

Initial screening of novel copolymer micelles for biocompatibility and effects on cell motility

Y. Yordanov1, D. Aluani1, B. Tzankov1, V. Tzankova1, R. Kalinova2, I. Dimitrov2, V.

Bankova3, M. Popova3, B. Trusheva3, K. Yoncheva1; 1Faculty of Pharmacy, Medical University of Sofia, 1000 Sofia, Bulgaria

2Institute of Polymers, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria 3Institute of Organic Chemistry with Center for Phytochemistry, Bulgarian Academy of

Sciences, 1113 Sofia, Bulgaria

Polymer micelles are effective drug delivery systems capable of introducing highly

lipophilic therapeutic molecules in the bloodstream. The present study applied fast

and reliable in vitro assays for initial toxicological evaluation of newly synthesized

copolymeric micellar carriers, in particular poly(oxyethylene)-b-poly(D,L-lactide-co-

carbonate) (BA) and cynnamyl-grafted poly(oxyethylene)-b-poly(D,L-lactide-co-

carbonate) (BCyn). The structure of these carriers was designed to be compatible

with that of caffeic acid phenethyl ester (CAPE), a lipophilic active molecule found in

propolis and known with variety of pharmacological activities.

An immediate toxicological hazard is direct damage to blood cells, which

mediates subsequent immune reactions. Furthermore, liver metabolites could exert

cytotoxicity or have subtler effects, affecting functions as cell motility. We tested the

hemolytic potential of the copolymer micelles on isolated human erythrocytes. The

micelles were tested for cytotoxicity using the standard MTT-dye reduction assay (24

and 48 h) on two cell lines – HepG2, which retains some of the metabolic activity of

human hepatocytes, and L929, a fibroblast cell line, prescribed for in vitro

biocompatibility studies. Changes in structure and function, affecting cell motility,

were evaluated visually and quantitatively by scratch assay on L929 cells. The

experimental study of both copolymers revealed that they did not exert hemolysis,

cytotoxic effects, or changes in L929 motility. Noteworthy, BA-treated cells tend to

migrate more slowly than BCyn-treated ones, with a statistically significant difference

in migration rates.

In conclusion, the initial evaluation of both copolymeric micelles qualified them

as safe, biocompatible and suitable for further studies as CAPE-delivery vehicles.

Acknowledgement: Support from National Science Fund of Bulgaria (Grant

DN 09/1) is greatly acknowledged.

100 101

Cell on cell – functionally immortalized smooth muscle cells as building blocks for 3D tissues

Aileen Bleisch1, Tobias May1, Susann Dehmel2, Sam Wadsworth3 1 InSCREENeX GmbH, Braunschweig, Germany

2 Fraunhofer ITEM, Hannover, Germany 3 Aspect Biosystems, Vancouver, Canada

Pulmonary hypertension and respiratory diseases as COPD and asthma are highly

prevalent and represent a major health problem worldwide. Currently-available drugs

mainly focus on relieving disease symptoms. For the development of more

efficacious drugs, the emergence of lung diseases needs to be even better

understood. Therefore in vitro test systems mimicking in vivo airways and vessels are

highly desirable. For this purpose smooth muscle cells are indispensable as they

regulate the bronchomotor tone and contribute to and regulate airway mucosal

inflammation as well as blood flow. The aim of the present study is to generate

authentic smooth muscle cell lines that can be used as building blocks to create

functional 3D muscle tissues in vitro.

Therefore, in a first step pulmonary artery and bronchial smooth muscle cell lines

were established by the CI-SCREEN technology. In this technology, a lentiviral gene

library is introduced into the respective primary cell and induces unlimited cell

expansion and at the same time maintains the primary phenotype. Primary smooth

muscle cells transduced with this gene library showed doubling times ranging from

2,3 - 3,1 days and reached 30 cumulative population doublings after 70-95 days. In

contrast primary cells doubled circa every 7,5 days and stopped proliferation

completely after reaching 12 population doublings. Characterization of the novel cell

lines displayed a typical spindle-like morphology and showed the expression of

smooth muscle specific markers as alpha smooth muscle actin and calponin 1.

Furthermore, intracellular calcium signaling could be induced in smooth muscle cell

lines by agonist stimulation – an initial step for contractions.

These robustly proliferating smooth muscle cell lines are promising tools to create

novel in vivo-like assay systems and will be used to establish 3D in vitro tissue

models.

A tissue engineered Full Thickness Skin Equivalent based on a non-contracting, biophysical optimised collagen type-I hydrogel

Philipp Fey, Fraunhofer ISC, Würzburg/Germany

Christian Reuter, Julius-Maximilians-Universität Würzburg, Würzburg/Germany

Tamara Finger, Fraunhofer ISC, Würzburg/Germany

Markus Engstler, Julius-Maximilians-Universität Würzburg, Würzburg/Germany

Heike Walles, Universitätsklink Würzburg, Würzburg/Germany

Florian Groeber-Becker, Fraunhofer ISC, Würzburg/Germany

Since the early work of Rheinwald and Green (1975), it is well known that the

development of epithelial and mesenchymal cells is highly codependent. Resulting

from this, a skin equivalent incorporating not only epithelial keratinocytes, but also

mesenchymal fibroblasts, might yield more accurate results, then just a reconstructed

human epidermis (RHE). Our full thickness skin equivalent (FTSE) is composed of a

stable murine collagen type-I matrix with incorporated primary human fibroblasts and

a well differentiated epidermis, composed of primary human keratinocytes. The used

insert system facilitates easy testing procedures such as toxicological testing, testing

of the skin’s barrier function using impedance spectroscopy or even infection studies

using trypanosome infected Tsetse flies (Glossina spec.). We use well defined in-

house isolated cells and collagen to control the key factors of the models and can

therefore produce a high-quality skin equivalent. The collagen hydrogel is

biophysically modified to minimise its contraction rate. Using a broad spectrum of

biochemical and biophysical measuring techniques, we can generate various

collagen hydrogels with defined stiffness to investigate different aspects of infections

or cell proliferation in the human skin. Due to the easy to-use preparation and

culturing process, the models guarantee a high standard and promise a highly

versatile testing platform for a broad variety of in-vitro applications. With the

incorporation of various cell types such as neurons, macrophages or endothelial cells

the application spectra will grow even more.

102 103

We demonstrated the feasibility to perform subcutaneous injections in the model.

Administration of a pro-inflammatory cocktail composed of TNFα and LPS induced

after 24 hours dermal collagen degradation and mild cell vacuolization in the

epidermis. Increased synthesis of pro-inflammatory cytokines such as IL-6 and IL-8

was also observed and RNAscope analysis revealed strong expression of genes

encoding for those cytokines in leucocytes, fibroblasts, adipocytes and endothelial

cells of the hypodermis.

In conclusion, we have developed a unique fully human ex vivo skin model that

allows for subcutaneous injection. Moreover, we showed the relevance of this model

to respond to injection site inflammatory reactions, demonstrating potential utility for

these models to support therapeutic development.

�

�Evaluation of local inflammatory reactions following subcutaneous

injection of a pro-inflammatory cocktail in a fully human ex vivo skin model

�Jardet C.1, Pagès E.1, Raude E.1,2, Seeliger F.3, Brandén L.3, Braun E.1, Ingelsten

M.3, Descargues P.4 �

1 Genoskin SAS, Toulouse, France 2 LAAS-CNRS, Toulouse, France 3 Drug Safety and Metabolism, iMED Biotech Unit, Astra Zeneca, Gothenburg,

Sweden 4 Genoskin Inc. Boston (MA), USA

�

Subcutaneous injections can evoke local reactions such as inflammation or necrosis.

Preclinical development of subcutaneous therapies relies on animal models to

characterize these responses, however, translation of these models to the clinic is

often limited, and no single model has emerged as an accepted standard.

Furthermore, animal testing is expensive, time consuming and poses ethical issues.

There are currently no in vitro human models to test toxicological effects of

subcutaneous injections.

We developed a new ex vivo human skin model called HypoSkin, containing all skin

layers including epidermis, dermis and hypodermis. The skin explant is embedded in

a proprietary gel-like matrix with epidermal surface left in direct contact with air. The

system is mounted into cell culture inserts and cultured in standard cell culture

conditions.

Histological analysis of the HypoSkin model showed preserved tissue integrity and

cell viability in all 3 skin layers for up to 5 days culture. Distribution of adipocyte sizes

across the tissue and total number of adipocytes was also similar before and after ex

vivo culture.

104 105

Automating 3D cell culture using a wood-derived hydrogel Lauri Paasonen, UPM-Kymmene Oyj, Helsinki, Finland

Introduction

In the pursuit of in vitro cell models that are biological relevant with improved

functionality, new materials and methods for creating of three-dimensional (3D) cell

culture systems are a key requirement. GrowDex®, nanofibrillar cellulose (NFC)

hydrogel, which is derived from the Birch tree, has been shown to provide an

effective support matrix for culturing cells in 3D. As such this hydrogel can play a vital

role in aiding the a simplified transition from 2D to 3D cellular models.

Results Studies have shown NFC hydrogel to be an effective support matrix for a variety of

cells including primary, ES, iPS and patient derived cells. Hepatic cells have been

shown to form 3D spheroids with polarized structures and better functionality of the

metabolic enzymes when compared to traditional 2D culture. Long term culture of

primary hepatocytes enables repeated dose drug toxicity testing. Proliferation of

embryonic stem cells and induced pluripotent stem cells without feeder cells has also

been successfully accomplished, as well as the proliferation and differentiation of

mesenchymal stem cells. The shear-thinning property of GrowDex, the fact that is not

temperature sensitive and requires no cross-linking step, permits its use with

automated dispensing systems at ambient temperature in high throughput screening

(HTS) –related 3D cell culture applications. Finally, grown 3D cell structures can be

collected for detailed downstream analysis by cellulase enzyme treatment.

Conclusions Nanofibrillar cellulose hydrogel, GrowDex., is biocompatible with both human and

animal cells and tissues. It is a xeno-free, ready-to-use hydrogel with shear-thinning

properties that enable injecting and simple manual or automated pipetting of the

material. 3D cell growth is made possible as GrowDex physically resembles

extracellular matrix (ECM) allowing free diffusion of small molecules, such as

nutrients and oxygen. GrowDex can be completely degraded to soluble glucose by

enzyme treatment. This preserves the 3D cellular structure with no adverse impact

on cells being observed. These properties make GrowDex a suitable matrix for a

culturing a wide variety cells for use in numerous 3D cell culture applications.

A microchip array-based 3D culture system for the in vitro differentiation of osteoblasts

Weiping Zhang1, Pascal Tomakidi2, Thorsten Steinberg2, Ralf-J. Kohal3, Eric

Gottwald4, Brigitte Altmann1,3, 1G.E.R.N., Department of Oral and Maxillofacial Surgery, 2Department of Oral

Biotechnology, 3Department of Prosthetic Dentistry, Medical Center, Faculty of

Medicine, University of Freiburg, Freiburg/D, 4300MICRONS GmbH, Karlsruhe/D

In order to integrate cell-instructive cues into bone tissue engineering scaffolds that

guide target cell behavior towards tissue regeneration it is imperative to extend our

knowledge on cell function modulation by the microenvironment on the molecular

level. In this context, we established an osteogenic 3D cell culture model to study the

effect of spatial and mechanical cues on morphogenesis and biomarker expression

of primary human osteoblasts under more in vivo-relevant growth conditions when

compared to conventional monolayer (2D). Therefore, osteoblasts were cultured for 7

and 14 days under static and microfluidic growth conditions in DYNARRAYS© and

analyzed for aggregate morphogenesis by live/dead-staining, scanning electron

microscopy, immunocytochemistry and real-time PCR. The results show that cells in

3D-static conditions self-assembled into multilayered cubic aggregates with high

viability in the chip cavities. Compared to 2D, osteoblasts in 3D-DYNARRAYS©

exhibited differential extracellular matrix deposition patterns and higher expression of

osteogenic genes. Among 3D-conditions, addition of fluid flow induced osteoblast

reorganization into rotund bony microtissue, comprising more densely packed

multicellular 3D-aggregates, while viability of microtissues was flow rate dependent.

Our data provide evidence for the superiority of 3D-culture devices over conventional

2D-systems, regarding the outcome of the osteogenic phenotype. The further

improvement of microtissue architecture by fluid flow emphasizes the inclusion of in

vivo-like biomechanical cues into bone-directed 3D-experimental setups. Hence, the

presented static and dynamic 3D osteoblast culture model provides a unique tool for

in vitro bone cell research with regard to cell function modulation on molecular level.

106 107

Microspheres-based scaffolds from poly(3-hydroxybutyrate) for 3D cell growth

Chesnokova Dariana V.1, Zharkova Irina I.1, Bonatsev Anton P.1, Voinova Vera V.1

1Moscow State University, Moscow, Russia.

Advanced models for substance testing Poly(3-hydroxybutyrate) (PHB) is a biocompatible and biodegradable polymer

produced by a microbiological way. The devices from this biomaterial can be used for

long-time cultivation and substance testing on cells. Mesenchymal stem cells (MSCs)

have a potential to proliferate to different cell types, so the use of MSCs opens up

great opportunities for testing drugs. The shape and microstructure of the substrate is

known to exert a strong influence for growth and differentiation of MSCs, which

imposes special requirements on the design of 3D scaffolds. The aim of this work

was to study the possibility of using 3D scaffolds based on porous microspheres of

different diameter from PHB to create models of 3D cell growth and further use for

testing substances.

To produce porous microspheres the double water-oil-water emulsification technique

using ammonium carbonate as a blowing agent was used. To produce 3D scaffolds

the obtained porous microspheres were immobilized on the PHB film by gluing with

chloroform. MSCs isolated from bone marrow of newborn rats and fibroblasts COS-1

were used. For investigation of the growth pattern of MSCs and COS-1 on scaffolds

they were analyzed by scanning electron microscopy after three weeks of cells

cultivation. The produced 3D scaffolds were able to support growth of MSCs and

COS-1 on the scaffolds, on scaffolds from small microspheres cell grew on the

surface of microspheres and between them; on scaffolds from large microspheres

MSCs grew preliminary on the surface of microspheres. The activity of alkaline

phosphatase was studied to test possible MSCs osteogenic differentiation. In

osteogenic media the activity of alkaline phosphatase in MSCs isolated from bone

marrow was elevated 30-times in comparison with control. However, even in

osteogenic medium the activity of alkaline phosphatase of MSCs grown on scaffolds

didn’t differ from the control that allow to use PHB microspheres-based scaffolds for

long-term cultivation of MSCs of their native phenotype and testing substances on

them.

This work was supported by Russian Science Foundation, project # 17-74-20104.

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108 109

A tissue engineering approach to model Primary Ciliary Dyskinesia

Nina Lodes1,2, Heike Walles1,3, Stephan Hackenberg2, Helge Hebestreit4, Maria

Steinke1,3 1 University Hospital Würzburg, Chair of Tissue Engineering and Regenerative Medicine, Röntgenring 11, 97070 Würzburg,

Germany 2 University Hospital Würzburg, Department of Otorhinolaryngology, Plastic, Aesthetic and Reconstructive Head and Neck

Surgery, Josef-Schneider-Str. 11, 97080 Würzburg, Germany 3 Fraunhofer Institute for Silicate Research, Translational Center Regenerative Therapies, Röntgenring 11, 97070 Würzburg,

Germany 4 University Hospital Würzburg, Department of Paediatrics, Josef-Schneider-Str. 2, 97080 Würzburg, Germany

Primary ciliary dyskinesia (PCD) is a rare disease with recurrent upper and lower

airway infections due to abnormal function of cilia and, therefore, impaired

mucociliary clearance. PCD has an autosomal recessive inheritance pattern with

mutations in at least 32 genes. The generation of complex 3D tissue models either

based on individual tissue biopsies, or genetically modified human airway epithelial

cells could support personalized treatment strategies.

The aim of this study is the establishment of a 3D PCD disease model that reflects

the in vitro in vivo correlation better than 2D cell culture and subsequently, substance

testing for personalized medicine.

Healthy 3D airway mucosa tissue models, which serve as controls, were generated

based on a decellularized porcine jejunal scaffold, using human primary airway

epithelial cells and fibroblasts for co-culture.

To verify the mucociliary phenotype, histological analysis was carried out. High speed

video microscopy analysis is performed for ciliary beating frequency analysis and

particle transport studies are exerted for investigation of mucociliary clearance. Due

to lacking donor material and in order to model individual phenotypes of PCD, the

establishment of genetically modified cell lines will be done by using the

CRISPR/Cas9 technology. Different therapeutical substances will be tested on both,

the healthy and the PCD models.

In vitro 3D bladder cancer model using PDX-derived cells Robson Amaral1, Ai-Hong Ma2, Hongyong Zhang2, Kamilla Swiech1, Chong-Xian Pan2

1University of Sao Paulo, Ribeirao Preto, Brazil; 2University of California Davis,

Sacramento, USA

The search for an effective and efficient model for drug-screening assays is one

of the main goals in the pharmaceutical area, both in academia and industry1. Using

patient-derived xenograft (PDX) models in immune-compromised mice it is possible to

keep human-derived tumors in vivo and allows compound testing during preclinical

phase. However, PDX models present technical disadvantages, such as long

engraftment time and low success rate, and high maintenance cost2, as well as ethical

restrictions. The present work aimed to develop a simple, reproducible and low-cost

3D in vitro culture method for PDX-derived bladder tumor cells that mimic the in vivo-

like cell behavior and response to therapeutics. We used two PDX-derived cells,

BL0293 and BL0808, previously established from advanced bladder cancer3. After

digestion, different concentrations of single cells were cultured in 96-well round-bottom

Ultra-Low Attachment (ULA) plates with 5% of Matrigel in RPMI 10% SFB. Tumor

spheroids were characterized regarding size (Diameter) and shape parameters using

ImageJ software. Both PDX-derived cells formed regular and round-shaped spheroids

(roundness>0.8) with a diameter higher than 400 �m, size that can be considered

ideal to use in drug response assays due to the presence of a hypoxic core that is

related to drug resistance in solid tumors in vivo4, 5. The response of the spheroids to

10 �M of antineoplastic drugs, Cisplatin, Gemcitabine and the combination of both,

were evaluated using viability assay kit 3D Cell Titer Glo during 3 days. BL0293

spheroids were more resistant to Cisplatin and partial resistant to Gemcitabine while

BL0808 spheroids were partial resistant to both. However, both were sensitive when

treated with the combination Cisplatin + Gemcitabine. These preliminary results were

similar to those observed in in vivo studies with BL0293 and BL08083. Therefore, the

in vitro 3D PDX-derived model established may predict the outcome of in vivo drug-

screening assays representing a low-cost strategy to perform high-throughput

screening.

References: (1) Waring et al., 2015. Nat. Rev. Drug Discov.14: 475-486. doi: 10.1038/nrd46092. (2) Gheibi et al.,

2017. Scientific Reports 7:12277. doi:10.1038/s41598-017-12543-9. (3) Pan et al., 2015. PLoS ONE 10(8):

e0134346. doi:10.1371/journal.pone.0134346. (4) Hirschhaeuser et al., 2010. J. Biotechnol. 148, 3–15.

doi:10.1016/j.jbiotec.2010.01.012. (5) Francia et al., 2005. Mol. Cancer Ther. 4, 1484–1494. doi:10.1158/1535-

7163.MCT-04-0214 110 111

Characterisation of Bordetella pertussis virulence mechanisms using engineered human airway tissue models

David Komla Kessie1, Maria Steinke 2, 3, Heike Walles2, 3 and Roy Gross1

1. Chair of Microbiology, University of Wuerzburg, Am Hubland, 97074,

Wuerzburg, Germany.

2. Translational Centre and Regenerative Medicine, University Hospital of

Wuerzburg, Roentgenring 11, 97070, Wuerzburg, Germany

3. Fraunhofer Institute for Silicate Research (ISC),Translational Center “Regenerative Therapies for Oncology and Musculoskeletal Diseases“

Roentgenring 11, 97070 Wuerzburg, Germany

Bordetella pertussis is the obligate human pathogen that causes whooping cough. It

expresses various virulence factors that enable it to adhere and colonise the ciliated

airway mucosa. Many of these factors also directly interfere with host signal

transduction systems causing damage to the ciliated airway mucosa and increased

mucous production. Incubation of human tracheal biopsies with Bordetella culture

supernatants caused tissue degeneration and ciliated cell extrusion similar to live

bacterial infection. Tracheal cytotoxin (TCT) is reported to be responsible for the

observed ciliated cell extrusion and epithelia damage in hamster tracheal rings. TCT

is a 921Da muramyl peptide which is released during peptidoglycan cell wall

modification. Using a biological scaffold (SISser®), a 3D airway mucosa test system

was engineered using human tracheobronchial epithelia cells and fibroblasts. The

aim is to use this novel 3D tissue engineered airway mucosa to elucidate the

mechanisms underlying TCT effect and the virulence mechanism of Bordetella

pertussis. These 3D models have a high in vitro/in vivo correlation with human

tracheal epithelia. The 3D models were with TCT for 24 hours. The supernatant was

assessed for nitric oxide production and the models for inflammation and

morphological damage due to the toxin. Furthermore, the interaction of the bacteria

with the airway mucosa will be assessed using high throughput techniques.

The 3D airway models show a well differentiated and polarized epithelium containing

all important cell types, like basal, ciliated and goblet cells as well as mucus on the

epithelial surface. An increasing relative barrier over 21 days of air liquid interface

(ALI) culture is detectable. We identified a suitable cell line for gene editing

experiments, showing the mucociliary phenotype in ALI culture.

The establishment of ciliated 3D airway mucosa models enables to introduce specific

gene knockouts in order to generate disease models for individual phenotypes of

primary ciliary dyskinesia. This approach provides the requirements for individual

therapy and leads to personalized medicine.

112 113

Novel 3D tumour models with stromal components to evaluate the efficacy of immunotherapy with gene-engineered ROR1-specific

CAR T cells

Johanna Kühnemundt1, Claudia Göttlich1, 3, Lars Wallstabe2, Lena Nelke1, Thomas

Schwarz1, 3, Hermann Einsele2, Heike Walles1,3, Sarah Nietzer1, Michael Hudecek2,

Gudrun Dandekar1, 3

1Chair of Tissue Engineering and Regenerative Medicine, University Hospital

Wuerzburg, Roentgenring 11, 97070 Wuerzburg, Germany

2Department of Medicine II - Hematology and Medical Oncology, University Hospital

Wuerzburg, Josef-Schneider-Str.2, 97080 Wuerzburg, Germany 3Fraunhofer Institute for Silicate Research (ISC), Translational Center ‘Regenerative

Therapies’ (TLC-RT), Roentgenring 11, 97070 Wuerzburg, Germany

Introduction During the last years, T cells expressing a synthetic tumour targeting chimeric

antigen receptor (CAR) have shown their potential as a novel therapeutic tool

especially in CD19+ B-cell malignancies. One of the key objectives in this field is to

transfer CAR T-cell therapy from haematological malignancies to solid epithelial

tumours. However, solid tumours pose additional challenges for T-cell therapy, since

CAR T cells have to migrate into tumour lesions, maintain their effector functions and

survive long enough in the immunosuppressive tumour microenvironment to mediate

tumour destruction or regression. To mimic parts of the tumour microenvironment, we

incorporated primary stromal cells into our three-dimensional (3D) breast and lung

tumour models based on a biological tissue matrix. In previous work, ROR1-CAR T

cells displayed substantial reactivity against lung and triple negative breast cancer

(TNBC) cell lines in 2D cultures and xenograft models in immunodeficient mice

(Hudecek et al. 2013 & 2015). Thus, we applied ROR1-CAR T cells in our

physiologically more relevant 3D models and investigate T-cell function and tumour

cell killing.

Methods To generate 3D tumour models of ROR1+ lung carcinoma (A549) and TNBC (MDA-

MB-231), tumour cells were seeded on a decellularised scaffold derived from porcine

small intestine. In addition to cancer cells, human dermal fibroblasts (hDF) or cancer-

Establishment and initial characterization of a simple 3D organotypic wound healing model

Sabine Hensler, Molecular Cell Biology Lab, Institute of Technical Medicine, HFU

Furtwangen University, Villingen-Schwenningen, Germany, Claudia Kuehlbach,

Molecular Cell Biology Lab, Institute of Technical Medicine, HFU Furtwangen

University, Villingen-Schwenningen, Germany, Jacquelyn Dawn Parente, Institute of

Technical Medicine, HFU Furtwangen University, Villingen-Schwenningen, Germany,

Sabine Krueger-Ziolek, Institute of Technical Medicine, HFU Furtwangen University,

Villingen-Schwenningen, Germany, Knut Moeller, Institute of Technical Medicine,

HFU Furtwangen University, Villingen-Schwenningen, Germany, Margareta M.

Mueller, Molecular Cell Biology Lab, Institute of Technical Medicine, HFU

Furtwangen University, Villingen-Schwenningen, Germany,

Poor wound healing resulting in chronic wounds affects millions of people worldwide.

While the complex biological repair process requiring the regulated interaction of

numerous cells in a healing wound is well understood, the number of therapies

available to successfully treat chronic wound is still very limited. Development of new

therapies is costly and time consuming as it requires tests in a complex tissue

context. Thus, defined but simple to use 3D systems reflecting the in vivo tissue

complexity are urgently needed. A 3D organotypic model (OTC) containing the major

cellular component active during wound healing i.e. keratinocytes, fibroblasts and

inflammatory cells – specifically macrophages and neutrophils - was established and

its use in wound healing studies employing standardized wounding procedures was

demonstrated. The model is characterized histologically and by immunofluorescent

staining allowing the localization of specific cell types and matrix structures. Soluble

mediators of wound healing like MMP-2 and -9 and IL-1β are determined and exhibit

an in vivo like kinetics of secretion. The system provides a reproducible, simple to

use yet sufficiently complex basis that will allow the analysis of molecular effects of

therapeutic regimen used in the management of chronic wounds.

114 115

Evaluation of pharmacological responses in InflammaSkin®, a fully human full-thickness ex vivo skin model reproducing key features

of psoriatic lesions Lovato P.1, Jardet C.2, Pagès E.2, David A.2, Braun E.2, Norsgaard H.1, Descargues

P.3 1 LEO Pharma, Ballerup, Denmark 2 Genoskin SAS, Toulouse, France 3 Genoskin Inc. Boston (MA), USA

We have recently established a new T cell-driven skin inflammation model for

psoriasis based on the activation and differentiation of skin resident T cells in

NativeSkin® skin explants. We have demonstrated that intradermal injection of anti-

CD3 and -CD28 antibodies promotes in situ activation of resident T cells and culture

in a chemically-defined medium supplemented with IL-1b, IL-23 and TGF-� induces

Th17/Th1 T cell polarization.

T-cell activation and differentiation into a Th17/Th1 phenotype were supported by the

secretion of T cell-derived cytokines detected from day 3, including IL-17A, IL-22,

IFN-� and TNF-� but no IL-4. At day 7, we observed a sustained expression of these

cytokines and a deterioration of tissue integrity and cell viability at the histological

level. Secretion of T cell-derived cytokines led to overexpression of the epidermal

activation markers S100A7 and Keratin 16 in suprabasal layers. Furthermore, skin

barrier integrity in the inflamed model was assessed by topically applied Lucifer

yellow.

In order to assess pharmacological responses of the model to topically applied

treatments, PDE-4 inhibitor or betamethasone were topically applied either at the

start of ex vivo culture (prophylactic setting) or after 3 days of in situ activation and

polarization (therapeutic setting). Results showed that both treatments led to a

decrease in secretion of inflammatory cytokines and preservation or restoration of

epidermal integrity and viability, indicating anti-inflammatory effects.

associated fibroblasts (CAFs) were incorporated. Models were cultured statically in

so called cell crowns or transferred to a bioreactor system for dynamic culture

conditions. (Moll et al. 2013, Göttlich et al 2016). Immunotherapy was performed

during the last 3 days of static and 5 days of dynamic culture with T cells expressing

a 2nd generation ROR1-CAR (4-1BB costimulation) generated by lentiviral

transduction. The efficacy of CAR T-cell therapy was evaluated by staining and

microscopy of tissue sections, measurement of tumour cell apoptosis as well as and

flow cytometry of T cells.

Results and Outlook After treatment, CD45+ ROR1-CAR T cells were detected in the tumour tissue

indicating that they are capable of actively adhering and migrating throughout the 3D

tumour and matrix.

Treatment with ROR1-CAR T cells resulted in specific destruction of both A549 lung

cancer and MDA-MB-231 TNBC cell lines independent of the presence of either hDF

or CAFs. ROR1-CAR T cells induced rapid tumour cell apoptosis, whereas

untransduced control T cells derived from the same donor did not exert an anti-

tumour effect. This was accompanied by higher proliferation and stronger expression

of the activation markers CD25 and CD69 in ROR1-CAR T compared to control T

cells.

Taken together, these data demonstrate that ROR1-CAR T cells are capable of

destructing 3D solid tumours selectively in the presence of primary fibroblasts and

suggest they have significant therapeutic potential in ROR1+ lung carcinoma and

TNBC. In the future, it will be tested if the presence of fibroblasts impairs the T cell

function in long term treatment of tumor cells by immunoregulatory mechanisms such

as suppressive cytokines or the upregulation of checkpoint molecules. Additionally,

other primary cells like endothelial cells or tumour cells from biopsies will be

incorporated into the models to examine the influence on T cell function. Since the

here presented 3D tumour models for efficacy determination of CAR T cells are

modular, standardised and physiological relevant, they have the potential to reduce

animal testing.

116 117

Generation of human induced pluripotent stem cells (hiPSc)-derived hepatocyte organoids to study liver size control

E.Saponara1 , R. Ungricht1, A. Wenner1, T. Doll1, T. Herper2, M. Mueller1, H. Ruffner1

1 Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland. 2 Novartis Institutes for BioMedical Research, Novartis Pharma AG, Cambridge, Massachusetts 02139, USA Background: While pathways initiating liver regeneration are widely being investigated, mechanisms involved in sensing liver growth still remain unclear. Our team has recently demonstrated that abrogation of Wnt signaling, in a murine model, leads not only to aberrant liver regeneration but also reduction in liver size. Interestingly, the latter phenotype correlated with a dysregulation of several extracellular matrix (ECM)-associated genes and a more abundant collagen deposition in animal livers.

Aim: At present, ECM is recognized as a dynamic structure actively regulating several biological functions, including cell growth. Therefore, prompted by our in vivo evidences, we aim now to generate suitable in vitro liver-mimicking models (organoids) in order to study the influence of Wnt signaling on ECM remodeling and liver size control.

Material and Methods: A hiPSc to induced hepatocyte (iHEP) differentiation protocol was optimized for both laminin-521-cultured hIPSc cultures and self-aggregating, matrix-free, organoids (3D). Molecular and imaging techniques were utilized to assess hepatocyte markers expression and organoids morphology. CRISPR/Cas9 genome editing technology was utilized to inactivate Wnt signaling pathway components.

Results & Outlook: Current and yet preliminary results have confirmed the applicability of the differentiation protocol both on 2D and on 3D systems. Furthermore, efficient CRISPR/Cas9-mediated genome editing was achieved (>85%) in hIPSc clones and maintained throughout the entire differentiation process in 2D. Functional and phenotypic studies are now planned to better dissect the cellular complexity of the iHEP-organoids and, importantly, future work is expected to elucidate how modulation of Wnt signaling will affect ECM deposition, organoid formation and size.

In conclusion, we developed a new fully human ex vivo skin model that successfully

reproduces key features of skin inflammation observed in psoriatic lesions and

showed the use of this model to assess efficacy of topically applied test compounds.

Further development of the model will aim at investigating different treatment

administrations, such as subcutaneous injection in an InflammaSkin® model with

adipose tissue.

118 119

Towards a three-dimensional microfluidic in vitro model to assess efficacy & safety of immune-stimulatory antibody drugs

Ramona Nudischer1,3, Cristina Bertinetti-Lapatki1, Christina Claus2, Kasper Renggli3 ,

Christian Lohasz3, Olivier Frey4, Andreas Hierlemann3 , Adrian B. Roth1

Roche Pharma Research and Early Development, 1Roche Innovation Center Basel,

Switzerland, 2Roche Innovation Center Zurich, Switzerland, 3ETH Zürich, D-BSSE,

Basel, Switzerland, 4InSphero AG, Schlieren, Switzerland

Immune-stimulatory therapies, designed to

engage the adaptive immune system as

part of the pharmacological mode of

action, have gained increasing importance

in drug development and offer a promising

option for therapeutic intervention to treat,

for example, various types of cancers. This

field of “Cancer Immunotherapy” (CIT)

often includes engineered antibodies,

which are highly specific for their targets

and typically reside on different cell types

at distant sites in the human body. These

highly dynamic organ-immune cell

interactions cannot be recapitulated in

simple single-cell-based in vitro systems,

and in vivo tests in rodents would require

the generation of surrogate molecules.

Thus, more comprehensive in-vitro models are needed, which more reliably mimic

the situation in the human body in that they allow for co-culturing of different tissue

types as well as for circulating immune cells under physiological conditions. The goal

of this project is to establish a novel in vitro microfluidic platform, which enables

Figure 1: Schematic of the microfluidic in vitro

system for testing efficacy & safety of immune-

stimulatory antibody drugs. iMAB – immuno-modulatory antibody

Using the Real Architecture For 3D Tissue (3D RAFT™) System as a Versatile Tool to Build in vitro Epithelial Barrier Models

Therese Willstaedt1, John Langer1, Sabine Schäpermeier2, Stefanie Büsch2, Theresa

D’Souza1, Lubna Hussain1, Jenny Schröder2 1Lonza Walkersville Inc., Walkersville, MD, USA; 2Lonza Cologne GmbH, Cologne,

Germany

Conventional in vitro assays are based on cells grown on two-dimensional (2D)

substrates, which are not representative for the true in vivo cell environment. In

tissue environments, cells interact with neighboring cells and with the extracellular

matrix (ECM). Three-dimensional (3D) cell culture methods mimic these interactions

and allow cells to grow in structures resembling more the in vivo environment.

The RAFT™ 3D Culture System uses a collagen matrix at physiologically relevant

concentrations. Cells and neutralized collagen are mixed and dispensed into wells of

standard cell culture plates or transwell inserts, and subsequently incubated at 37°C

to allow the formation of a hydrogel. Specialized RAFT™ Absorbers are placed on

top of the hydrogels. These absorbers gently remove abundant medium and compact

the hydrogel to a layer approximately 100 �m thick. The cultures are then ready to

use, but additional epithelial or endothelial cells may be added on top.

The resulting models provide valuable tools to investigate barrier tissues in an in

vivo-like micro-environment, potentially for use in pre-clinical efficacy and safety

testing. This presentation focuses on different epithelial barriers.

A full-thickness skin model was generated by embedding primary human dermal

fibroblasts within the RAFT™ Collagen and seeding and differentiating human

primary keratinocytes on top of the air-lifted cultures. Histological and immuno-

histochemical evaluation confirmed the resemblance to native skin.

A RAFT™ 3D lung co-culture model containing normal or asthmatic bronchial

epithelial and smooth muscle cells was compared to 2D cultures with respect to cell

proliferation and morphology as well as growth factor and cytokine secretion.

We also demonstrate the feasibility of co-culturing primary human mammary

epithelial cells or the mammary gland adenocarcinoma cell line MCF-7 with human

mammary fibroblasts in a 3D breast cancer model.

120 121

MSCs Isolation in 3D cell culture conditions: challenges, modeling and perspectives.

Dominik Egger1; Marline Kirsch2; Thomas Scheper2; Antonina Lavrentieva2; Cornelia

Kasper1

(1) Department of Biotechnology, University of Natural Resources and Life Sciences,

Vienna, Austria;

(2) Institute of Technical Chemistry, Leibniz University Hanover, Hanover, Germany;

Mesenchymal stem cells (MSCs) are widely used in clinical trials for cell based

therapies and as the biological part of diverse tissue engineered constructs. Plastic

adherence is the oldest method for MSCs isolation and one of the minimal criteria

which must be fulfilled for MSCs characterization. However, isolation and expansion

of MSCs in non-physiologic two-dimensional (2D) environment can influence cell

biology and, later, treatment outcome.

In the present study we developed different approaches for the isolation of MSC from

adipose tissue in 3D culture conditions. First, MSC aggregates were used as a model

to study cell migration from aggregates into three different hydrogels (human platelet

lysate (HPL), GelMA, PEG-fibrinogen). Second, the influence of the aggregate and

pore size on the cell migration rate in hydrogels was investigated. And third, the

migration of MSCs from small pieces of adipose tissue into the three hydrogels was

studied. Initial results showed that MSCs start to migrate from adipose tissue into

HPL gel after 2 days and fully populate the hydrogel after 8 days of cultivation. The

cell population was found to maintain a MSC-like surface marker profile. Future

studies will concentrate on the optimization of 3D conditions for the isolation of

MSCs. Furthermore, the isolation of MSCs from other sources such as umbilical cord

will be considered.

The presented 3D isolation method/system provides a more physiological approach

for stem cell and primary cell isolation (3D to 3D), avoiding 2D conditions in between.

studying the interaction of organs and circulating immune cells for CIT approaches

employing immunomodulatory antibodies (iMABs).

The system, based on an established microfluidic device platform1, was designed to

allow for culturing of circulating immune cells in the same medium that interacts with

different resident 3D organotypic spheroids, so that organ–immune cell interaction

within a body could be mimicked. The system has been tested with different

antibody-constructs to address efficacy and safety questions.

References:

1: Kim, J.-Y., Fluri, D. A., Kelm, J. M., Hierlemann, A. & Frey, O. 96-well format-

based microfluidic platform for parallel interconnection of multiple multicellular

spheroids. J. Lab. Autom. 20, 274–82 (2015).

Figure 2: The in vitro platform was designed to allow for continuous flow of circulating immune cells,

which can interact with different 3D microtissues. MT- microtissues

122 123

WAT-on-a-Chip: Microphysiological systems integrating white adipose tissue

Julia Rogal1,2, Carina Binder1, Elena Rubiu1, Christopher Probst1, Katja Schenke-

Layland1,2, Peter Loskill1,2

1Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Stuttgart,

Germany; 2Research Institute for Women’s Health, Eberhard Karls University

Tübingen, Tübingen, Germany

White adipose tissue (WAT) constitutes about one fourth of a healthy human’s body

mass. Besides being the main depot for excess dietary energy, WAT is now

recognized as a major endocrine gland regulating events like appetite, and aberrant

WAT function is a risk factor for disorders like type 2 diabetes or cardiovascular

disease. Despite WAT’s significance, there is still plenty of research to be done on

the underlying mechanisms associating WAT to human pathophysiology. One of the

main limitations are animal models, which are poor representatives of human

physiology; they differ strongly from human biology especially with regard to

hormone dynamics. Organ-on-a-chip systems have become a promising alternative

to animal models; they integrate engineered tissues into microfluidic platforms

generating physiological relevant in vitro models capable of revolutionizing

personalized medicine, disease modeling and drug development.

We developed a human microphysiological system (MPS) integrating 3D adipose

tissue based on human primary adipocytes. The MPS is based on a micropatterned

multilayer device featuring tissue chambers specifically designed for human WAT

and vasculature-like media channels ensuring the tissue’s nourishment; it provides a

precise control of media composition and the capability to induce temporal variations

such as drug or hormone content. The MPS enables the maintenance of viability and

functionality of the 3D adipose tissue for over 14 days. Physiological functionality of

the WAT was validated by visualization of its fatty acid metabolism and examination

of its response to compounds with established lipolysis-inducing effect on adipose

tissue.

The potential applications of our developed WAT-on-a-chip systems are eminently

multifarious and expand from drug testing platforms to possibilities of disease

modelling.

Retina-on-a-Chip: Merging Organoid and Organ-on-a-Chip technology for complex multi-layer tissue models

Johanna Chuchuy1, Kevin Achberger2, Christopher Probst1, Jasmin Haderspeck2,

Julia Rogal1, Stefan Liebau2, Peter Loskill1; 1Fraunhofer Institute for Interfacial

Engineering and Biotechnology IGB, Stuttgart, Germany; 2Institut für Neuroanatomie

und Entwicklungsbiologie, Eberhard Karls Universität Tübingen, Tübingen, Germany

The human retina is essential for the sensation of light and hence crucial for the

human vision. Retinal damages or diseases such as age-related macular

degeneration or retinitis significantly impact the everyday life of affected individuals,

very often leading to blindness or severe vision impairments. However, for many eye

diseases there is currently no cure or treatment available, partly due to a lack of

suitable model systems. In vivo animal models do have similarities with the human

retina, but very often differ significantly in structure and functionality such as missing

macula or trichromacy and hence are poor representatives of human retinal

diseases. Ex vivo models based on human retinal tissue extracted post mortem are

problematic due to their limited availability. In vitro cell culture models mostly utilize

individual cell types and are not able to recapitulate the complex physiological

structure and functionality of retinal tissue. In recent years, human iPSC based retinal

organoids have emerged as a potential alternative, comprising all retinal cell types,

including photoreceptors, retinal neurons and glia cells, in a self-assembled 3D

structure. However, they are still limited by the lack of retinal pigmented epithelium

(RPE) and by general disadvantages of static well plate culture. Simultaneously,

Organ-on-a-Chip (OoC) systems have evolved to a powerful alternative for classical

cell culture and animal models by providing microphysiological environments

embedded in a vascular-like perfusion. By merging OoC and organoid technologies,

we have developed a physiological Retina-on-a-Chip, which enables the

recapitulation of the complex stratified structure of the human retina in a microfluidic

environment. Combining the self-assembly in organoids with the precise micro

engineering of OoCs, we were able to co-culture RPE layers with organoids in a

defined structure enabling a so-far unmatched functional interplay of photoreceptor

segments with the epithelial cells, which is essential for understanding the continuous

lifecycle of photoreceptors. The developed Retina-on-a-Chip is versatile and

amenable for disease modeling, personalized medicine and toxicity screening.

124 125

3D co-cultivation of beta cells and mesenchymal stromal/stem cells

for diabetes therapy

Florian Petrya, Peter Czermaka,b,c,d,*, Denise Salziga

a Institute of Bioprocess Engineering and Pharmaceutical Technology, University of

Applied Sciences Mittelhessen, Wiesenstraße 14, 35390 Giessen, Germany

b Dept. of Chemical Engineering, Kansas State University, Manhattan KS, USA

c Faculty of Chemistry and Biology, University of Giessen, Germany

d Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Project group

Bioresources, Winchesterstr. 3, 35394 Giessen, Germany

* Corresponding author: Prof. Dr.-Ing. Peter Czermak, Institute of Bioprocess

Engineering and Pharmaceutical Technology, University of Applied Sciences

Mittelhessen, Wiesenstraße 14, 35390 Giessen; Tel.: +49-641-309-2551 Fax: +49-

641-309-2553; e-mail: [email protected]

The number of diabetic patients grows rapidly every year. Diabetes is characterized

by failure of the insulin-producing beta cells and cannot be cured, but only treated

symptomatically with insulin application. The functionality of beta cells in vivo can be

restored with the transplantation of whole pancreatic islets, beta cell pseudoislets or

the pancreatic-differentiated induced pluripotent stem cells (iPSCs). However, this

therapeutic concept bothers from the facts that beta cell grafts do not survive at the

transplantation site and beta cells lose functionality when expanded/cultivated in

vitro. Beta cells can be strengthen and remain their functionality by a co-cultivation

with human mesenchymal stem/stromal cells (hMSCs). hMSCs are known for their

therapeutic properties transmitted by direct cell contact and/or the secretion of trophic

factors. Due to their origin from the islets of Langerhans, beta cells prefer a 3D

environment in form of cell agglomerates/spheroids. We aim to develop a 3D co-

cultivation process in bioreactors, which ensures the high amounts of cells needed

for cell therapy (106-1010 cells per dose) and fulfills the requirements of good

manufacturing practice (GMP) and process analytical technology (PAT). Therefore,

we investigated the effects of hMSCs on beta cells in direct and indirect co-cultivation

set-ups.

High content screening of intestinal organoid cultures to visualize and quantify immune responses

Mariusz Madej, Bram Herpers, Lucia Salinaro, Kuan Yan, Lidia Daszkiewicz, Leo

Price. OcellO BV. Leiden, the Netherlands.

Background Recent advances in establishing organoids from human material have enabled

further development of 3D cell culture models for the intestinal epithelium to

investigate physiological and pathological mechanisms. Here we present a high

throughput 3D human intestinal organoid culture platform combined with high content

phenotype-based analysis that allows visualization and quantification of compounds

and treatment conditions effects on the epithelial integrity.

Methods To investigate the effect and role of the immune response on the function, formation

and integrity of the gut epithelium, we set up the following intestinal organoid

systems: 1. Human normal vs. inflamed ileum or colon organoid assays for chronic

gut inflammatory studies and 2. Co-culture assays of intestinal organoids with

immune cells in diseased and healthy conditions.

Results

High-content 3D image analysis of these organoid models enables measurement of

changes in growth, development, lumen formation, polarity and cell death as well as

the investigation of direct cell-cell interactions in co-culture models (e.g. infiltration of

immune cells). Morphological measurements are complimented with detection of

secreted factors (e.g. cytokines, chemokines) in response to compound exposure.

Conclusion High-content analysis of in vitro cultured organoids represents a rapid and cost-

effective approach to the testing of compounds for the treatment of intestinal

disorders such as Inflammatory Bowel Disease and colon cancer and flag

compounds that may induce adverse effects. Our intestinal organoid technology

platform represents a significant advance on conventional in vitro models and helps

bridge the translational gap between in vivo studies.

126 127

Development of a 3D spheroid SK-MEL-28 tumor model and its characterisation

Julia Klicks, Rüdiger Rudolf, Mathias Hafner; Institute of Molecular and Cell Biology,

Department of Biotechnology, Mannheim University of Applied Sciences

Malignant melanoma is an aggressive type of cancer that tends to metastasize

beyond its primary site. Already before this stage, melanoma cells locally influence

the differentiation pattern of keratinocytes in the epidermis. In particular, the

epidermis surrounding nodular melanoma becomes hyperplastic in 90 % of cases

accompanied by an enhanced expression of the undifferentiated keratinocyte marker

ck14 and a loss of the differentiated keratinocyte marker ck10. Here, we have

established a complex 3D spheroid melanoma tumor model by co-cultivating

fibroblasts together with keratinocytes and melanoma cells. This shows formation of

distinct ”dermal”, “epidermal” and “melanoma-like” zones. Similar to the in vivo

situation, keratinocyte differentiation was impaired at the interaction site with

melanoma cells and melanoma cells were found to spread through the “epidermal”

into the “dermal” layer, mimicking the situation in stage II melanoma. Treatment with

the cytostatic drug, docetaxel, killed external melanoma cells and reconstituted a

normal differentiation pattern of the “epidermal“ cell layers, but did not affect viability

of melanoma cells that had migrated into the “dermal“ layer.

Modeling tumor microenvironment to address the dynamics of tumor, stromal and immune cell interactions

Sofia P. Rebelo1,2, Catarina Pinto1,2, Tatiana R. Martins1,2, Marta Estrada1,2, Nathalie Harrer3, Paula M. Alves1,2, Wolfgang Sommergruber3, Catarina Brito1,2

1 iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal; 2 Instituto de

Tecnologia Química e Biológica António Xavier, Universidede Nova de Lisboa, Oeiras, Portugal; 3 Boehringer Ingelheim, Wien, Áustria; Tumor initiation, progression and treatment are modulated by its complex microenvironment. In addition to the stromal cells and extracellular matrix (ECM), the interaction between tumor and immune cells plays a critical role in all steps of tumor development. Macrophages, part of the innate immune system, are among the most abundant infiltrating cells and may represent up to 50 % of the tumor mass. These polarize into a tumor promoting M2-like phenotype, with low antigenic presenting capacity and cytotoxic function. In this work, we developed a cell model in which the crosstalk between cell compartments is recapitulated, emulating TME dynamics and allowing interrogation of efficacy of TME-targeting agents and their mechanisms of action. This model combines alginate microencapsulation and bioreactor technology for long-term 3D co-culture of tumor cell aggregates (NSCLC and breast cancer), fibroblasts and monocytes.

In triple co-cultures with NSCLC aggregates, monocytes differentiate into macrophages expressing M2 markers that infiltrate the tumor mass. The secretory profile indicates the presence of a tumor-supportive and immunosuppressive TME (e.g, accumulation of G-CSF, CCL22, CCL24, IL4, IL10, MMP1/9). Moreover, we challenged model with chemotherapeutic and immunomodulatory drugs to evaluate the potential of the triple co-culture to be used as a therapeutic screening platform. We show that the treatment with standard of care drugs for NSCLC, Paclitaxel and Cisplatin, led to different outcomes and the contribution of each cell compartment to the drug response was depicted. Challenging the model with an immunomodulatory agent which inhibits CSF1R, Blz945, induced a repolarization of macrophages into a M1-like tumor suppressive phenotype.

In conclusion, the crosstalk between stroma, immune and cancer cells in the developed model promotes the activation of monocytes into tumor associated macrophages, mimicking more aggressive stages of cancer. This constitutes a novel tool to study macrophage plasticity and repolarization in response to chemotherapeutic and immunomodulatory drugs in a tumor microenvironment relevant context.

Acknowledgements:

We acknowledge support from the IMI Joint Undertaking (grant agreement no.115188) and FCT (iNOVA4Health—UID/Multi/04462/2013, SFRH/BD/52202/2013, SFRH/BD/52208/2013).

128 129

Our combined data support the hypothesis of an essential role of autocrine signaling

factors for β-cell activity and insulin secretion and that functional autocrine signaling

might be a key factor for 3D cell culture systems to converge towards in vivo models.

Trace Amines and Fatty Acids are Essential Endogenous Signaling Factors for β-Cell Activity and Insulin Secretion

Sebastian Hauke1, Kaya Keutler1,2, Mireia Andreu Carbo1, Dmytro A. Yushchenko1

and Carsten Schultz1,2*/

1 European Molecular Biology Laboratory (EMBL), Heidelberg, Germany. 2 Oregon Health & Science University (OHSU), Portland (OR), USA.

The secretion of insulin from β-cells depends on extracellular factors, in particular

glucose and other small molecules, such as fatty acids (FAs) and trace amines (TAs),

which act on G-protein coupled receptors (GPCRs).

FAs have been discussed as exogenous secretagogues of insulin for decades,

especially after the FA-receptor GPR40 was discovered. However, FAs have not

been considered as essential endogenous factors, so far. We show that lowering FA

levels in β-cell medium by fatty acid-free bovine serum albumin (FAF-BSA)

immediately reduced glucose-induced oscillations of cytosolic Ca2+ ([Ca2+]i

oscillations) in β-cells, as well as insulin secretion. Mass spectrometry confirmed

FAF-BSA -mediated removal of FAs, with C16-C20 fatty acids identified as most

abundant species. [Ca2+]i oscillations in MIN6 cells recovered, as FAF-BSA was

replaced by buffer or as FA-levels were artificially restored by lipase action or

photolysis of caged FAs.

First identified in the neural system, the trace amine associated receptor 1 (TAAR1)

is suspected to play a functional role even in peripheral tissue. We show that

increased TA levels, selective TAAR1 agonism or the inhibition of TA-inactivating

monoamine oxidases stimulated [Ca2+]i oscillations and insulin secretion from β-cells.

Opposite effects were observed when endogenous TA levels were lowered by

recombinant monoamine oxidase action, by the inhibition of amino acid

decarboxylase, or by cyclodextrins. Selective TAAR1 antagonism shut down [Ca2+]i

oscillations from glucose or TA-stimulated β-cells. The modulation of biochemical

pathways of TAs directly translated into changes of β-cell activity and insulin

secretion. From this we inferred high metabolic turnover rates and an essential role

of TAs as autocrine signaling factors for β-cell activity and insulin secretion.

130 131

Development and characterization of PDX-derived 3D tumor microtissues as platform for screening targeted molecular

therapeutics �

Francesca Chiovaro1, Nicole Buschmann1, Irina Agarkova1, Armin Maier2, Simon

Messner1, Julia Schueler2, Patrick Guye1

�1 InSphero AG, Wagistr. 27, 8952, Schlieren, Switzerland, 2 Charles River DRS

Germany GmbH Am Flughafen 12, 79108 Freiburg im Breisgau, Deutschland

�

A major hurdle for improved success rates in drug development is the lack of

accurate experimental human in vitro models. Patient-derived xenograft (PDX)

models are extensively used in pre-clinical cancer research and are known to

preserve principal biological and histological features, as well as genetic

characteristics. Here we tested the retainment of such criteria in in vitro 3D InSightTM

Tumor Microtissues derived from PDX cell suspensions. The resulting 3D PDX

Tumor Microtissues mimic tumor microenvironment and allow to assess candidate

drugs for novel therapeutic approaches.

Charles River provided InSphero with PDX derived cell suspensions of lung (LXFA

1647, LXFA 677, LXFL 1121), breast (MAXF 2500) and melanoma (MEXF 2106)

origin. 3D Microtissues were generated in AKURA™ 96-well format and

characterized over 10 days in culture. Morphology, tumor proliferation and

phenotypic resemblance of 3D Microtissues were assessed by

Immunohistochemistry. The growth rate and viability of PDX-derived Microtissues

were determined by size analysis (3D Imager) and ATP assay. Based on the distinct

molecular signatures of PDX tumor cells, we have applied selected, targeted

therapies for efficacy testing and validated the correlation to in vivo efficacy.

In this study we have demonstrated that the morphological and molecular features of

the parental tumors are well retained in in vitro 3D PDX Tumor Microtissues. We

suggest that in vitro 3D PDX models offer a more suitable and robust approach to

expedite faithful efficacy assessment and approval of optimal drug candidates.�

In vitro vascularization of a human bone marrow model. Kübrah Keskin, Technische Universität Berlin, Berlin/D; Stefan Sieber, Technische

Universität Berlin, Berlin/D; Uwe Marx, TissUse GmbH, Berlin/D; Roland Lauster,

Technische Universität Berlin, Berlin/D; Mark Rosowski, Technische Universität

Berlin, Berlin/D

Background The bone marrow is, as a harbour of the endosteal and perivascular niche of

haematopoietic stem and progenitor cells (HSPCs), an important organ in the human

body. The stem cell niche is a heterogenous composition of cells and extracellular

matrix (ECM) which serves as a home for HSPCs. To properly understand this part of

the human body, a suitable model of said organ with its niche has to be generated.

Sieber et al. (2017) developed a dynamic bone marrow model harbouring HSPCs in

co-culture with mesenchymal stromal cells (MSCs) for up to 28 days on a

hydroxyapatite coated zirconium oxide based ceramic, mimicking the endosteal

niche. To further improve the model, endothelial cells (ECs) have to be added to the

model since they are also described as a part of the perivascular stem cell niche. Results It could be shown that a one week pre-culture of MSCs is essential to induce pre-

vascular growth of the ECs on the ceramic. It is anticipated that the ECM build up by

the MSCs is essential for the ECs. However, there seems to be a maximum for the

effective pre-cultivation. An eight week pre-culture of MSCs could not support the

pre-vascular formation of the ECs. First tri-culture approaches with MSCs, ECs and

HSPCs, showed the survival of all three cell types in a serum-free medium.

Outlook The developed model, with its endothelial pre-vascular structures, is a promising first

step into a vascularized organoid. This mimics the natural environment of the HSPC

niche more realistically. After a proper lumen formation could successfully be

induced, a linkage to the channel system of the “Multi-organ-chip” is planned.

132 133

Development of a Cardiac Organoid Culture System with hiPSC-derived Cardiomyocytes

Mirja L. Schulze, University Medical Center Hamburg-Eppendorf, Hamburg,

Germany; Bärbel Ulmer, University Medical Center Hamburg-Eppendorf, Hamburg,

Germany; Marc Lemoine, University Medical Center Hamburg-Eppendorf, Hamburg,

Germany; Alexander Fischer, University Medical Center Hamburg-Eppendorf,

Hamburg, Germany, Thomas Eschenhagen, University Medical Center Hamburg

Eppendorf, Hamburg, Germany

Introduction: Biological pacemakers could be an alternative to electronic pacemakers by providing a lifelong approach without battery and by their principal ability to adapt to heart function during adolescence and to physical exercise. The advantages of hiPSC-derived cardiomyocytes together with our established 3D engineered heart tissue (EHT) model were used to generate an in vitro cardiac organoid model suitable to study pacemaker function. Methods and Results: Irregular beating rat EHTs were used as a substrate to study the potential of spontaneously and regularly beating (~1 Hz) human iPSC-derived embryonic bodies (EBs; ~80% cardiomyocytes) to take over pacemaker function. Incorporation of one human EB per rat EHT during casting was sufficient to revert the usual burst beating (mean 4.5 Hz in the burst, 20-30 sec pause between bursts) into a slow (~1 Hz) and regular beating over the entire culture time of the rat EHTs. Connectivity between the human EB and rat tissue was substantiated by optical calcium transient recordings from whole EHT, immunohistochemistry and action potential measurements by both video-optical recording and sharp microelectrodes. Although video-optical recording revealed contraction kinetics of the whole EHT to be rat-like as expected, sharp microelectrode measurements showed human-like action potential kinetics in the part of the EHT in which the human EB is located and rat-like kinetics in the surrounding rat tissue. Conclusion: Taken together, we generated a 2-component cardiac organoid culture system which can be used to study biological pacemaker function.

3D culture model, coupling mechanisms, biological pacemaker model, trigger-substrate interactions

Imitation of the long-lived plasma cell survival niche of the human bone marrow in vitro

Zehra Uyar, Technische Universität Berlin, Berlin/D; Stefan Sieber, Technische

Universität Berlin, Berlin/D; Uwe Marx, TissUse GmbH, Berlin/D; Roland Lauster,

Technische Universität Berlin, Berlin/D; Mark Rosowski, Technische Universität

Berlin, Berlin/D

Plasma cells are generated during immune response in secondary lymphoid organs

and play an important role for humoral immune protection against certain antigens by

continuously secreting antibodies. They reside in specialized niches in the human

bone marrow. The aim of this project is to develop an in vitro model imitating the

long-lived plasma cell survival niche of the human bone marrow. Previously

published data show that a three-dimensional model based on a hydroxyapatite

coated zirconium oxide-based ceramic could be successfully used to culture

hematopoietic stem and progenitor cells (HSPCs) up to 28 days within a microfluidic

Multi-Organ-Chip (MOC). The HSPCs maintained their primitive state (CD34+CD38-)

when co-cultivated in this three-dimensional model with human mesenchymal

stromal cells (MSCs) isolated from human femoral head bone marrow. Based on this

data, the model will be expanded with further cell types to establish a

microenvironment with the required cell-cell interactions and survival signals to

support a plasma cell survival niche. Thus, a complex model imitating the in vivo

human bone marrow regarding the parallel existence of the different niches will be

developed. This survival niche model would serve as a system to study specific niche

interactions and could also help to reduce animal testing.

Human plasma cells (CD138+CD38+) were successfully isolated and enriched via

magnetic activated cell sorting (MACS) from human femoral head bone marrow. The

next step comprises their introduction into the above mentioned and established

human bone marrow model. Essential factors and the microenvironment enabling a

long-term cultivation of plasma cells in vitro will be determined and the bone marrow

model optimized accordingly. Afterwards, the survival and antibody secreting

capacities of the plasma cells cultivated in the bone marrow model will be assessed.

134 135

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Neuronal differentiation of human iPSCs in 3DProSeed hydrogel well plate and establishment of glia co-cultures

Sherida de Leeuw [1, 2], Vincent Milleret [3,4], Benjamin Simona [4], Riccardo Urbanet

[3], Martin Ehrbar [3] and Christian Tackenberg [1,2]. [1] University of Zurich, Schlieren,

Switzerland; [2] Zentrum für Neurowissenschaften Zürich, Zürich, Switzerland. [3]

University Hospital Zurich, Zurich, Switzerland; [4] Ectica Technologies AG, Zurich,

Switzerland.

The iPSC-derived neuronal model is widely used to study neurodegenerative diseases

with a relevant human physiological background in vitro. However, conventional two-

dimensional (2D) in vitro models poorly recapitulate the tissue microenvironment and

therefore three-dimensional (3D) systems are of high interest. Synaptic maturation and

expression of pathological markers in disease models were enhanced in 3D hydrogel-

based systems compared to conventional 2D system, but unfortunately current 3D

techniques are laborious, and do not allow for sequential seeding.

Here we have developed a protocol for neuronal differentiation of human iPSCs in

3DProSeed hydrogel well plates and established co-cultures with glia cells by

sequential seeding. The 3DProSeed well plates feature a high-content analysis-

compatible format (96-wells), with pre-assembled synthetic PEG-based hydrogels that

are stored hydrated and ready for cell seeding. The engineering surface of the

3DProSeed hydrogels enhances cell growth and 3D migration.

iPSCs were seeded onto 3DProSeed gels and directed to neurons by the forced

expression of Ngn2. Sequential co-culture with glial cells was performed to promote

neuronal survival and maturation. After 21 days the iPSCs successfully differentiated

into neurons with comparable survival to conventional 2D co-cultures. Analyses of

synaptic and neuronal maturation by immunocytochemical and Westernblot indicate a

difference in neuronal maturation markers in 3D cultures compared to 2D. In the near

future we aim to establish Alzheimer’s disease-relevant cultures, in order to create a

human in vitro model compatible with pharmacological screening.

136 137

Patient-derived 3D tumor cultures for clinical diagnostics and pre-clinical drug development.

Sander Basten1, Bram Herpers1, Kuan Yan1, Torsten Giesemann2, Julia Schueler2,

Willemijn Vader3, and Leo Price1, 1OcellO B.V. Leiden, The Netherlands 2Charles

River, Freiburg, Germany 3Vitroscan B.V. Leiden, The Netherlands

Background

Ex vivo cultures of patient-derived tumor enable functional testing of treatment

options and optimization of pre-clinical drug development. Patient biopsies or tumor

from patient-derived xenografts (PDX) mouse models are a valuable source of

human tumor material that can be cultured in 3D for screening of drug efficacy, drug

resistance and cellular processes such as proliferation, survival and invasion. A high

throughput approach using 384 well plates enables evaluation of multiple drug

treatments and dose ranges in parallel. After compound exposure, cultures are fixed

and stained with cellular markers. 3D image stack acquisition is followed by ultra-high

content multiparametric analysis using the OMiner platform to profile drug responses

and quantify tumor spheroid volume, apoptosis and tumor invasion.

Results

Dissected PDX material and tumor biopsy material, were used to establish 3D tumor

cultures derived from various indications, including breast, ovarian, cervix,

endometrium, stomach, pancreatic, colon, bladder and lung cancer. These were

exposed to standard-of-care chemotherapeutics (e.g. 5-FU, taxanes, platinum

compounds, anthracyclines, alkylating agents), small molecules (e.g. erlotinib,

lapatinib, trametinib), targeted therapies (PARPi; niraparib, olaparib, rucaparib),

antibodies (e.g. cetuximab, trastuzumab) and antibody-drug-conjugate (ADC, T-DM1)

dose ranges. The tumor culture response was measured, generating dose-

dependent profiles for relevant features.

Conclusions

Patient-derived 3D tumor cultures were tested with standard-of-care and novel

therapeutic agents and high content analysis was used to evaluate drug sensitivity.

This method enables both the in vitro selection of drug candidates in a pre-clinical

Development of microvascular structures inside porous fibrin coated polydioxanon and PLLA/PLGA scaffolds Sebastian Heene1, Stefanie Thoms1, Dr. Rebecca Jonczyk1,

Prof. Dr. Thomas Scheper 1, Prof. Dr. med. Cornelia Blume1 1Institute of Technical Chemistry, Leibniz Universität Hannover, Hannover/Germany

A vascular network is a key requirement for the engineering of tissues of a clinically

relevant size. Hence, pre-vascularization is a crucial process during the development

of an artificial tissue to enable an optimal supply with oxygen and nutrients.

Patches out of different dissolved biocompatible scaffold materials such as

polydioxanon (PDO) and PLLA/PLGA (50/50) were produced using a porogen-

leaching method with a surplus of NaCl. Afterwards, the porous scaffold patches

were decorated with human fibrin for better cell adhesion. Human umbilicial vein

endothelial cells (HUVECs) were cultured on these fibrin coated PDO- and

PLLA/PGLA-scaffolds. In a transwell system human adipose-tissue-derived

mesenchymal stem cells (hMSCs) were used for a conditioning of the medium with

native angiogenetic factors. Cell cultivation was performed for maximally two weeks

under either hypoxic (2.5% pO2) or normoxic (21% pO2) conditions. For visualization

of actin-filaments, the cells on the scaffolds were stained with phalloidin. By immune-

histochemical analysis on 8-10 µm cryo-sected tissue slights, the expression of the

vascular markers CD31 and VE-cadherin was analyzed after 7 and 14 days of cell

cultivation.

We observed a development of microvascular networks under hypoxic conditions on

PLLA/PLGA as well as on PDO. Porous scaffold structures as well as suitable

adhesion factors such as fibrin favor the successful formation of capillaries. This is an

important step towards the development of clinically relevant tissue constructs.

Complex and multi-cell type models Successful development of microvascular structures.

138 139

Detailed Cell-Material Interactions in 3D Cell Culture Systems R. Harjumäki1,2, R. Nugroho1, J.J Valle-Delgado1, Y-R Lou2, M. Yliperttula2, M.

Österberg1, 1Aalto University, Espoo, Finland 2University of Helsinki, Helsinki, Finland

Extracellular matrix plays a vital role in regulating cell morphology, viability, growth

and differentiation. Because the interactions between cells and materials occur at

nanoscale in a liquid environment, it has been difficult to study them in detail and little

is known about them so far. For example, what cell-biomaterial interactions are

needed for successful 3D cell culture is still a question. The most common 3D cell

culture material is Matrigel. Unfortunately, it is undefined, has great batch-to-batch

variability and the spheroids are trapped inside the hydrogel. Many groups studies

new, suitable materials to tackle these problems. Nanofibrillar cellulose (NFC)

hydrogel is one of these novel materials. It owns great physical properties and ECM-

like morphology. This material is suitable for culturing a big variety cells lines from

carcinoma cells to demanding human pluripotent stem cells (hPSCs). These stem

cells have a great tendency to spontaneous differentiations, but in this environment,

they maintained their pluripotency exceptionally. This material allows cells to move

very freely in this material. Similar movement can be seen when cells are cultured on

laminins. On the other hand, they have also very different properties; laminins are

excellent in 2D cultures and triggers stem cell differentiation into certain cell types,

but it allows cells to form spheroids only as mild concentrations.

In this study, we wanted to see how these two materials differs in cell interactions.

We wanted to study more detailed the interactions occurring between the cells and

biomaterials and with this information to find an explanation for cell behavior in

different materials and the reason why NFC hydrogel allows spheroid formation and

hPSCs to be undifferentiated. Atomic force microscopy (AFM) is an excellent tool for

this kind of studies. Our study shows that NFC hydrogel has only very low and non-

specific interactions with cells, when cell-laminin interactions are specific and strong.

This indicates that NFC hydrogel gives only physical support for the cells and thus

allows cells to move freely in hydrogel and does not give cues for stem cell

differentiation. The lack of cell-material interactions makes cell-cell interactions to be

setting as well as efficient selection of PDX models for in vivo follow-up in the same

tumor. This highly translational in vitro-in vivo PDX pipeline is expected to reduce

attrition and increase efficiency in early drug-discovery. Correlation of drug sensitivity

in 3D cultures from fresh patient tumor biopsies, on the other hand, can be used for

development of predictive diagnostics and also provides a unique source of patient

material for drug discovery and development.

140 141

References [1] U. Marx, T.B. Andersson, A. Bahinski, et al., “Biology-Inspired Microphysiological

System Approaches to Solve the Prediction Dilemma of Substance Testing,” ALTEX, 33, 272–321, 2016.

[2] S. Rismani Yazdi, A. Shadmani, S.C. Bu�rgel, et al., “Adding the “Heart” to Hanging Drop Networks for Microphysiological Multi-Tissue Experiments,” Lab Chip, 15, 4138–4147, 2015.

Figure 1: (a) Schematic of a hanging-drop network and (b) 3D model of a drop with the model parameters and experimentally measured flow rate.

Figure 2: (a) Schematic of the forces on beads at the ALI and (b) superimposition of the modelled net tangential force vectors (white vector field) and their magnitudes (colored contour map) on the experimentally observed stagnation of beads at the bottom of the drop.

Figure 3: Controlled stagnation and flow of beads at the bottom of the hanging drop for various drop heights, h, for a drop aperture diameter of 3.5 mm.

Towards controlling the mobility of flowing cells in a hanging-drop network for microphysiological systems

N. Rousset, M. de Geus, A. J. Kaestli, K. Renggli, A. Hierlemann

ETH Zürich, Department of Biosystems Science and Engineering, Basel, Switzerland

Designing microfluidic devices for cell cultures, especially multi-tissue cultures, has

led to approaches, which combine 2D or 3D assemblies of different cell types (tumor,

liver, heart, etc.), interconnected by microchannels in a physiologically relevant order.

These microphysiological systems are often considered the next step towards more

comprehensive and representative in vitro assays [1]. However, to study tissue-

liquid-phase or tissue-blood interactions, e.g., to mimic cellular response to

immunotherapeutic drugs, a liquid-phase transport system with circulating cells is

needed. Emulating such a circulatory system is not trivial, as it requires flowing single

cells around a closed loop. Open microfluidic systems – such as the hanging-drop

network (HDN) – are particularly well suited for this purpose due to their air-liquid

interface (ALI) that reduces cell interaction with microfluidic structures and surfaces

(e.g., PDMS). These HDNs are composed of sequences of drops, connected by

microchannels, where each drop acts as a tissue trap (Figure 1). An integrated

peristaltic pump enables liquid circulation around a closed loop [2].

Numerical models To model cell flow through a HDN, a finite-element method (COMSOL Multiphysics®)

was used to calculate the forces acting on cells at the ALI of a hanging drop (Figure

2). Assuming spherical particles have settled on the ALI, we determined the range of

drop heights, for various drop aperture diameters, which allow cells to circulate

continuously through the system. The numerical models suggest that cell flow or

stagnation can be controlled by modulating/actuating the HDN drop height.

Experimental observations The model was tested experimentally by flowing polystyrene beads (8 µm diameter

and 1.05 g·cm–3 density) in cell culture medium (RPMI 1640 + 10% FBS) around the

closed-loop HDN. With drops of 3.5 mm diameter and an experimentally measured

flow rate of 1 µL·min–1, the flow or stagnation of beads was successfully controlled by

actuating the drop height (Figure 3). The ability to manipulate cell flow will enable

indirect control over cell-microtissue interaction by allowing cells to stagnate near –

or flow away from – microtissues trapped at the bottom of the hanging drop.

142 143

Microphysiological system based on human liver microtissues for intrinsic clearance prediction

Fabrizio Hürlimann1, Salvatore Mannino2, Christian Lohasz3, Kasper Renggli3,

Andreas Hierlemann3, Laura Suter-Dick2, Olivier Frey1 1InSphero AG, Schlieren, Switzerland

2University of Applied Sciences and Arts Northwestern Switzerland (FHNW),

Muttenz, Switzerland 3ETH Zurich, Dept. of Biosystems Science and Engineering, Basel, Switzerland

Medicines given to a patient are in 70% of the cases metabolized through

biochemical processes in the liver. Thus, the concentration of the active compound

decreases over time. Therefore, the therapeutic concentration of drugs with high

metabolic turnover is only maintained for a short period of time, which requires

frequent administration to ensure a therapeutic effect. To circumvent this issue, the

pharmaceutical industry is developing so-called “low clearance”-compounds, which

are more stable and require less frequent dosing. Key for this drug optimization

process is an accurate estimate of the drug metabolism in the liver. Current assays

relying on primary hepatocytes in suspension or monolayers are short-lived and,

therefore, not suited to measure metabolism over longer periods of time (> 6-24 h).

The prediction of in-vivo clearance using in-vitro data is central in drug discovery and

important to set drug doses in the clinic.

Here, we present a low-clearance assay based on 3D primary human liver

microtissues showing long-lasting and stable metabolic activity over more than 4

weeks. Microtissues are cultured in a new microfluidic system with the following

features: (a) Up to 10 microtissues are inter-connected through microfluidic perfusion

and allow for obtaining a substantially higher cell-to-medium volume ratio compared

to conventional assays; (b) Continuous flow and the appropriate microenvironment

provide more physiological conditions; (c) Downscaling of the system by using

microfabrication techniques reduces the use of human donor material and reagents;

(d) Microtissue handling allows for parallelization, automation and thus compatible

with high throughput screening.

By using microfluidic devices, we could show an increase of up to two-fold in

metabolic activity of the liver microtissues compared to the same microtissues

Three-dimensional in vitro co-culture model for

nanoparticle-mediated transfection

Viktoriya Sokolova, Nataniel Bialas, Leonardo Rojas and Matthias Epple

Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE),

University of Duisburg-Essen, Essen, D-45117, Germany

In order to accurately mimic the in vivo environment, 3D cell culture models

without scaffolds can been generated, such as the spheroid model [1]. In this

study, we investigate a co-culture 3D model to mimic heterogeneous tumors

without the use of a scaffold while allowing for homotypic and heterotypic cell-cell

interactions. The transfection (expression of the corresponding protein),

cytotoxicity and the uptake studies of calcium phosphate nanoparticles in the 3D

cell culture model were carried out. Calcium phosphate is a well-suited delivery

system for biomolecules due to its high biocompatibility and good

biodegradability because it is the inorganic component of human bone and teeth

[2-3]. By using appropriate synthetic methods, multi-shell nanoparticles, loaded

with nucleic acids (e.g. plasmid DNA), were prepared and characterized. The

proposed 3D cell culture model can serve as bridge between in vitro and in vivo

studies. We could show that calcium phosphate nanoparticles represent a well-

characterized delivery system in 3D cell culture and are great candidates for

future in vivo application.

References: [1] M. Zanoni, F. Piccinini et al., Sci. Rep., 2016, 6, 19103.

[2] S. Chernousova, M. Epple, Gene Ther. 2017, 282.

[3] B. Neuhaus, B. Tosun, O. Rotan, A. Frede, A.M. Westendorf, M. Epple, RSC

Adv. 2016, 6, 18102.

144 145

Silencing GALNT1 or GALNT2 suppresses malignant phenotypes of

pancreatic cancer cells Ting-Chih Yeh and Min-Chuan Huang

Graduate Institute of Anatomy and Cell Biology, National Taiwan University College of

Medicine, Taipei, Taiwan.

Pancreatic cancer is a highly lethal cancer being the seventh most common cause of

cancer-related deaths worldwide. At the time of diagnosis, most patients present with

metastasis to the regional lymph nodes and distant organs. O-glycosylation is a common

posttranslational modification of proteins and can regulate functions of cancer behaviors.

Aberrant O-glycosylation in cancer cells is often attributed to altered expression of

polypeptide N-acetylgalactosaminyl transferase (GALNT) family proteins which initiate the

GalNAc-type O-glycosylation. Data retrieved from The Human Protein Atlas indicated that

high expression of GALNT1 or GALNT2 was associated with poor survival of patients with

pancreatic cancer. However, the roles of GALNT1 and GALNT2 in pancreatic cancer are

largely uncharacterized. Results from real-time RT-PCR showed that, among 20 GALNT

family members, higher levels of GALNT1 and GALNT2 were expressed in HPAC and

PANC-1 pancreatic cancer cells. Knockdown of GALNT1 or GALNT2 with siRNA in HPAC

and PANC-1 cells was confirmed by real-time RT-PCR or Western blot analysis. MTT

assay showed that knockdown of GALNT1 or GALNT2 reduced viability of pancreatic

cancer cells. In addition, Transwell migration and Matrigel invasion assay showed that

both GALNT1 and GALNT2 knockdown suppressed cell migration and invasion. In

conclusion, these results suggest that silencing GALNT1 or GALNT2 suppresses the

malignant phenotypes of pancreatic cancer cells and that targeting O-glycosylating

enzymes could be a promising strategy to treat pancreatic cancer.

cultured in standard wells. Moreover, metabolic activity per microtissue was not

decreased by a 10-fold higher cell-to-media ratio. Viability and functionality of the

hepatocyte microtissues remained stable over at least 7 days without any medium

exchange. In summary, we were able to substantially increase the metabolic

competence of a microtissue-based assay, which turned out to be suited for

predicting intrinsic clearance of stable compounds.

146 147

Establishment of a Novel Functional in Vitro Assay to Investigate the Angiogenic Potential of Colonic Adenocarcinomas

Sarah Line Bring Truelsen1, Grith Hagel1, Nabi Mousavi2, Henrik Harling3, Klaus

Qvortrup4, Ole Thastrup1 and Jacob Thastrup1

12cureX, Birkerød, Denmark. 2Department of pathology, Rigshospitalet, Copenhagen

University, Copenhagen, Denmark. 3Abdominal center K, Bisbebjerg Hospital,

Copenhagen Universitet, Copenhagen, Denmark. 4Core Facility for Integrated

Microscopy, The Panum Institut, Copenhagen University, Copenhagen, Denmark.

In the treatment of colon cancer only few tools are assisting the clinicians in the

choice of treatment and despite aggressive multidisciplinary therapy, the overall 5-

year relative survival was only reported to be 63% for Danish patients in 2009-2012

[1]. The metastatic process is dependent on neo-vascularization (angiogenesis) of

the tumor by endothelial cells lining the blood vessels. The aim of this project was to

establish a functional assay to quantify the angiogenic potential of 3D micro-tumors

(tumoroids) established from primary colon cancer tumors and liver metastases.

Such a model would provide the oncologists with an in vitro tool to choose the most

effective anti-angiogenic treatment.

The novel model is an advancement of the in vitro angiogenesis assay where Human

Umbilical Vein Endothelial Cells (HUVEC) reorganize into tubules when grown in co-

culture with fibroblasts [2]. By modification of the culture conditions, we have

established a cancer angiogenesis assay, where HUVEC, fibroblasts and tumoroids

(>200 µm in diameter) are able to be co-cultured. In the angiogenesis assay, we can

induce the formation of tubes in a dose-dependent manner by treating with the pro-

angiogenic factor Vascular Endothelial Growth Factor (VEGF) and reduce tube

formation by subsequent treatment with the anti-angiogenic agents Bevacizumab,

Regorafenib, Sunitinib and Sorafenib. Furthermore, we have used the cancer

angiogenesis assay to investigate the induction of tubes upon co-culture with

tumoroids established from five colon tumors and five liver metastases. We found

that tumoroids from seven out of ten patients induced a significant increase in tube

formation and that this induction could be abolished by treatment with Bevacizumab.

Production of clinical grade temporary epidermal substitute obtained from hESC derived keratinocytes for the treatment of

sickle cell leg ulcers: a challenge for regenerative medicine

Domingues S1*, Masson Y1*, Poulet A1, Saïdani M1, Polentes J1, Lemaître G1,

Peschanski M1, Baldeschi C1

1) I-STEM, INSERM/UEVE U861, CECS, Evry, France

* Equally contributing authors

Skin is the largest organ of the body involved in self-protection against external

damages. Epidermis, the upper layer of the skin is mainly composed of keratinocytes

organized to form a physical barrier at the interface of the environment. Some of

diseases associated to genetic mutations or not could weaken this protection and

lead to the disruption of skin integrity. Cell therapy approaches using adult

keratinocytes are currently envisaged however these cells present limited

proliferative capacities and variability in genetic background. Access to an unlimited

source of embryonic pluripotent stem cells (hESC) will aim at overcoming these

limitations since these cells are available in unlimited quantities thanks to their

unlimited proliferation capacity and their pluripotency.

In this context, a protocol allowing the generation of keratinocytes from hESC able to

perform functional pluristratified epidermis was developed. In the perspective of a

human clinical application, the entire protocol have been optimized and adapted

following good manufacturing practice (GMP) conditions from a clinical grade hES

cell line (RC9) obtained at the Biotech Company Roslin Cells. A quality control of the

keratinocytes was established. These controls include the checking for

contaminations, karyology, and viability. Specific controls such as the analyses of the

expression of keratinocytes markers and the absence of pluripotency markers were

performed to verify the quality of the keratinocytes cells bank. In addition, a clinical

grade support was selected for this capacity to allow the formation of a pluristratified

epidermis in vivo.

148 149

Imaging oxygen gradients in cell aggregates and in spheroids G. Liebscha, R.J. Meiera, J. Wegenerb

aPreSens GmbH, Regensburg/Germany; b Universität Regensburg, Institut für

Analytische Chemie, Chemo- und Biosensorik, Regensburg/Germany

3D tissue models are considered as useful in vitro models in biomedical and clinical

research. Multicellular tumor spheroids consist of aggregates of cells and mimic

physiological conditions normally found in tissue more closely compared to standard

2D cell culture. Multicellular tumor spheroids are currently used in many cancer

studies. They are an attractive model system in studies on (chemotherapeutic) drug

efficacy and delivery, cell responses to radiotherapy, angiogenesis, tumor growth and

proliferation, as well as invasion and migration processes. Because 2D cell

monolayers behave differently compared to cells in a 3D environment, spheroids

possess a more complex network of extracellular matrix and cell�cell contacts. Their

3D structure usually comprises an outer region of viable, proliferating cells followed

by an intermediate quiescent cell layer. Depending on the spheroid size, deprivation

of nutrients leads to the formation of an inner necrotic core. This structure with

different isocentric layers leads to the formation of metabolic gradients from the

inside to the outside of the spheroid which also occur in tissue. Due to diffusion limits

and metabolic activity of the outer cell layers, metabolic waste products accumulate

in the inner spheroid region, where levels of nutrients and oxygen are low. However,

studies about oxygenation and hypoxia in live 3D spheroids are scarce because of

the lack of biocompatible measurement techniques. Standard methods are invasive

(Clark-Electrodes) and consume oxygen during the measurement.

Immunohistochemical detection with markers such as misonidazole allows only an

end�point, semiquantitative determination of oxygenation. Nanoparticle�based

detection of oxygen inside spheroids can overcome these drawbacks to a certain

extent. However, incorporation of highly oxygen permeable particles inside spheroids

can alter spheroid structure or physiology, thus influencing oxygen values or

physiological relevance.

We present the novel imaging system VisiSens TD mic for quantitative monitoring of

oxygenation levels in live MCF�7 tumor spheroids under standard growth conditions

with good spatio�temporal resolution. Furthermore, the set�up was used to image the

influence of drugs on oxygen gradients formed by metabolically active spheroids.

Several clinical trials have failed to identify a correlation between the level of VEGF

and treatment response to Bevacizumab [3]. Similarly, when investigating the amount

of tumoroid secreted VEGF-A165 by ELISA, we found no correlation between VEGF

and the measured in vitro tube formation. These results indicate that other pro- and

anti-angiogenic factors, secreted from the cancer cells, are affecting the overall

induction of tubes and support the importance of using a functional assay, such as

the cancer angiogenesis assay, when investigating whether a treatment should be

recommended to a patient.

1. Iversen LH, Green A, Ingeholm P, Osterlind K, Gogenur I: Improved survival of colorectal cancer in

Denmark during 2001-2012 - The efforts of several national initiatives. Acta Oncol 2016, 55 Suppl

2:10-23.

2. Bishop ET, Bell GT, Bloor S, Broom IJ, Hendry NF, Wheatley DN: An in vitro model of

angiogenesis: basic features. Angiogenesis 1999, 3(4):335-344.

3. Maru D, Venook AP, Ellis LM: Predictive biomarkers for bevacizumab: are we there yet? Clinical

cancer research : an official journal of the American Association for Cancer Research 2013,

19(11):2824-2827.

150 151

Microfluidics: a powerful tool to recreate in vivo environment Clémence Vergne, Benjamin Rouffet, Simon Renard & Marine Verhulsel

Fluigent, France

Enabling technologies The importance of the composition and stiffness of the matrix to induce relevant

phenotype and genetic expression to sitting cells is now globally admitted. However,

such models are essentially static whereas most living cells are constantly exposed

to mechanical and chemical stimuli which affect cell differentiation and self-

organization into functional tissue. To overcome this issue, Fluigent has developed

effective instruments to reproduce physiological stimuli. Reproducing aortic pressure

variations is essential to generate a functional endothelium (Figure 1). Fluigent

instruments also recreate smooth gradient typical of paracrine secretions (Figure 2)

and cyclic injection of molecule characteristic of hormonal signaling. Enclose blood

circulation can be modeled through medium recirculation at constant flow rate. As all

instruments are software driven, specific or standard protocols (sequential injection of

different solutions for cell seeding and cell staining), complex flow pattern and

periodic dosing and sampling can be fully automated (Figure 3).

Fig1: Reproduction of aortic pressure variations Fig 2: Stable smooth gradient

Fig 3: Set up for sequential injections and periodic sampling at controlled flow rate

Application of video analysis for the evaluation of cardiac contractility in different in vitro model systems including freshly

isolated adult rat cardiomyocytes and human iPSC-derived cardiomyocytes in 2D- and 3D-culture

Philippe Beauchamp1, Adrian Segiser2, Sarah Longnus2, Thomas M. Suter1,

Christian Zuppinger1

1 Bern Univ. Hospital, Department of Cardiology, Bern, Switzerland, 2Cardiovascular

Surgery Department, Bern Univ. Hospital, Bern, Switzerland

Purpose: Besides the use of existing methods for the measurement of electrical and

calcium signals in cultured cardiomyocytes, the development of methods for non-

invasive and repeated, long-term measurements of contractile function by digital

video analysis may be instrumental for drug development and cardiac disease

modelling. Methods & Results: Adult ventricular cardiomyocytes (ARC)

representing fully mature cells in this project were isolated by retrograde

Langendorff-perfusion of hearts from Wistar rats and were used on the same day or

after overnight incubation. Human iPSC-derived cardiomyocytes (hiPSC-CM) were

purchased from Ncardia and either cultured as a monolayer or in 3D-culture.

Myocardial microtissues (MTs) with and without the addition of human cardiac

fibroblasts (HCF) were generated by scaffold-free self-assembly in hanging drops

(InSphero) for 4 days and cultured for another 10 days in non-adhesive wells or on

glass-bottom dishes (Ibidi). We then used a modified GoPro camera (Ribcage-

H6PRO) to record short movie sequences at high frame rate (240 frames per

second) of cells in a heating chamber (Ibidi) on the stage of an inverted microscope

(Nikon). HiPSC-CM were beating spontaneously while freshly isolated ARC were

field-paced using a MyoPacer (IonOptix). The open-source macro for NIH-Image

"Musclemotion" by Sala et al. was then used to extract kinetic data from the video

sequences after format conversion and data reduction steps. As a first application,

we have compared ARC and 2D-cultured hiPSC-CM with MTs and found significantly

slower kinetics of contractions in the 3D-cultured microtissue. Conclusions: The first

results of this ongoing project have already demonstrated the feasibility of repeated

digital video image analysis of different cardiomyocyte in vitro models using

comparably low-cost equipment.

152 153

Minimalistic hydrogel matrices to direct early neural progenitors from pluripotent stem cells in 3D culture

Andrea Meinhardt, Leibniz Institute of Polymer Research Dresden, Max Bergmann

Center of Biomaterials Dresden, Dresden/Germany; Adrian Ranga, KU Leuven,

Leuven/ Belgium; Elly M. Tanaka, Research Institute of Molecular Pathology,

Vienna/Austria, Matthias P. Lutolf, Ecole Polytechnique Fédérale de Lausanne,

Lausanne/Switzerland; Carsten Werner, Leibniz Institute of Polymer Research

Dresden, Max Bergmann Center of Biomaterials Dresden, and Center for

Regenerative Therapies Dresden, TU Dresden, Dresden/Germany

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An assay to characterize the impact of cigarette smoke exposure on mucociliary clearance in-vitro.

Stefan Frentzel, Laura Ortega Torres, Shoaib Majeed, Patrice Leroy, Filippo Zanetti,

Marco van der Toorn, Manuel C. Peitsch and Julia Hoeng

PMI R&D, Philip Morris Products S.A., Neuchâtel, Switzerland (part of Philip Morris

International group of companies)

Abstract Mucociliary clearance (MCC) constitutes a first-line defense mechanism to remove

inhaled particles or pathogens from the respiratory tract. Impairment of MCC

contributes or plays a causative role in the etiology of various respiratory diseases

and is associated with an increased risk for pulmonary infections. Cigarette smoke

(CS) has been reported to impact all functional elements required for an effective

MCC. This includes the observation that respiratory epithelia of smokers show fewer

cilia and with abnormal morphology. Smoking can lead to mucus hypersecretion or

changes in the biophysical properties of mucus. CS may also influence the hydration

of the periciliary surface liquid (PCL). While there are established tests to measure

MCC (mucociliary transport) rates in humans (e.g. Saccharine transit test), standard

in-vitro assays are lacking that can be used to characterize CS (whole smoke)

effects. We have setup an assay to measure mucociliary transport rates in an in-vitro

setting on nasal MucilAirTM 3D-organotypic air-liquid interface cultures by

determining velocities of polystyrene microbeads. We observed a dose-dependent

decrease of bead transport rates upon exposure of MucilAirTM to 3R4F reference CS.

Concomitant with a decreased transport, cilia beating, as determined at various post-

exposure time points, was similarly impaired in the cultures. This assay is a useful

addition to match clinical reports on CS effects on MCC in humans and may be used

for comparative studies using potential modified risk tobacco products.

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154 155

Real-Time Assay for Apoptosis using Complementation of Annexin V Luciferase Fragments

Terry Riss1, Kevin Kupcho1, John Shultz1, Jim Hartnett1, Robin Hurst1, Wenhui

Zhou2, Ryutaro Akiyoshi3 and Andrew Niles1

1Promega Corporation, Madison WI/USA; 2Promega Biosciences, San Louis Obispo,

CA/USA; 3Olympus Corporation, Tokyo/Japan

We have developed a homogeneous real-time assay for detecting apoptosis that is

recorded using a standard plate-reading luminometer. The assay is based on binding

of annexin V to phosphatidyl serine (PS) which becomes exposed on the outer leaflet

of the cell membrane during the process of apoptosis. We have engineered two

genetic fusion proteins composed of annexin V linked to a small or large subunits of

luciferase. The purified fusion proteins and a luciferase substrate are added as a

reagent to the medium of cultured cells. When exposed to apoptotic cells, the

annexin V-luciferase fragment fusion proteins bind to PS in close proximity to

reconstitute an active luciferase enzyme and generate a luminescent signal. The

reagent can be added to cells for extended periods of incubation enabling detection

of the onset of apoptosis in real time. The homogeneous luminescent assay has

been multiplexed with a fluorogenic DNA binding dye to demonstrate the onset of

annexin V binding precedes loss of membrane integrity and secondary necrosis in

vitro. The assay has been validated using a number of anchorage dependent and

suspension cell lines as well as 3D spheroids and has been shown to correlate with

activation of caspase-3/7 activity as an orthogonal marker of apoptosis. Imaging the

luminescent signal enables creation of time lapse movies showing individual cells

among a population undergoing apoptosis. This new homogeneous apoptosis assay

method represents a simplification and improvement over flow cytometry and

endpoint assay methods by providing kinetic data from the same sample of live cells

in real time using a standard plate reading luminometer.

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156 157

Volume Regulation of HaCaT Spheroids in Response to Hypotonic Stimuli

Elena von Molitor1, Janina Trothe2, Tiziana Cesetti1, Torsten Ertongur-Fauth2,

Rüdiger Rudolf1, and Mathias Hafner1; 1Mannheim University of Applied Sciences,

Institute of Molecular and Cell Biology, Mannheim, Germany; 2BRAIN AG,

Zwingenberg, Germany

The capability to tightly control cell volume is crucial to cope with osmotic fluctuations

and it is required for numerous physiological processes such as apoptotic cell death,

migration and cell division. Hypotonic conditions cause cell swelling, which is

counteracted by regulatory volume decrease (RVD). Key players in this process are

volume-regulated anion channels (VRACs) which allow efflux of Cl- and small organic

osmolytes leading to cell shrinkage. Just recently, LRRC8A and at least one

additional LRRC8 family member were shown to form functional VRACs. Despite the

important function of the human skin in protecting from osmotic stress, the role of

LRRC8A has only been partially addressed in keratinocytes.

In this work, we present a spheroid model composed of HaCaT keratinocytes that is

suitable to study LRRC8A-mediated RVD in a 3D environment. HaCaT spheroids

were formed by using ultra-low attachment plate culturing and then characterized. A

distinct distribution of the differentiation markers keratin 10, keratin 14 and involucrin

was found, suggesting a separation of HaCaT keratinocytes into inner basal and

outer, more differentiated layers. To monitor keratinocyte swelling behavior and

VRAC activity, spheroids were formed with HaCaT cells expressing the halide-

sensitive fluorescent biosensor hsYFP, which is quenched by iodide influx through

activated chloride channels. HaCaT spheroids were perfused with hypotonic buffer

and simultaneously imaged with live cell confocal microscopy to determine cellular

response in real time. Strikingly, upon hypotonic stimulation, cell swelling and

subsequent shrinkage of HaCaT spheroids was observed. Moreover, in parallel to

spheroid swelling, we were able to quantify an osmolarity-dependent decrease of

hsYFP fluorescence indicating activation of VRACs. In summary, we here present a

novel spheroid-based model for the analysis of RVD in skin cells that, due to its 3-

dimensionality, is closer to the in vivo situation than traditional 2D approaches.

Benefits of Real-Time Measurements of Cell Health in 2D or 3D Using a Plate Reader

Terry Riss, Promega Corporation, Madison, WI/USA

Recording data from the same sample of cells at different incubation times during a

treatment protocol provides many advantages compared to using endpoint assays

that kill cells and result in only a single measurement. In addition to eliminating error

from pipetting into replicate assay plates to measure different endpoints or the same

parameter at different times, “real time” assay chemistries that do not kill cells enable

many opportunities to use the remaining live cell population for multiplexing with

other assay chemistries. We will describe example real time assays applied to 3D

cultures to measure cell health parameters (e.g. live cells, dead cells, apoptosis,

metabolites) and provide recommendations for how to overcome challenges applying

“off the shelf” assays to 3D culture models.

158 159

Scaffold-Free Aggregate Cultivation of Mesenchymal Stem Cells in a Stirred

Tank Bioreactor Dominik Egger 1, Ivo Schwedhelm 2, Jan Hansmann 2 and Cornelia Kasper 1

1 Department of Biotechnology, University of Natural Resources and Life Sciences,

Muthgasse 18, 1190 Vienna, Austria

2 Translational Center, University Hospital Wuerzburg, Roentgenring 11, 97070Wuerzburg,

Germany

For the use of mesenchymal stem cells (MSCs) in cell based therapies extensive

expansion is necessary to obtain sufficient cell number for patient treatments. This

expansion phase still remains one of the key challenges since long-term cultivation

and excessive passaging in two-dimensional conditions result in a loss of essential

stem cell properties. These functional damages and alterations often result in low

survival rate of cells, changes of surface marker profiles, and reduced differentiation

capacity, which also influences the success rate in clinical applications.

The cultivation of MSCs in three-dimensional aggregates positively influences stem

cell properties, thus large scale cultivation of MSC aggregates is highly desirable. It is

well known that MSC physiologically reside in 3 D microenvironment under hypoxic

conditions. “Classical” cell culture protocols do not mimic these requirements

resulting in loss of functional properties and genetic instability. Therefore, we

developed strategies for cultivation of adipose derived human MSC aggregates in a

stirred tank reactor under hypoxic conditions. Aggregates were cultivated in a stirred

tank bioreactor and according to computational fluid dynamics calculations were

exposed to comparatively high average shear stress of 0.2 Pa. The viability of MSC

was 78–86% and cells maintained their surface marker profile and differentiation

potential after cultivation.

From these initial experiments, we postulate that expansion of MSC in 3D

aggregates in stirred tank bioreactors is suitable for large-scale production of MSCs

for cell based therapy applications.

Bioengineering 2017, 4, 47; doi:10.3390/bioengineering4020047

Calcium signals in taste-bud like 3D cultures Tiziana Cesetti1, Elena von Molitor1, Paul Scholz2, Katia Riedel2, Rüdiger Rudolf1,

and Mathias Hafner1; 1Mannheim University of Applied Sciences, Institute of

Molecular and Cell Biology, Mannheim, Germany; 2BRAIN AG, Zwingenberg,

Germany

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160 161

Straining of this lattice results in anisotropic movement of the cell similar to the

experimental result.

Our model shows that a geometric anisotropic stiffening of the meshwork on the

microscale due to the macroscopically applied strain, can act as a guidance cue for

directed cell migration. This work highlights that it is crucial to consider the network

properties on the cellular scale when trying to understand the mechanical guidance of cell

migration.

Guiding 3D cell migration in deformed synthetic hydrogel micro-structures

Miriam Dietrich1,2, Hugo Le Roy3, David Brückner4, Hanna Engelke5, Roman Zantl2,

Joachim O. Rädler1, Chase P. Broedersz4

1 Faculty of Physics and Center for NanoScience, Ludwig-Maximilians-University, Munich,

Germany 2 ibidi GmbH, Martinsried, Germany

3 École Normale supérieure Paris-Saclay, France 4 Arnold-Sommerfeld Center for Theoretical Physics and Center for NanoScience, Ludwig-

Maximilians-University, Munich, Germany 5 Department of Chemistry and Center for NanoScience, Ludwig-Maximilians-University,

Munich, Germany

The mechanical properties of the extracellular matrix are fundamental guidance cues for

cell migration in 3D. To prevent cross-signaling of different stimuli present in naturally

derived hydrogels, synthetic hydrogels with a defined composition and therefore reduced

complexity are often used to analyze the underlying mechanisms of directed cell migration.

We are interested in how static strains in these matrices influence migration of embedded

HT-1080 cells.

We use a novel method to introduce uniaxial static strain in matrices. In a photo-induced

polymerization reaction, a polyethylene glycol (PEG) based hydrogel, functionalized with

small peptide sequences that provide cell adhesion ligands and allow proteolytic migration

within the gel, are formed. We micro-structure the gel in thin strips via simple

photolithography inside a channel slide. Due to the confinement of the channel, hydrogel

strips swell anisotropically, thereby inducing uniaxial strain in the network. Embedded HT-

1080 cells show a highly anisotropic migration response parallel to the strain direction, with

maximal anisotropy at intermediate strain levels. Surprisingly, cell migration is less

anisotropic for higher strains. We can account for this non-monotonic response with a

theoretical model of a durotactic cell on a 2D lattice performing proteolytic migration.

162 163

Development, Characterization and Application of a Parallelizable Perfusion Bioreactor for 3D Cell Culture

Dominik Egger 1, Monica Fischer 1, Andreas Clementi 1, Jan Hansmann 2 and Cornelia Kasper 1




Germany

The Development and optimization of expansion and differentiation strategies for

cultivation of stem cells in dynamic bioreactor systems in 3D is of utmost importance

for the manufacturing of cell based therapy products. The main reason for the use of

bioreactor systems is to provide a closed system for safe and efficient manufacturing

under well-defined and controllable conditions.

Therefore, we developed a miniaturized, parallelizable perfusion bioreactor which

were equipped with pressure sensors to determine the permeability of cell-

biomaterial-constructs inside the perfused chamber also allowing us to approximate

the shear stress conditions. Flow velocity and shear stress profile of a porous

scaffold was determined and computational fluid dynamics analysis was performed.

Furthermore, the mixing behavior was characterized by acquisition of the residence

time distributions. Finally, the effects of the flow and shear stress profiles on

osteogenic differentiation of human mesenchymal stem cells were evaluated in a

proof of concept study. The bioreactor system was operated in a tailor-made

incubator to be flexible and modular in terms of instrumentation and cultivation

modes, also allowing cultivation under defined gas phase compositions e.g. hypoxia.

Integrated pressure sensors allow the estimation of fluid shear stress that cells

experience on a scaffold and as a result permit the screening of the effects of

different mechanical culture conditions.

From our study we suggest that the system is beneficial for parallel dynamic

cultivation of multiple samples for 3D cell culture processes and thus can be a

valuable tool for tissue engineering applications as well as for the generation of 3 D

constructs for cell-based testing systems.

Scaffold-Free Aggregate Cultivation of Mesenchymal Stem Cells in a Stirred

Tank Bioreactor Dominik Egger 1, Ivo Schwedhelm 2, Jan Hansmann 2 and Cornelia Kasper 1




Germany

For the use of mesenchymal stem cells (MSCs) in cell based therapies extensive

expansion is necessary to obtain sufficient cell number for patient treatments. This

expansion phase still remains one of the key challenges since long-term cultivation

and excessive passaging in two-dimensional conditions result in a loss of essential

stem cell properties. These functional damages and alterations often result in low

survival rate of cells, changes of surface marker profiles, and reduced differentiation

capacity, which also influences the success rate in clinical applications.

The cultivation of MSCs in three-dimensional aggregates positively influences stem

cell properties, thus large scale cultivation of MSC aggregates is highly desirable. It is

well known that MSC physiologically reside in 3 D microenvironment under hypoxic

conditions. “Classical” cell culture protocols do not mimic these requirements

resulting in loss of functional properties and genetic instability. Therefore, we

developed strategies for cultivation of adipose derived human MSC aggregates in a

stirred tank reactor under hypoxic conditions. Aggregates were cultivated in a stirred

tank bioreactor and according to computational fluid dynamics calculations were

exposed to comparatively high average shear stress of 0.2 Pa. The viability of MSC

was 78–86% and cells maintained their surface marker profile and differentiation

potential after cultivation.

From these initial experiments, we postulate that expansion of MSC in 3D

aggregates in stirred tank bioreactors is suitable for large-scale production of MSCs

for cell based therapy applications.

Bioengineering 2017, 4, 47; doi:10.3390/bioengineering4020047

164 165

Gelatin-based hydrogels for 3D cell culture: stability at physiological temperatures by UV-crosslinking or nanoparticles Katharina Kruppa, Antonina Lavrentieva, Thomas Scheper, Iliyana Pepelanova

Institute of Technical Chemistry, Leibniz University of Hannover, Hannover, Germany

In many ways, gelatin is very attractive biomaterial due to its availability, low cost,

and multiple bioadhesion sequences. However, the low melting point of gelatin at ca.

30°C limits the use of the unmodified material in 3D cell culture. One way to obtain

stability at physiological temperatures is modification of the gelatin with methacryloyl

groups, resulting in gelatin-methacryloyl hydrogels (GelMA), which can be covalently

crosslinked under UV-light. GelMA hydrogels are stable at 37 °C and retain the

desired properties of the gelatin molecule. Drawbacks of GelMA include the toxicity

of the photo initiator and UV-exposure. These parameters must be carefully

controlled in cell experiments to avoid reduction in cell viability. Moreover, the use of

UV light is critical in many clinical applications and, therefore, completely out of the

question as an option. Another strategy to control stability of gelatin at physiological

temperature is the use of silicate nanoparticles (SiNP).

The above-mentioned strategies for obtaining gelatin stability at 37 °C were

evaluated in this study. GelMA materials were prepared with different degree of

functionalization (DoF). The mechanical properties of the UV-crosslinked hydrogels

were analyzed by rheology. The effect of GelMA concentration and DoF on the

proliferation and spreading of mesenchymal stem cells was also evaluated. In the

alternative method, silicate nanoparticles were mixed with gelatin, leading to stable

hydrogels at 37 °C. The mechanical properties of this composite hydrogels can be

controlled by variation of the SiNP proportion or the gelatin concentration.

Concentrations of the used nanoparticles should, however, not be too high since it

leads to opaque materials, which make microscopic examination of the material

challenging.

A modular perfusion microbioreactor system for oxygen level control and optimization for bone tissue engineering

Schmid Jakob, University of Applied Sciences Munich, Germany

Schieker Matthias, Ludwig-Maximilians-University Munich, Germany

Huber Robert, University of Applied Sciences Munich, Germany

In tissue engineering, the oxygen concentration within a 3D cell culture is of high

importance. A proper oxygen supply is crucial to ensure homogenious tissue quality.

In addition, not only cell growth, but also cell differentiation can depend on the

oxygen level in the 3D cell culture.

To determine optimal oxygen concentrations in 3D cell cultures, we developed a

modular perfusion microbioreactor system with integrated oxygen sensing. The

system is manufactured using 3D-printing technology and consists of up to 4

microbioreactors which can carry individually shaped 3D cultures. Oxygen is

controlled independently in each microbioreactor using a feedback mechanism to

adjust pump speed – according to the oxygen concentration in the geometric center

of the 3D cell culture. Furthermore, the microbioreactors were optimized to minimize

death volumes and to remove air bubbles.

In order to demonstrate the correct operation mode of the system, scaffolds were

seeded with preosteoblastic cells and cultivated at the following oxygen levels: 15 %,

10 %, 5 %, 0 % (21 % accords to air saturated medium). After cultivation, the 3D cell

cultures were analysed regarding cell viability and homogeneity. During the

cultivation, oxygen was properly adjusted to setpoint level +/- 0.5 % in each

bioreactor. Furthermore, it was found, that a proper oxygen level is crucial for the

quality of the cultured tissue.

Thus it was shown, that the control of oxygen levels and the screening for optimal

oxygen conditions is feasible using the developed microbioreactor system, allowing

for an automated investigation of one of the most crucial parameters in 3D cell

culture.

166 167

�Figure 1: Design and operation of the microfluidic tilting platform. (a) Channel layout of the device with the asymmetric Y-junction, a mixing structure and spheroid compartments. (b) Tilting of the platform by an angle α induces gravity-driven perfusion of the channels according to the height difference Δh. (c) Close-up top view and (d) side view of a spheroid compartment. (e) Schematic depicting the unequal flow through the differently sized channels (indicated by arrow thickness) and the changing concentrations within the three reservoirs.

A tubing-free, microfluidic tilting platform for the realization of in vivo-like drug exposure scenarios for three-dimensional

microtissues Christian Lohasz1, Olivier Frey2, Kasper Renggli1, Andreas Hierlemann1

1ETH Zurich, Dept. of Biosystems Science and Engineering, Basel, Switzerland

2InSphero AG, Schlieren, Switzerland

During the last decades, screening methods for efficacy and toxicity increasingly

focused on human in vitro cell culture models to replace animal testing. Doing so,

constant compound concentrations over a defined time are routinely applied. These

profiles, however, do not represent the situation in a human body. Upon repeated

uptake of certain substances, its concentration is characterized by a gradual increase

until a steady state is reached. Such dynamic concentration profiles enable the cells

to develop defense mechanisms against a specific substance over time, so that

simplified protocols often lead to false positive results during toxicity and efficacy

testing [1]. To realize physiologically relevant conditions in vitro, multiple pipetting

steps or experimental setups with complex pumping schemes are applied to date.

We present a tubing-free, microfluidic tilting platform with gravity-driven flow that

enables culturing of three-dimensional microtissues under in vivo-like drug dosing

regimens (Figure 1). In contrast to traditional, constant dosing regimens, the chip

allows for gradual changes in substance concentrations. Dosing gradients are

generated through an asymmetric Y-junction of microfluidic channels of different

widths (Figure 1e). Changes in the chip operation parameters, e.g., different tilting

angles, enable to alter the drug dosage on demand. The modulation of the tilting

angle changes the slope of the dosing curve, allowing to mimic curves that resemble

the pharmacokinetic characteristics of common substances. Our experimental results

evidenced that in vivo concentration curves of acetaminophen could be approximated

with the device. Furthermore, drug exposure experiments with a duration of up to

seven days can be performed. The chip represents an easy-to-handle tool for toxicity

and efficacy testing of dynamic drug concentrations changes.

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168 169

Fig. 1: Image of 3D tubular scaffolds produced from short electrospun nanofibers and shaped via freeze-casting method. From left to right polyamide (PA), pullulan/polyvinyl alcohol (PUL/PVA), polyacrylonitrile (PAN) mixed with PUL/PVA and PAN.

Porous, ultralight 3D tubular scaffolds from short electrospun nanofibers

Markus Merk 1, Christian Adlhart 1

1 Institute of Chemistry and Biotechnology, Zurich University of Applied Sciences

ZHAW, Einsiedlerstrass 31, 8820 Wädenswil, Switzerland

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INTRODUCTION: Tubular organs are ubiquitously found within

the human body and include blood vessel,

trachea gastrointestinal tract and urinary tract.

These complex tubular tissues are composed

of different types of cells, extracellular matrix

and proteins but share the functional purpose

of transporting nutrients in liquid or solid

form. Repair and regeneration of these

tubular organs is of great interest due to the

high amount of surgeries performed annually.

However, the design and fabrication of

synthetic scaffolds that mimic the mechanical

and structural characteristics of their natural counterparts remains challenging.[1]

MATERIALS AND METHODS: All nanofibers were received from the University of Liberec except of

pullulan/polyvinyl alcohol (PUL/PVA) which was produced using the Elmarco

NanoSpider as reported previously.[2] Tubular structures were formed by freezing

homogenized nanofiber-slurries using a self-constructed cryo-rotator. Subsequent

freeze-drying and crosslinking formed the porous, ultralight 3D scaffolds.

RESULTS: Figure 1 shows the porous, ultralight 3D tubular scaffolds produced by a novel freeze

casting method. Scaffolds were crafted using various polymers in order to create a

wide range of mechanical properties.

CONCLUSIONS:

Organ-on-a-Disc – Enabling technology for the parallelization and automation of microphysiological systems

Stefan Schneider, Oliver Schneider, Florian Erdemann, Christopher Probst,

Peter Loskill; Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB,

Nobelstraße 12, 70569 Stuttgart, Germany

In recent years, Organ-on-a-chip (OoC) technology has emerged from a conceptual

idea to a feasible alternative to animal models and traditional cell assays. OoCs in

combination with human induced pluripotent stem cells (hiPSCs) allow for drug

screening and disease modelling using human (disease-specific) organ/tissue

models with great potential for personalized medicine. So far, a variety of OoC

systems have been introduced based on similar concepts, thereby mostly sharing the

same limitations. Typically, OoCs culture cells in monolayers on two sides of an inert

membrane and therefore do not sufficiently represent a large number of tissue types

that are composed of 3D stacks of closely packed cells mixed with extracellular

matrix molecules. Additionally, current OoCs mostly consist of individual units

integrating only one single tissue and rely on pump technology. These systems

require manual, error-prone handling based on specialized and time-consuming

training.

To advance the idea of microphysiological systems to the next level, we developed

the Organ-on-a-Disc (OrganDisc) technology based on the combination of concepts

from centrifugal microfluidics, OoCs, and tissue engineering. OrganDiscs consist of

two layers of circular polymer slabs – a tissue and a media layer – sandwiching a

porous membrane. The tissue layer features a large number of parallel tissue-

specific µ-chambers, located at the outer part of the disc and connected to inlet ports

in the central region of the disc. For initial tissue generation, cell suspensions are

pipetted into the inlet ports and the disc is subsequently rotated. The cells are

thereby transported into the tissue chambers and form densely packed 3D pellets

replicating their tissue-specific shapes due to precisely controllable centrifugal forces.

By using well-known, cell-specific g-forces and no fluid flow, the damaging of cells is

prevented. Moreover, a slow rotation during the subsequent culture phase enables a

defined pump-free media supply to individual tissues or multi-organ-combinations.

The user-friendly, standardizable processes and the rotational symmetry of the discs

170 171

Enhanced cardiomyocyte maturation in a microfluidic system as a potential platform for pharmacological screening

T. Kolanowski 1*, M. Busek 2*, S. Grünzner 2,3, F. Sonntag 2+, K. Guan 1+ 1 TU Dresden, Faculty of Medicine Carl Gustav Carus, Institute of Pharmacology

and Toxicology, Dresden, Germany; 2 Fraunhofer Institute of Material and Beam

Technology IWS, Dresden, Germany; 3TU Dresden, Faculty of Manufacturing

Technology, Dresden, Germany

* Authors contributes equally to this work; + Authors share senior authorship

Cardiovascular diseases (CVDs) are among the main causes of death

worldwide. One of the limitations in the development of therapies for CVDs is the lack

of an adequate human model that could be used for drug screening and toxicity.

Eligible models should resemble human, adult heart tissue especially in terms

of physiological parameters observed in patients.

Currently, the most efficient

technology for in vitro derivation of cardio-

myocytes relies on differentiation of induced

pluripotent stem cells (iPSC-CMs). However,

these CMs remain immature in static 2D

culture, which leads to differences in

pharmacological response to many drugs

tested thus far when compared to adult CMs.

Establishing the adult phenotype in these

cells would potentiate the progress in the

field. However, to drive maturation of iPSC-

CMs in vitro, different types of stimuli need

to be applied.

We developed a microfluidic platform

for perfused cell cultivation that features a

micro pump, micro channels and

an oxygenator for gas exchange (a). The

oxygen level can be non-invasively measured and controlled in the cell culture

chamber allowing efficient switch between normoxic and hypoxic states. We have

The design and fabrication of new materials is essential to keep up with the

increasing demand for scaffolds in regenerative medicine. Here, we show the

construction and comparison of different tubular structures fabricated via the

combination of two-dimensional (2D) electrospinning and the freeze-casting

method.[2] Together these techniques allow us to create 3D tubular scaffolds with

high open porosity and adaptable mechanical properties depending upon the

polymer used.

REFERENCES: [1] Góra, A., Pliszka, D., Mukherjee, S., Ramakrishna, S. (2016). Journal of

Nanoscience and Nanotechnology, 1, 19-39.

[2] Deuber, F., Mousavi, S., Hofer, M., Adlhart, C. (2016). ChemistrySelect, 1, 5595.

ACKNOWLEDGEMENT:

The authors thank the University of Liberec for providing the electrospun nanofibers.

172 173

Autonomous Plug&Play Multi-Organ-Chips with Integrated Pumping and Sensing

F. Sonntag,1 C. Probst,2 S. Grünzner,1,3 M. Busek,1 P. Loskill2,4 1Fraunhofer Institute for Material and Beam Technology IWS, Dresden, Germany; 2Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Stuttgart,

Germany; 3Technische Universität Dresden, Dresden, Germany;

4Research Institute for Women’s Health, Eberhard Karls University Tübingen,

Tübingen, Germany

Multi-organ platforms have an enormous potential to lead to a paradigm shift in a

multitude of research domains including drug development, toxicological screening,

personalized medicine as well as disease modeling. Integrating multiple organ–

tissues into one microfluidic circulation merges the advantages of cell lines (human

genetic background) and animal models (complex physiology) and enables the

creation of more in vivo-like in vitro models. In recent years, a variety of design

concepts for multi-organ platforms have been introduced, categorizable into static,

semistatic and flexible systems. Static as well as semistatic multi-organ integration

concepts feature a number of restraints on the success rates of those systems, which

can be avoided by flexible integration.

We have developed a plug&play multi-organ system that combines a microfluidic

base chip, featuring integrated micro pumps, valves, reservoirs and oxygenators,

with ultra-compact microphysiological tissue modules, comprising various types of µ-

tissues. Both systems can be interconnected with temporal flexibility, whereby pre-

cultivation of the individual modules is possible. Since both the individual modules

and the base chip are transparent, it is possible to monitor the µ-tissues as well as

the flow architecture using (fluorescence) microscopy. The concept moreover allows

the integration of multiple individual �-tissue modules with the superordinate basis

chip. This approach creates for the first time a fully customizable multi-organ-chip

platform within a closed circulation system. For this purpose, defined fluidic interfaces

have been developed and established.

proved that cells cultured within our system contract more efficiently, develop more

mature physiological features (for example, increased sarcomere length) and create

more aligned structures (b) during culture when compared to the static culture (c).

Taken together, our results demonstrate that the microfluidic system can help

driving cardiomyocytes towards more mature phenotypes. Moreover, with completely

isolated culture system and easy-to-automatize handling, our platform holds great

potential for high-throughput applications in pharmacological screening.

174 175

vasQchip: A blood vessel scaffold for the reconstruction and 3D bioprinting of 3D-tissues in vitro

Christoph Grün, Vanessa Kappings, Eva Zittel, Ute Schepers

Institute of Toxicology and Genetics Karlsruhe Institute of Technology (KIT),

Hermann von Helmholtz Platz 1, 763131 Karlsruhe

Observations of single cell interactions in complex tissues often require the

manipulation of the respective cells and their microenvironment. However, the

complexity of tissues and the interplay with the vasculature and other tissues often

hampers this investigation at the molecular level. Three-dimensional (3D) in vitro

models bridge the gap between two-dimensional cell cultures and whole-animal

systems. Many of the in vitro tissues are based on 3D- cell cultures from different cell

types. However, as the tissue thickness of a 3D-model increases oxygenation and

nutrition will be

limited. As the

nutrition supply of

almost all tissues is

ensured via a network

of blood vessels

containing an intact

endothelium, which is

also very important to

study the migration of immune cells and metastases, we developed a disposable

microfluidic 3D-vascular microstructure (vasQchip). The microvascular curvilinear

channel structure, which can be coated with a confluent endothelial layer is derived

by microthermoforming of a porous polycarbonate film and is connected to

microfluidic circuit. It is surrounded by a second microfluidic compartment, which can

be used for 3D-cultures of different tissues or supports 3D bioprinting of tissues

surrounding the porous microchannel. It also supports the removal of lymph fluid.

This disposable microfluidic device was so far used as a model system to study

inflammatory migration of immune cells and the transendothelial transport of drugs.

We are currently developing a variety of 3D vascularized tissues such as the blood

brain barrier, the neurovascular unit, vascularized skin, kidney, bone marrow niche,

tumor-models, retina, and colon for academic and pharmaceutical applications.

Figure 1: Schematic illustration of a base chip with integrated micro pumps, valves,

reservoirs and oxygenators (top) coupled with four ultra-compact microphysiological

tissue modules (bottom).

176 177

Comparison of 2D and 3D cultures of primary hepatocytes on hepatocellular functions and hepatotoxicity

Heiko Dinter1,2, Anett Ullrich1, Dieter Runge1 1PRIMACYT Cell Culture Technology GmbH, Schwerin, Germany

2Hochschule Biberach, University of Applied Sciences, Biberach, Germany

Primary hepatocytes of human and animal origin are the gold standard for all

pharmacological-toxicological studies in drug development. They play a major role in

ecotoxicological evaluation as well. Three dimensional (3D) cultures became more

popular in the last years since they might mimic the in-vivo cell morphology, polarity

and cell-cell interactions better than traditional two dimensional (2D) cultures.

Here, we used primary hepatocytes of Cynomolgus monkey and Beagle dog to

compare two different 3D culture systems, molded hydrogel discs consisting of a

collagen mimetic peptide and a 3D spheroid system, with the standard 2D culture

system. Hepatocellular detoxification functions like urea release and CYP450 activity

as well as the response to the hepatotoxin Diclofenac were analysed in these three

culture systems. The results were normalized to the corresponding volume of culture

medium or to protein content.

The secretion of urea was improved and maintained at higher levels in both 3D

culture models compared to the conventional 2D culture on collagen-coated plates.

CYP1A activity was inducible by ß-Naphthoflavone in a similar manner in 3D

hydrogels and 2D culture, but could only be marginally induced in Cynomolgus but

not in Beagle using the 3D spheroid system. Diclofenac, a known and well described

hepatotoxic compound, did not show any toxic effect on hepatocytes cultured on 3D

hydrogels. However, in 3D spheroid systems and in 2D culture, Diclofenac lead to a

decrease in cellular ATP content and to an increased LDH release.

In summary, our results indicate that major differences may exist between different

3D culture systems and in comparison to standard 2D culture methods. These

differences may lead to different and conflicting results in the assessment of drug

toxicity and drug-drug interaction. While one 3D culture system (hydrogel discs)

showed similar results with regard to cytochrome induction, it failed to detect the

hepatotoxicity of Diclofenac. On the other hand the second 3D culture system

(spheroids) revealed the Diclofenac toxicity but failed to express CYP1A enzyme

activity and did not respond to prototypical CYP1A inducer.

A non-invasive microscopy platform for the online monitoring of human induced pluripotent stem cell aggregation in suspension

cultures in small-scale stirred tank bioreactors Ivo Schwedhelm, University Hospital Würzburg, Würzburg, Germany;

Dominik Egger, University of Natural Resources and Life Sciences, Vienna, Austria;

Philipp Wiedemann, Mannheim University of Applied Sciences, Mannheim, Germany;

Thomas Schwarz, Fraunhofer Institute for Silicate Research ISC, Würzburg,

Germany;

Heike Walles, University Hospital Würzburg, Würzburg, Germany;

Jan Hansmann, Fraunhofer Institute for Silicate Research ISC, Würzburg, Germany

Human induced pluripotent stem cells (hiPSCs) bear potential to generate large

quantities of lineage-specific precursors and are thus of high interest for applications

in the tissue engineering field. The steady production of sufficient numbers of high-

quality pluripotent and homogenous hiPSCs, however, still represents a major aspect

to consider, as the format of conventional two-dimensional cell culture is limited to

lab-scale production. With the aid of scalable suspension culture setups in finely

adjusted continuously stirred tank reactors (CSTR), the production of sufficient

amounts of hiPSCs is feasible. Further, automated process control becomes

available which allows for defined culture conditions, as well as the use of a variety of

monitoring options. Next to standard process parameters of interest such as oxygen

consumption, pH, and metabolite turnover, a uniform formation of hiPSC aggregates

is desirable. In this regard, we developed a microscopy platform that can be

connected to any suspension culture vessel and thus facilitates the online

observation of hiPSCs during aggregation without the need for sampling. In our

studies, we were able to expand hiPSCs in custom-made miniature CSTRs that were

optimized in accord to preceding computational fluid dynamics to guarantee gentle,

yet thorough mixing at low shear. During expansion, the average aggregate diameter

and aggregate size distribution was determined by microscope imaging and

subsequent automated image analysis throughout the culture. The data was

harnessed to detect the optimal time point at which aggregates were either ready for

differentiation or required passaging. To provide evidence for hiPSC pluripotency

178 179

A novel 3D microwell array for the analysis of adhesion independent micro-tumours

Thomsen ARa,b, Aldrian C a,, Thomann Y c,, Grosu A-La,b, Bronsertb,d, Leu Me, Lund Pa,f

aDepartment of Radiation Oncology, Medical Center – University of Freiburg, Germany.

bGerman Cancer Consortium (DKTK), Partner Site Freiburg and German Cancer Research Center

(DKFZ), Heidelberg, Germany cFreiburg Material Research Center and Institute for Macromolecular Chemistry, Department of

Chemistry, University of Freiburg, Germany dInstitute for Surgical Pathology, Medical Center – University of Freiburg, Germany

eabc biopply ag, Solothurn, Switzerland fDepartment of Radiation Oncology, Ortenau-Klinikum, Offenburg, Germany

Background:

Multicellular organoids, namely micro-tumours, represent a well-established 3D model to study

tumour response to radiation and anti-neoplastic treatment in vitro. The analysis of certain cancer

cell lines was impeded by the inability of those cells to form stable spheroidal structures.

Furthermore, co-culture experiments represent an important tool to understand key questions in

tissue development and disease biology. Yet, the use of feeder cells together with test cells in the

generation of 3D organoids led to heterogeneous cell aggregates whose treatment response could

be interpreted ambiguously. Here we present a novel and innovative 3D microwell array that

circumvents those issues by offering a number of unique features. It´s conical geometry favours

the formation of spheroids from cell lines and cell types that did not form spheroids under other

approaches. Furthermore, feeder and test cells can be cultivated in contact co-culture as

previously, or in a distance co-culture setup separated by a thin layer of agarose, only.

Material and Methods

The microwell arrays are generated from agarose by using a high-precision replica moulding

technique. The resulting arrays comprise several hundreds of cone-shaped microwells each and fit

into 6 well plates. To generate 3D micro-tumours, a cell suspension is seeded into the

3D CoSeedisTM micro-well array. Cells then sediment into microwells, where aggregate formation

is supported by the confined space within the non-adhesive hydrogel. For co-culture, the microwell

array is placed on top of a layer of adherent stromal cells, so that both cell types remain in

separate compartments, but may communicate via soluble factors that diffuse through the

permeable hydrogel. As a readout, light and fluorescence microscopy, optical scanning or even

paraffin histology is used.

Funnel-Guided Positioning of Multi-cellular Microtissues to Build Macrotissues

Kali L. Manning, Sc.M.1, 2, Andrew H. Thomson1, Jeffrey R. Morgan, Ph.D. 1, 2 1 Department of Molecular Pharmacology, Physiology and Biotechnology

2 Center for Biomedical Engineering

Brown University, Providence, RI, USA

The field of tissue engineering is developing new additive manufacturing

technologies to fabricate 3D living constructs. These constructs can be used for in

vitro drug testing platforms, or to restore lost function in vivo. We have developed a

new additive manufacturing strategy, the funnel-guide, which can be used to build

macrotissues layer-by-layer using non-contact manipulation and positioning of multi-

cellular microtissues. Microtissues were self-assembled into toroid and honeycomb

shapes using agarose micro-molds. We observed that when falling in cell culture

medium, the microtissues spontaneously righted themselves to a horizontal

orientation. We fabricated a funnel to harness this spontaneous righting, and guide

these falling toroids and honeycombs into precise positions to create a stack. Once

stacked, they fused to form tubular structures. We tested multiple cell-types and

toroid sizes, and ultimately used the funnel-guide to create a stack of 45 toroids that

fused into a tube 5mm long with an inner diameter of 600µm. The funnel-guide is a

new principle for the manipulation of microtissues and is a platform for the layer-by-

layer positioning of microtissue building blocks to form macrotissues. The

fundamental principal of the funnel-guide can be used to create funnel-guides for a

variety of microtissue parts in order to build more complex macrotissues. In addition,

funnel-guide facilitated macrotissue construction is amenable to automation and

conducive to perfusion, which is necessary to sustain the viability of complex

macrotissues of high cell density.

180 181

Integration of 3d printed hollow hydrogel fiber with microfluidic system to develop a perfusable nephron model.

Ashwini Rahul Akkineni1; Deborah Förster2; Jan Sardnick2; Florian Schmieder3;

Frank Sonntag3; Michael Gelinsky1; Anja Lode1 1 Centre for Translational Bone, Joint and Soft Tissue Research, Technische

Universität Dresden, Germany

2 Division of Nephrology, Department of Internal Medicine III, University Hospital Carl

Gustav Carus, Technische Universität Dresden, Dresden, Germany 3 Fraunhofer Institute for Material and Beam Technology IWS, Dresden, Germany

Introduction : Biopolymer hydrogels provide an excellent basis for engineering various tissue

models for application in regenerative therapies. Translation of additive

manufacturing methods, such as 3D extrusion printing for hydrogels, has now

enabled fabrication of complex volumetric tissue constructs1. Recently fabrication of

complex hydrogel constructs, composed of hollow fibers was developed using

coaxial extrusion method2. The lumen of the hollow fiber constructs can be filled with

a core material and serve as a depot of growth factors or living cells3. Furthermore,

the lumen of the hollow fibers can be perfused mimicking natural tubular structures.

In the present work, a single layer hollow fiber construct was fabricated, with the aim

to integrate them in a microfluidic chip and colonize the lumen with renal cells.

Materials and methods : BioScaffolder 3.1 by GeSiM, Radeburg; with a 3 channel plotting unit and custom

made core shell needles were used to fabricate hollow fiber scaffolds. A 20 wt%

alginate paste (supplemented with 50 µg/g fibronectin) and 50 mM BaCl2 were

extruded from the outer needle and inner needle, respectively. A continuous hollow

fiber was extruded by the print head according to a CAD design. The fiber was

crosslinked for 10 min using 50 mM BaCl2 after which was cannulated to a 200 µm

cannula. The inner lumen of the hollow fiber was seeded with Renal Proximal Tubule

Epithelial Cells (RPTEC) using the perfusion system. Cell attachment and

proliferation was observed over a period of 7 days.

Results The 3D microwell array allows the formation of spheroidal and non-spheroidal cell aggregates and

consequently enables the examination of tumour cells in 3D structures that have so far been

elusive. Seeding of feeder cells underneath the 3D-microwell array enables to assess the

aggregate growth in presence of secreted feeder cell-derived factors omitting a direct physical

contact (distance co-culture). However, different cell types may also be assembled as mixed cell

aggregates (contact co-culture). Validated read-out methods, namely volumetric and histological

protocols, enable a rapid and reliable analysis and warrant reproducible results over different

experiments and treatments.

Conclusions The 3D CoSeedis™ micro-well array is well-suited to grow cells as 3D aggregates that would

normally not do so, e.g. MIA PaCa-2 cells, thus extending the range of cells to be used in analyses

of the micro-tumour treatment response.

Special emphasis has been put on the interaction between stromal and cancer cells. Both, contact

and distance co-culture effects can be easily addressed with the presented 3D microwell array.

3D CoSeedis™ allows to monitor dose-dependent effects of complex treatments using both cell

aggregate volume and marker expression as a read-out.

Fig.1: Geometry of the 3D CoSeedis™ microwell array.

Upper row: Scanning electron microscopy of the master

structure. Bottom row: Micro computer tomography (µCT).

Fig.2: human pancreatic cancer cells in 3D CoSeedis™.

These cells - as many other cell lines - do not grow as 3D

aggregates under standard spheroid culture conditions

(e.g. hanging drop method).

Left: brightfield, Right: Life-dead staining (fluoresceine

diacetate -green- and propidium iodide - red).

182 183

Impedance analysis of viability of Schistosoma mansoni larvae for drug screening application

Mario M. Modena*, Ketki Chawla*, Flavio Lombardo†, Sebastian C. Bürgel*, Gordana

Panic†, Jennifer Keiser†, Andreas Hierlemann*

*ETH Zurich, Dept. of Biosystems Science and Engineering, Bio Engineering

Laboratory, Basel, Switzerland †Swiss Tropical and Public Health Institute, Department of Medical Parasitology and

Infection Biology, Basel, Switzerland

High-throughput and automatisation

Human schistosomiasis is a neglected tropical disease caused by trematodes,

affecting almost 250 million people worldwide. For the past 30 years, treatment has

relied on the large-scale administration of praziquantel. However, concerns regarding

the appearance of drug-resistance parasites require efforts in identifying novel

classes of suitable drugs against schistosomiasis. The current drug screening system

is manual, slow and subjective. We present here a novel microfluidic platform for the

assessment of the viability of Schistosoma mansoni larvae (Newly Transformed

Schistosomula, NTS) by using an impedance-based readout of NTS motility. NTS

viability after exposure to a test drug is detected by measuring the current

fluctuations caused by NTS when confined in a compartment between a pair of

coplanar electrodes, patterned at the bottom of a microfluidic channel. The

combination of microfluidics with an electrical-readout scheme enables a massive

sample reduction, as only ~5 NTS and 20 �L of solution are required per

measurement, as compared to 50-100 NTS in 200 �L solution for the standard

evaluation. Finally, an electrical readout of the viability paves the way to devising

parallelized and automatic platforms for the identification of drug candidates against

schistosomiasis.

Results : A continuous hollow fiber was successfully fabricated according to the CAD design

and tightly connected to 200 µm cannulas at its two ends using Histoacryl®. During

perfusion, no leakage at the hollow fiber-cannula interfaces was observed at flow

rates of 5 to 50 µl/sec. Islands of spread RPTEC monolayer cells was observed

attached to the inner lumen of hollow fiber. Progressive increase in the cell

colonization was observed in later days of the culture.

Conclusion and outlook: A perfusbale continuous hollow hydrogel fiber was fabricated using coaxial 3D

extrusion printing. RPTEC cells could be colonized in the lumen of hollow tubes. And

potentially to mimic the renal tubule, other cell types could be seeded on both the

surfaces. Versatility of 3D extrusion printing can enable inclusion of hollow fiber of

various diameters and sizes to be integrated into microfluidic systems.

References : 1. D. Kilian et al. MRS Bull. 2017, 42, 582 - 592

2. Y. Luo et al., Adv. Healthcare Mater. 2013, 2, 777-783

3. AR Akkineni et al., Biofabrication. 2016, 8, 045001

184 185

Magnetic 3D Bioprinting for High-Throughput and Automated Hepatotoxicity Testing

Glauco R. Souza1,2 and Brad Larson3 1University of Texas Health Science Center at Houston, Texas, USA

2Nano3D Biosciences, Inc., Houston, Texas, USA 3Biotek Instruments, Inc., Winooski, Vermont, USA

High-Throughput Screening and Automation

Prescription medications, environmental toxins, and non-prescription herbal remedies

together form the major causes of hepatic injury. When looking at drugs alone, induced

hepatic injury is the most common reason cited for warnings or withdrawal of an approved

drug depending on the severity of the induced hepatotoxicity. Due to this reality, a paradigm

shift has taken place in the way toxicology studies are being performed, including

determination of hepatotoxicity.

Hepatotoxicity studies historically have been performed by repeatedly dosing

hepatocytes cultured on the bottom of a microplate with multiple concentrations of a test drug

or compound. Because hepatocytes rapidly de-differentiate, lose metabolic activity, and lack

the communication networks found in vivo when cultured in this manner, results may be

inaccurate and yield misleading claims regarding the safety of the test agent. To combat this

shortfall, three-dimensional (3D) spheroidal models, incorporating hiPSC-hepatocytes, can be

incorporated that allow cells to aggregate and retain typical long-term viability, functionality

and communication found in vivo; allowing for the generation of repeatable, accurate data. To

meet the demand for increased hepatotoxicity testing, automation has been incorporated to

streamline the procedure and reduce the need for large scale manual manipulations. Typically,

included liquid handling systems have been large, expensive, and many times required

placement into clean rooms for sterile processing. While this type of solution is suitable for

pharmaceutical, biotech, and even larger core facilities, the size and cost can be prohibitive to

the typical academic research lab. Therefore, a smaller, less expensive instrumentation set,

which can still provide accurate and repeatable results, is necessary.

Here we demonstrate the ability to combine magnetic 3D bioprinting, liquid handling,

and a novel cell imaging multi-mode reader to perform 3D hepatotoxicity studies. hiPSC-

hepatocytes were aggregated into spheroids easily and efficiently using magnetic bioprinting.

Fig. 1. Comparison between the standard evaluation of motility of NTS through visual

inspection in well plates (a) and our microfluidic platform relying on impedance changes

induced by larvae motility (b). Live NTS induce fluctuations in the voltage-converted recorded

signal, whereas a stable and flat signal is recorded for dead worms (c). The images in the

insets show three screenshots of the worms in the chip during the measurements. The black

regions at the top and bottom are the platinum electrodes, an pillar structures used to contain

the larvae in the sensing area are clearly visible.

186 187

Cytotoxicity Evaluation of Nanoparticles using Automatic 3D Cell Culture System �

Min Beom Heo

Center for Nano-Bio Measurement, Industrial Metrology, Korea Research Institute of Standards and Science,

Daejeon, South Korea E-mail address: [email protected]

�The cells that make up the human body exist in three-dimensions (3D). However, most of

cell experiments in the laboratory currently have used two-dimensional (2D) form cells. In fact, 2D cell culture method is difficult to carry out the realistic role of diffusion / transport conditions as well as intercellular interactions [1]. On the other hand, the 3D cell culture method has an advantage of overcoming the limitation of the 2D cell culture technique and minimizing the animal experiment by providing an environment similar to a living body. In order to overcome these limitations worldwide, 3D cell culture methods have been actively studied. According to a study by BCC research, the market for 3D cell culture technology is expected to grow from $1.4 billion in 2017 to an estimated $6.5 billion by the end of 2020, and CAGR (the compound annual growth rate) is 36.2% [2]. Today, a variety of functional nanoparticles are being developed and applied to products and

environments around us. Nanomaterials are most important to use safely and must be well equipped with a method to assess their toxicity. Conventional methods of assessing cytotoxic of nanomaterials mostly use 2D cell culture. However, an assessment of cytotoxicity through 2D cell culture may not accurately reflect the actual toxicity of the nanomaterial. The 3D cell culture method, which plays a role in connecting the intermediate steps in vitro 2D and in vivo, would extend the nanotoxicity assessment method at the cellular level to the tissue level and improves nanotoxicity prediction ability in vitro. In this study, we compared the results of nanomaterial toxicity with 3D cell culture method and 2D cell culture method.

References

1. J. Lee, M. J. Cuddihy, N. A. Kotov, Tissue Eng. B 14, 61 (2008). 2. https://www.bccresearch.com

Multiple known hepatotoxicants were tested with the automation and cell model to validate

the ability of the combined solution to be used to meet the need to perform automate

hepatotoxicity studies.

188 189

References [1] L. Gutzweiler et al., “Large scale production and controlled deposition of single

HUVEC spheroids for bioprinting applications,” (eng), Biofabrication, vol. 9, no. 2,

p. 25027, 2017.

[2] S. Kartmann, P. Koltay, R. Zengerle, and A. Ernst, “A Disposable Dispensing

Valve for Non-Contact Microliter Applications in a 96-Well Plate Format,”

Micromachines, vol. 6, no. 4, pp. 423–436, 2015.

[3] W. Streule, T. Lindemann, G. Birkle, R. Zengerle, and P. Koltay, “PipeJet: A

simple disposable dispenser for the nano- and microliter range,” Journal of the

Association for Laboratory Automation, vol. 9, no. 5, pp. 300–306, 2004.

Automated large-scale production and deposition of spheroids Kevin Tröndle, Sabrina Kartmann, Ludwig Gutzweiler, Peter Koltay and Stefan

Zimmermann, University of Freiburg, Germany

As spheroids are of increasing interest in manifold 3D cell culture applications there

is a growing demand for reproducible large-scale production of spheroids as

preformed micro tissue models or building blocks for 3D-Bio-Printing. Here, we

present a rapid automated solution for (A) the formation of size defined cell spheroids

by the hanging drop method and (B) the spatially controlled non-contact deposition of

single spheroids onto target coordinates [1].

System characteristics The system is based on a movable printhead with two liquid dispenser types. All

fluid-carrying components are sterilizable and disposable. (A) For spheroid

production a single-use solenoid dispensing valve [2], connected to a cell suspension

reservoir, is used for the contact-free ejection of droplets with adjustable volumes

(840 nl to 5.3 �l). By applying actuation pressure (150 to 300 mbar) to the reservoir,

and opening the dispensing valve for short times, free flying droplets can be ejected

with high frequency. Thus, an array of 1.000 droplets can be produced in 3 minutes

and during upside down incubation spheroids form within hanging droplets. The

droplet volumes are adjusted by the actuation pressure and the opening time of the

valve. (B) For controlled deposition of single spheroids a piezo-actuated nanoliter

dispenser (P9TM, Biofluidix, Germany, [3]) is combined with an optical setup and

particle detection software. Spheroid suspension is filled into a reservoir connected to

the dispenser. Thus, free fyling micro droplets (with volumes 5 to 20 nl) containing

single spheroids are produced and deposited in spatially defined locations.

Applications (A) With the automated process, arbitrary cell spheroids with defined size and cell

number were produced with a throughput of up to five spheroids per second.

Spheroids with approx. 250, 500 and 1000 cells each, with a respective size

distribution of 115 ± 10, 153 ± 12 and 244 ± 28 �m were achieved with different cell

types, including primary cells and stem cells. (B) Arrays of single spheroids deposited

at defined distances (down to 500 µm) were produced on standard culture plates and

in artificial 3D fibrin matrices. Cultivation of produced arrays revealed excellent cell

viability, as well as cellular interactions between spatially separated cell spheroids.

190 191

Engineering bio-mimetic vasculature with photolithographic fabrication techniques

Alexander Thomas, Technische Universität Berlin, Germany; Katharina Schimek,

Technische Universität Berlin, Germany; Gerry Giese, Freie Universität Berlin,

Germany; Anna-Elisabeth Kreuder, Technische Universität Berlin, Germany; Tobias

Grix, Technische Universität Berlin, Germany; Lutz Kloke, Cellbricks GmbH,

Germany

The field of tissue engineering – the fabrication of bio-mimetic tissues and organs –

has made considerable progress to develop three-dimensional, multi-cell type

constructs that can in part recapitulate the physiological functions of native tissues

and organs. The majority of fabrication approaches used in tissue engineering, e.g.

spheroid formation and gel casting, rely more on cellular self-organization than on

deliberate compartmentalization and positioning of cell populations according to a

master design. Although the complexity and functionality of tissue and organ models

created with these techniques is astounding, the main problem remains in the lacking

understanding of intricate cellular processes on molecular level to deliberately control

cellular behavior and thus, tissue formation.

Critical for the further evolution of current tissue and organ models is the presence of

an integrated, bio-mimicking vascular system. Nevertheless, conventional fabrication

approaches mostly have failed to achieve vascularization of tissue and organ models

due to the aforementioned shortcomings. The rise of 3D printing as a powerful tool

for a directed design approach has the potential to create tissue and organ models

with a designed, integrated vasculature. In this work, we present a

photopolymerization-based fabrication system for the creation of different vascular

constructs, with or without combination of a relevant organ model. The biofabrication

process of these constructs is presented from design to 3D culture and analysis. The

complementation of the vascular constructs with a microfluidic culture platform is

explored and their use for in vitro studies of vascularization is discussed.

Characterization of GelMa and alginate hydrogels for bioprinting: printability, polymerization and biocompatibility

Lukas Raddatz1, 2; Carola Schmitz1; Pia Gellermann1; Marline Kirsch1; Dominik

Geier2; Sascha Beutel1; Thomas Becker2; Thomas Scheper1; Iliyana Pepelanova1;

Antonina Lavrentieva1

1 Institute of Technical Chemistry, Leibniz University of Hannover, Hannover,

Germany;

2 Institute of Brewing and Beverage Technology, Forschungszentrum

Weihenstephan, Technical University Munich, Munich, Germany

Hydrogels are widely used in 3D cell culture due to their high variability, transparency

and biocompatibility. Hydrogels are water-retaining polymers, which act as adhesion

matrix for cells allowing 3D cell growth. Bioprinting represents an advanced

technique of 3D cell culture, allowing spatial control of cells and material

arrangement. Major challenge in bioprinting nowadays is the development of

appropriate bioinks. Besides optional properties like integrin-binding sites, the

materials used should fulfill four mandatory features: appropriate viscosity and shear-

thinning properties for extrusion-based bioprinting, quick crosslinking, stability over

prolonged cultivation under physiological conditions and biocompatibility.

In our study, we investigated gelatin methacryloyl (GelMA)-based and alginate

hydrogels as bioinks for extrusion-based bioprinting. The effect of hydrogel

concentration on viscosity and printability was studied in detail, as well as

possibilities to improve printing resolution by the employment of various rheological

additives. The investigation concluded with experiments of whether the optimal

conditions for bioprinting compromise the biocompatibility of the biomaterial.

192 193

3D Bioprinting of hydrogels for viral Infection and transduction with viral gene vectors

Hiller, T.*; Berg, J.*; Röhrs V.; Al-Zeer, M.; Kissner, M.; Kurreck, J.

Technische Universität Berlin, Institute of Biotechnology, Department of Applied

Biochemsitry, Gustav-Meyer-Allee 25, 13355 Berlin, Germany, Email:

[email protected]

*Shared first authors

Aims To overcome the shortcomings of current animal models we currently print lung and

liver models for the study of viral Infections and transduction with viral gene vectors.

The models will be employed to develop new antiviral strategies and new drug

candidates in humanized models.

Methods Human lung (A549) and liver (HepaRG) cells were bioprinted in different types of

hydrogels with a pneumatic extrusion printer. The generated 3D models were

infected with influenza virus (A549) or transduced with AAV vectors (HepaRG). Cell

distribution, metabolic cell activity, viral response and replication as well as silencing

of an endogen target mediated by shRNA containing AAV vectors were analyzed.

Results All tested types of printed hydrogels supported the cell viability of A549 and HepaRG

up to three weeks. Specific liver markers such as albumin secretion and Cyp3A4

activity showed a steady increase up to two weeks. We could achieve an adequate

three-dimensional distribution of the cells in the printed models with differences in cell

distribution between the types of hydrogel formulation. It was possible to infect and

transduce the 3D models with Influenza virus and AAV6 and to confirm proper virus

replication, as well as the functionality of the AAV vectors by efficient shRNA

mediated knockdown of human cyclophilin b (hCycB).

Conclusion We could show that bioprinted 3D models can be used to study infection biology and

viral gene vectors as the basis to develop new antiviral strategies. These humanized

models could overcome some of the shortcomings of current animal models that do

not support replication of human pathogens and they may also help to reduce the

number of animals in the drug development process.

Characterisation of bioprinted mandibular osteoblasts for engineering an in vitro jaw bone model

Anna-Klara Amler, Technische Universität Berlin, Germany; Alexander Thomas,

Technische Universität Berlin, Germany; Tobias Grix, Technische Universität Berlin,

Germany; Roland Lauster, Technische Universität Berlin, Germany; Lutz Kloke,

Cellbricks GmbH, Germany

Jaw bone is unique in its capability to form and harbour teeth. This intriguing property is

governed by a complex interplay of matrix- and cell-associated factors. Osteoblasts

constitute the main cellular osteogenic component of jaw bone by building up its specific

extracellular matrix, including organotypic cytokine gradients. So far, our understanding

of tooth development mostly stems from in vivo studies, while relevant in vitro models are

sparse. Bioprinting gives the unprecedented opportunity to engineer tissue models as

complex as the maxillo-dental microenvironment, from which teeth arise. It is an additive

manufacturing technique that embeds biologicals in a supportive matrix – the so called

bioink. A suitable bioink has to fulfil certain criteria, e.g. cytocompatibility and stiffness,

which are essential to cellular behaviour.

In this work, we present an approach to engineer a jaw bone model via stereolithographic

bioprinting. To study the cellular behaviour in the prospective printing matrix, primary jaw

bone osteoblasts were embedded in different bioinks and cultivated over 28 days.

Morphology changes and cellular interactions were assessed by sequential microscopy.

Analyses of osteoblast-specific markers on RNA and protein level were conducted to

reveal differences between 2D and 3D cultures. These experiments show the feasibility

of developing an osteoblast-compatible bioink for stereolithographic bioprinting.

194 195

Imaging of O2 concentration and spatial distribution in 3D bioprinted hydrogel scaffolds using O2 sensing nanoparticles

Ashwini Rahul Akkineni1, Anja Lode1, Erik Trampe2, Klaus Koren2, Felix Krujatz3,

Michael Kühl2, Michael Gelinsky1

1 Centre for Translational Bone, Joint and Soft Tissue Research, Technische

Universität Dresden, Germany 2 Marine Biology Section, Department of Biology, University of Copenhagen,

Denmark 3 Institute of Natural Materials Technology, Technische Universität Dresden,

Germany

Introduction: One of the major limitations of cell-based regenerative approaches is the insufficient

supply of the cells with oxygen due to a lack of a functional vascular system. In

complex 3D culture systems, such as bioprinted constructs with clinically relevant

dimensions, the local O2 concentration depends on the macroporosity of the hydrogel

scaffold as well as on the micro/nanoporosity of the hydrogel material which oxygen

has to pass through via diffusion. A better understanding and therefore new tools are

needed to monitor oxygen distribution dynamics within bioprinted constructs.

Materials and methods: Alginate/methylcellulose [1,2] was used as bioink. O2 sensitive nanoparticles [3] as

well as oxygen consuming and/or producing cells, i. e. mammalian cells and

photosynthetically active microalgae (Chlorella sorokinania) were mixed into the

paste. The BioScaffolder 3.1 (GeSiM, Radeberg) was applied for extrusion-based

bioprinting. 3D constructs, containing either of a single cell type or both cell types in a

spatially defined pattern, were fabricated to image spatio-temporal dynamics of O2

concentration. The constructs were cultivated in media specific for mammalian cells

or microalgae or in an adapted medium suitable for both, at 37°C, 5% CO2 and

defined O2 concentrations (1-21%) with and without illumination. A SLR camera and

custom-built 445 nm LED was used to image O2 sensitive nanoparticles.

3D-printed drug delivery systems for cell therapy: A new approach for the treatment of Diabetes Mellitus

Axel Pössl, Peggy Schlupp, Thomas Schmidts and Frank Runkel

Technische Hochschule Mittelhessen – University of Applied Sciences,

Giessen/Germany

Diabetes mellitus is associated with a disorder in the glucose metabolism. In

normoglycemic organisms, β-cells exactly dose the required amount of insulin for

glucose uptake into tissue by biochemical signals. This function of the β-cells is

disrupted in Diabetes mellitus patients resulting in high blood glucose levels. The

so-called hyperglycemia can cause some issues and diseases, like kidney

dysfunction. To overcome hyperglycemia we seek for a cell therapy approach. We

will encapsulate the β-cells and stem cells in co-culture in a 3D-printed hydrogel

matrix. However, the encapsulation of cells in hydrogels involves several issues, for

example the limited nutrition supply and rejection by the immune system in case of

non-autologous cells [1]. Thus, the choice of the materials and the dimensions of the

cell-laden constructs are essential parameters.

Experimental design The goal of the investigations is the development of a well characterized cell-laden

three-dimensional device for long-term secretion of insulin. Therefore, we will

encapsulate β-cells and stem cells in a hydrogel via 3D-printing. The encapsulation is

subdivided into an inner micro- and a surrounding macroencapsulation. The

microencapsulation includes the β-cells and stem cells to protect these from induced

apoptosis by autologous immune cells. Macroencapsulation should support structural

properties and ensure sufficient supply of nutrients and secretion of insulin.

Consequently, materials have to be identified and characterized regarding mass

transfer, printability and the influence on the differentiation of the cells. A

mathematical model describing the mass transfer processes will be established. The

3D-printed cell-laden structures will be further investigated regarding insulin secretion

as a result of glucose stimulation, integrity of encapsulation and homeostasis of the

cells. Summarized, these studies should serve as basis for the long-term therapy of

Diabetes mellitus.

[1] BARKAI, Uriel; ROTEM, Avi; DE VOS, Paul. Survival of encapsulated islets: More

than a membrane story. World journal of transplantation, 2016, 6, 69-90.

196 197

Modelling of a microfluidic device to study tumor cell extravasation Claudia Kühlbach 1,2, Raffaela Glunz 3, Margareta M. Mueller 1, Frank Baganz 2 and

Volker C. Hass 2,3 1 Furtwangen University, Dept. Mechanical und Medical Engineering, Villingen-Schwenningen, Germany;

[email protected], [email protected] 2 UCL University College London, Dept. Biochemical Engineering, London, UK; [email protected] 3 Furtwangen University, Dept. Medical and Life Science, Villingen-Schwenningen, Germany;

[email protected]

Abstract: Cancer metastasis is a highly complex process, comprising intravasation, tumor cell

distribution by blood stream and extravasation into surrounding tissues. The extravasation is characterized by adhesion of tumor cells to the endothelium and

transendothelial migration. The exact mechanism and molecular conditions are still

not fully understood. Microfluidic devices are used to build up novel 3D models which

can take cell-cell interactions, structural and mechanical interactions into account.

The device consists of three parts and includes two parallel channels with a porous

membrane sandwiched in between. Channel and membrane acting as vessel equivalent are seeded with ECs and checked for monolayer confluency before

assembly. The endothelial cells showed in vivo like behavior under flow. GFP-

expressing tumor cells, both of epithelial and mesenchymal origin, can be introduced

to the system and observed by live cell imaging. The results show that the cancer

cells adhere tightly to the endothelium under different conditions. The device can be

used for studies of extravasation mechanisms, its inhibition and molecular requirements of the migrating tumor cells. For mathematical modelling, the

microfluidic device was described as a system of ordinary differential equations

(ODE). The microfluidic channel was subdivided into a cascade of ideal stir tank

reactors. The membrane was modeled as area for transmembrane transport of the

tumor cells into the reservoir of the second channel underneath. The aim of this work

is to determine if the permeability coefficient of the membrane is depending on the cell number within the medium introduced to the microfluidic channel, which acts as

the vessel equivalent. Three different kinetics are used to identify the permeability

coefficient for the membrane used in the device, either depending or independent of

cell numbers. To assess the best suitable model, experiments within the microfluidic

device are conducted.

By these results, effectiveness of anti-tumor pharmaceuticals, inhibiting the transendothelial migration, can be determined.

Results: The addition of the nanoparticles did not affect the rheological and printing properties

of the paste or the viability of mammalian cells and microalgae. Imaging of

microalgae-laden constructs revealed that the O2 concentration within the hydrogel

scaffolds shows high dynamics in response to light/dark cycles reflecting the

metabolic processes of photosynthesis and respiration. In case of mammalian cells,

oxygen consumption can indicate their metabolic and functional activity.

Conclusion: This simple method based on O2 sensitive nanoparticles is an excellent tool to study

local O2 consumption and/or production as well as diffusion processes by

measurement of spatio-temporal O2 concentrations in 3D bioprinted constructs. It can

help to identify suitable scaffold designs and culture conditions.

References: 1. K. Schütz et al., J Tissue Eng Regen Med 2017, 11, 1574

2. A. Lode et al., Eng Life Sci 2015, 15, 177

3. K. Koren et al., Sens Actuator B Chem 2016, 237, 1095

198 199

214 xxx

conference organiserDECHEMA e.V.Theodor-Heuss-Allee 2560486 Frankfurt am MainGermany

contactTel.: +49 69 7564-0Fax: +49 69 7564-201Email: [email protected]

DECHEMA FOCAL TOPIC

PHARMACEUTICALS> Development and e� cient production of innovative

(bio)pharmaceuticals and therapeutics

> Diverse working parties – from natural product research through to cell culture technology

> Event series: Irseer Natursto� age (Irsee Natural Products Conference), 3D Cell Culture

> Publications on (bio)pharmaceutical manufacturing

> German NanoBioMedicine Platform for nanotherapeutics

More information at:www.dechema.de/pharmaceuticals

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