Bioprinted 3D Primary Liver Tissues. PD Dr Adrian Roth Head Mechanistic Safety Roche Innovation Centre Basel, Switzerland

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1 Bioprinted 3D Primary Liver Tissues PD Dr Adrian Roth Head Mechanistic Safety Roche Innovation Centre Basel, Switzerland

2 Number of animals Drug Safety at Roche Reducing animal numbers - increasing Patient s Safety Human in vitro models to reduce attrition rate due to species-specificities. reduce pre-clinical animal testing. 2

3 Advanced Cell Models in Drug Safety Going into more dimensions Toxic Concentrations (µm) in vivo and in vitro (Q. Meng, Zhejiang University, China) 3

4 Our Vision Physiologically relevant human cell models matter Drug-induced organ toxicity is complex by nature Develops over time Depends on metabolism and bio-distribution Involves the interplay of different cell types Influenced by patient background and disease conditions Create multi-dimensional in vitro complexity to mimic organs and organ-interactions From 3D spheroids to microfluidic Organ-on-a-chip.. to bioprinting of organ structures 4

5 Our Mission Investigate toxicity from early to late development 1. Predictive screens 2. Address human relevance of pre-clinical in vivo findings 3. Assess mode of action of clinical findings 5

6 Our Approach The question defines the choice of the model Ease of use & Throughput Compound ranking Candidate Selection Complexity & Functionality Unknown MoT Longterm Effect Tissue Cross-talk PK/PD aspects (?) Unknown MoT Known, complex MoT Metabolites Organ-Organ interaction Address Specific known Mechanism Generate Hypothesis - Resolve unexplained Issue

7 Advanced Liver Cell Models Mimmicking human-relevant Drug response in Organ-like cell models 2D Micropatterned Co- Cultures 3D Transwell Co- Cultures 3D Spheroids Ability for longterm Incubation increases Predictivity for DILI Khetani et al.; Toxicol Sci (2013) 132 (1): Different Cell Types contributing to toxic Effect Kostadinova et al.; Toxicol Appl Pharmacol Apr 1;268(1):1-16 High Complexity in Semi-HT Format Messner et al.; Arch Toxicol Jan;87(1): Can Bioprinting lead to similar Improvements or even superior? 7

8 Biorpinting Cellular Models Organovo Layer-by-Layer Approach Primary or ips cells Normal or diseased Cell mixture Proprietary media ExVive Human Tissues 8

9 Biorpinting Cellular Models Organovo ExVive Human Liver Tissue Hepatocytes Stellates Endothelial Large scale tissue (2mm 2 X 0.5mm; >10 6 cells) Compartmentalized architecture Kupffer cells available on request 9

10 Biorpinting Cellular Models Organovo ExVive Human Liver Tissue Human Liver E-Cad+ junctions between Albumin+ hepatocytes Printed Human Liver CD31+ endothelial cells in proximity to Desmin+ hepatic stellate cells Vimentin staining (mesenchymal compartment) Hepatocellular polarization with evidence of mature bile canaliculi and junctional complexes 10

11 OrganovoExVive Human Liver Tissue Stable Functionality over Time 11

12 Bioprinted Liver Tissue Recapitulate Complex Drug Toxicity Trovafloxacin: use significantly restricted due to high potential for inducing serious, sometimes fatal liver damage MoT not clear, but suspected to be due to - Generation of Metabolite(s) - thrombin-antithrombin Dimers - Underlying inflammatory Stress -.. Recaptiulate Trovafloxacin-induced Liver Damage, compare to Levofloxacin 12

13 Trovafloxicin-Induced Liver Injury In Vitro Experiment ExVive Human Liver Tissue 0.5X Cmax* 1X Cmax 5X Cmax *Cmax ~ 4uM 7-Day Exposure 20X Cmax Journal of Antimicrobial Chemotherapy (1997) 39, Suppl. B, 87 92) ENDPOINTS ATP ALBUMIN Histology - H&E 13

14 Trovafloxacin DILI in Human Liver Tissue Differential Effect of Trovafloxacin vs. Levofloxacin Bioprinted tissues more sensitive than 2D culture Loss of cellular adhesion (arrow) and increased hepatocyte necrosis (arrowheads) * (p < 0.01) by ANOVA 14

15 Bioprinted 3D Liver Model Multicelltype Model stable over prolonged Period Could detect Trovafloxacin Toxicity without «preconditioning» «In Vitro» Histopathology shows distinct changes resembling in vivo 15

16 From Bioprinted to «Organs On A Chip»? Advanced human Cell Models for improved Drug Testing Vision for the Future Personalized Drug Safety Testing in Patient-derived Cell Systems Multi-Organs On A Chip

17 Summary Bioprinted 3D Cell Model shows improved physiological parameters Longterm culture, interplay of different cell types considered key for Drug Safety aspects Improved physiology allows better in vitro to in vivo correlations Detection of drug candidates with unfavourable characteristics Combination of improved in vitro models with state-of-the-art readout technologies suggested to significantly increase predictive power Selection of 3D model depending on question to be addressed Technical hurdles: Ease of use, Reproducibility, Throughput, Nonspecific binding 17

18 Where 3D Printed Tissue Models can win Building complex architectures under controlled conditions Vascularized tissue; e.g. systemic delivery Barrier tissue; e.g. oral delivery Multi-compartments; e.g. nephron structure with open lumen Long-term culture; e.g. late onset toxicity Connecting tissues... towards bioprinted body on a chip Liver-target tissue e.g. pro-drug, metabolites Immune cell infiltration e.g. tumor, auto-immunity Amenable to drug discovery platform Throughput & robustness Compatible readouts 18

19 Acknowledgements Roche Innovation Centre Basel Sabine Sewing Annie Moisan Franziska Boess Marcel Gubler Stefan Kustermann Crisitna Bertinetti Claudia McGinnis Liudmila Polonchuck Juergen Funk Franz Schuler Thomas Singer Organovo Deb Nguyen Sharon Presnell Stephanie Camacho 19

20 Doing now what patients need next 20