MPS development status, first industrial adoption and current challenges. Uwe Marx EMA

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1 MPS development status, first industrial adoption and current challenges Uwe Marx EMA

2 t4 - Report on biology-inspired MPS Marx et al ALTEX 33 (3) (CC 4.0 open access) MPS experts (16) Big industries (6) Microphysiological systems are microfluidic cell culture devices capable of emulating human biology in vitro at the smallest biologically acceptable scale. micro: at smallest biologically acceptable scale testing at relevant throughput minimum use of human tissue affordable assay economy physiological: truly emulating human biology human organ architecture healthy long term homeostasis damage, repair, regeneration, disease Regulatory bodies (14) systems: Devices, supporting human-like organ maintenance temperature, humidity, ph, O 2 -supply mechanical and electrical coupling optical imaging

3 Types of MPS and key commercial providers Marx et al. ALTEX 2016 Emulate Inc. Hesperos

4 Impact of MPS-tools on drug development Marx et al. ALTEX 2016 high content, systemic repeated dose testing, retrospective assessment of AoPs of candidates, which failed in late phases or post marketing years high throughput, single tissue, single dose testing, Discovery Lead Preclinics Phase I Phase II Phase optimization III Submission Patients Market laboratory animal studies - in vitro testing - clinical trials - Patient-on-a-chip >2030 Preclinical phase Validation in patients Submission Patients

5 A roadmap towards MPS-based decision-making Marx et al. ALTEX 2016 Regulatory acceptance none (MPS-based regulatory science) validated safety assays validated hazard identification, systemic efficacy assay Industrial adoption none (initial MPS evaluation) MoA assessment, toxicity testing systemic safety testing, disease modeling, on-chip clinical trials genotypic target discovery, personalized disease models Innovative solutions single- and multi-organ MPS tools human Body-ona-chip models You-on-a-chip precision medicine approaches special models, e.g. Pregnancy on-a-chip

6 MPS-based research highlights Pulmonary Edema in a Lung-on-a-Chip Four-Organ-ADMET-Chip A A Female Reproductive Tract-on-a-Chip D M E TRPV4 inhibitor blocks IL2-induced edema Huh et al. Science 2010 Huh et al. Sci. Transl. Med homeostatic 28-day downregulation of glucose Maschmeyer et al. LabChip 2015 modeling menstrual cycle and pregnancy Xiao et al. Nature Communications 2017

7 Downscaling a human body: How small can we go? 70 small intestine villi 4 mm 2 brain cerebral cortex 10 liver lobuli 18 mm 2 skin segment 1mm O 2 5 pancreatic islets 3000 lung alveoli 20 kidney nephrons 0.1 mg of ovary follicles Scaling factor: 1/ or 0.4 mg of testes seminiferous tubules 20 mg of bone-marrow units 300 mg of skeletal muscle fiber segments Bars: 100 µm Marx et al. (2012) ATLA 40, mg of adipose tissue clusters

8 The MOC technology touchscreen-based benchtop MOC features: area of a microscopic slide on-chip micropump enabling pulsatile flow suitable for primary cells, 3D tissues and cell lines compatible with live tissue imaging plug-in option for insert-based barrier models COMSOL Multiphysics 5.2. standard cell culture inserts (96-/12-/24-well format)

9 Multi-Organ-Chip Assays In Process Controls (Sampling) ~250µl/sample Blood Surrogate Analysis Metabolism (e.g.glucose/lactate) ~6µl Viability (e.g. LDH) ~12,5µl Repeated dose exposure regimen Histology various cell sources Model Preparation 1-4 weeks Day 1 Day 7 Day 14 Day 28 qpcr Metabolite Analysis -omics In Process Controls (non-invasive) Cell Morphology Molecular imaging Others Microarray

10 Preformed human organ equivalents V 200µm 100µm CK 8/18 Vimentin DAPI primary hepatic stellate cell and HepaRG cell spheroids Glucagon α Insulin β Somatostatin δ primary pancreatic islet cell spheroids 100µm V 200µm CK10 CK15 Dapi prepuce explants and primary keratinocyte/ fibroblast-based skin equivalents V V V - validated commercially available 3D models

11 In s u lin [m U /l] In s u lin [m U /l] Liver-islet chip: insulin responsiveness Bauer et al. Scientific Reports in press O C ts C u r e Islet-only culture (24h) 40 spheroids 10 spheroids 300µl T im e [h ] Liver 2 O C islet Is ts co-culture -L iv e r C u r(24h) e + liver pancreatic islets T im e [h ] Regulated physiological insulin secretion

12 G lu c o s e [g /l] In s u lin [m U /l] G lu c o s e [g /l] Liver-islet crosstalk to ensure glucose homeostasis Glucose tolerance test for liver-islet co-culture (48h) + G T T in 2 O C L iv e r -Is le t C u ltu r e T im e [h ] m e d iu m Glucose e x c h a n g e OGTT Oin G T T healthy in h e a y humans h u m a n s (4h) g o r a l g lu c o s e T im e [h ] 8-day liver islet co-culture Glucose-stimulated insulin secretion assay d 0 Long-term homeostasis and crosstalk + d ,8 mmm M glucose c o s e ,8 mmm M glucose c o s e Bauer et al. Scientific Reports in press

13 Industrial adoption of liver-islet MOC R&D, Innovative Medicines Rationale: establish Type 2 diabetes model for drug testing Model transfer and inter-lab validation Commercial cell source supply secured On-chip insulin resistance induction in progress tech transfer

14 Application of a skin-tumour model in the pharma industry Air 15 spheroids H292 cell line 10,000 ng/ ml 100 ng/ ml 10 ng/ ml Bay1a FT-skin healthy skin lung tumour Rationale Establish a combined safety and efficacy ( safficacy ) assay for cancer drug candidates Qualification tool d0 5-d skin-tumour co-culture readouts: histology, gene expression + d0 Bay1a, a drug effective on lung tumour cells but causing severe skin toxicity in primates Could an in vitro co-culture serve to screen for candidates with a better profile before going in vivo? d5 100 µm 100 µm + d5

15 Status quo of the safficacy assay control (D5) Bay1a 100 ng/ml (D5) HE-staining TUNEL/ Ki67 HE-staining TUNEL/ Ki µm reproducible human 3D tumour-skin co-culture in place skin side effects observed but no tumour effects yet detected various hurdles to be solved for feasibility completion with permission of Dr. Thomas Steger-Hartmann

16 Current challenges Secure reliable cell sources Increase level of similarity to human biology Regulatory acceptance to foster the paradigm shift

17 Our 3D organoid engineering pipeline 2017 Liver 1. Intestine 2. Kidney 3. Brain 4. Pancreas 5. Skeletal muscles 6. tissue explants islets primary cells V V cell lines selected context of use fit for purpose assays duration of 5 days to 8 weeks repeated dose exposure NCEs, NBEs, chemicals 12 multi-organ combinations Adipose tissue 7. Skin 8. subcutaneous explants punch biopsies V 13 single organ models (as of September 2017) Hair 9. follicular unit extraction Lung 10. V Bone-marrow 11. punch biopsies Lymph node* 12. Vasculature 13. 3D Lung tumor *in collaboration with ProBioGen, Germany V - validated commercially available 3D models

18 Potential of cell expansion Secure reliable cell sources Marx et al. ALTEX 2016 gut organoid neuronal organoid any other organoid neurons cardiomyocytes any other cell type immortalized human cell lines, e.g. HeLa, HepaRG, CaCo-2 ipsc s intestinal epithelia lung epithelia other epithelial cells endothelial cells hepatocytes other endoderm-derived cells reconstructed organ models, e.g. EpiDerm, EpiSkin + EpiAirway, MucilAir EpiIntestinal embryonal stem cells fetal stem cells tissue explants from adult donors adult tissue specific stem cells Human life span

19 Level of similarity to human biology segregate bile from liver equivalents and add entero-hepatic recirculation Materne et al. Lab Chip, 2013, Chip-based liver equivalents for toxicity testing organotypicalness versus cost-efficient high throughput vascularize channels and organ equivalents Schimek et al. Lab Chip 2013, Integrating biological vasculature into a multi-organ-chip microsystem. Hasenberg et al. J Biotechnology 2015, Emulating Human Microcapillaries in a Multi-Organ-Chip Platform. Knezevic et al. Frontiers in Bioeng Biotech 2017, 2017: Engineering blood and lymphatic microvascular networks in fibrin matrices. von Willebrand factor CD31 apply plasma/blood perfusion and integrate innate and adoptive immunity Giese and Marx, Adv. Drug Deliv. Rev., 2014, Immunity in vitro solving immunogenicity and more.

20 Regulatory acceptance = Marx et al. ALTEX 2016 Regulatory science + Validation strategy Segment: Aim: Drug Development Safety and Efficacy Testing Cosmetics, Chemicals Identification of Hazard Potential Process: Step 1 US FDA DDT Qualification Path US Animal models Biomarker add MPS-based tools feed step 1 data Step 2 OECD Guidance on Validation US Animal models In vitro assays Result: Drug-independent tool validation OECD guideline Eds. Eskes and Whelan: Validating Alternative Methods for Toxicity Testing. Vol 856 of the series Advances in Experimental Medicine and Biology pp Springer, 2016, Chapter 12: Rebelo et al; Validation of bioreactor and human-on-a-chip devices for chemical safety assessment

21 Thank you next workshop: Berlin, Germany, June 17th 19th 2019 Biology-Inspired Microphysiological System Approaches to Solve the Prediction Dilemma of Substance Testing