Developing a human chip based platform for repeated dose toxicity testing. Heike Walles Roland Lauster Gerd Lindner Uwe Marx

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1 Developing a human chip based platform for repeated dose toxicity testing Heike Walles Roland Lauster Gerd Lindner Uwe Marx

2 Repeated dose toxicity testing status quo Daily observation and sampling: hematology (blood) clinical biochemistry (blood) urine analysis 28 day: OECD 407 o, 410 d, 412 i, 419 ~40 rodents /substance 90 day: OECD 408 o, 411 d, 413 i dil daily exposure ~100 rodents per substance Death Organ weight Necropsy: liver, brain, heart kidney, spleen ovary, testes, uterus thyroid, adrenals epididymis dd others toxicity, dose response relationship d A roadmap for thedevelopment of alternative (non animal) methods for systemic toxicity testing 90d 12 mo OECD 453 integrated testing strategy (ITS) Basketter et al, ALTEX 29, 1/12, pp

3 Integrated testing strategies human in silico + human in vitro + animal in vivo Roadmap questions:.. what constitutes an appropriate model for each organ system, i.e., what makes a heart a heart, a liver a liver etc. Roadmap request: more physiological culture conditions such as homeostasis, oxygen supply, cell density SKIN LIVER INTESTINE primary cells HepaRG CaCo 2 Molecular initiation subcellular cellular organ organism Pathway of Toxicity (PoT) Mode of Action (MoA) Adverse Outcome Pathway (AOP) 3

4 Integrated testing strategies human in silico + human in vitro + animal in vivo SKIN primary cells Roadmap questions:.. what constitutes an appropriate model for each organ system, i.e., what makes a heart a heart, a liver a liver etc. Roadmap request: more physiological culture conditions such as homeostasis, oxygen supply, cell density LIVER INTESTINE HepaRG CaCo 2 epidermis dermis lobuli villi Molecular initiation subcellular cellular organ organism Pathway of Toxicity (PoT) Mode of Action (MoA) Adverse Outcome Pathway (AOP) 4

5 Maturation of human in vitro tests static cultures dynamic cultures primary cells of all relevant organs tissue slices spheroids miniaturization i i ti!? (organs on a chip) full thickness skin long term stability!? (28d, 90d, 12mo) cell line es hecs cell lines: cardiomyocyte, neuronal hepatocyte like NIH 3T3, CaCo2,, HepaRG, Fa32, HepG2, SH SY5Y, LLC PK1, HL60.. cell line spheroids LIINTOP CaCo2 HepaRG organotypicness!? combination!? 2D single type 3D single tissue 2D multi tissue 3D single tissue 3D multi tissue 5

6 Our development objectives dynamic 3D multi tissue culture (optimized oxygen supply, mechanical stress, dynamic protein gradients, shared medium..) miniaturization to smallest possible scale optimized fluid to tissue ratio standardized easy to handle bioreactor and chip format equally supporting cell line and primary tissue culture 6

7 The Multi Organ Chip (MOC) technology Laboratory bioreactor Transwell inserts two peristaltic itlti micro pumps per chip two media perfusion circuits per chip adopted to use of Transwell inserts two MOC s (4 circiuts) per bioreactor peristaltic micro pump Transwell inserts injection port MOC s of microscopic slide format 7

8 28 day MOC culture experimental setup 3D tissue preparation and chip loading HepaRG cells + stellate cells (12 : 1) Foreskin donor recruitment 24h aggregate formation in AggreWell plates tissue excision and transport to lab 4 days low attachment plate culture Preparation of sterile punch biopsies tissue volume: ~30µl channel volume: ~10µl total volume: 300µl liver model skin model 8

9 28 day MOC culture regimen day 0 (8 circuits) Process parameters and endpoints: n = 8 perfused liver skin circuits continuous perfusion at t = 37 C, humidified air with 5% CO2 daily feeding (50% medium exchange) daily sampling (metabolic analysis) day 14 day 21 day 28 n=2 n=2 n=4 Media composition: William s E Medium (Basalmedium) + 10%FCS, + 5µg/ml insulin, + 2mM glutamine + 5 * 10^ 5 mol/l hydrocortisone hemisuccinate + 1% Pen/ Strep interims tissue analysis at days 14 and 21 histology at completion of each circuit 9

10 28 day MOC culture preliminary results day 0 day 28 HepaRG stellate cells 3D culture IMMUNOHISTOSTAINING: CYP3A4 CYP7A1 Foreskin 3D tissue biopsies IMMUNOHISTOSTAINING: Tenascin ColIV 10

11 28 day MOC culture summary 28 day 3D multi tissue culture is feasible both cell lines and primary tissues can be used MOC format supports investigation into different non standard repeated dose toxicity test designs complex tissues still suffer from limited nutrition unlimited organotypic culture (90 day, 12 months ) is impossible at organ homeostasis add human vasculature! 11

12 Endothelialization of the BioVaSc A BioVaSc V seeded with human endothelial cells cultivation under shear stress Live Dead staining Living cells Dead cells add human liver tissue for long term dynamic bioreactor culture 12

13 Bio Vasculature = Unlimited Liver Maintenance human liver bioreactor metabolism study (n=3) 1 Albumin (mg/ml) Stable Synthesis of Albumin 3 2,5 2 1,5 1 0,5 1 2 Albumin synthesis achieves homeostasis & correlates lt to metabolism tbli shown in (2). 2 Metabolites (pmol/ml) Metabolism of dextometorphan (OTC cough 60 suppressant) to dextorphan glucuronidid (Phase II 40 metabolite) is stable during the entire experiment. 20 No signs of loss of stability or function for over 3 months! days Metabolism of Small Molecule Drugs Dextometorphan Dextorphan Glucuronidid days 13

14 Conclusion and outlook dynamic MOC bioreactors provide a tool for 28 day repeated dose human in vitro testing using cell lines and/or primary cells experimental throughput does not fit industrial needs Further combination of 3D tissues within one perfusion circuit may provide additional data partners for multi tissue model dlresearch, involving i human cardiomyocytes, neuronal cells, intestine, kidney, lung or others tissues arewelcome for 90 day repeated dose or chronic toxicity testing unlimited homeostasis can be ensured only by vascularized MOC s 14

15 Acknowledgements Institute of Biotechnology Eva Materne Ilka Wagner Reyk khorland Gerd Lindner Roland Lauster Fraunhofer IWS, Dresden Frank Sonntag Niels Schilling Udo Klotzbach Funding BMBF: GO BIO Fraunhofer IGB, Stuttgart Heike Walles 15