Can Organ-on-a-Chip Technology Really Replace Animal Testing of Drug and Chemical Safety?

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1 1 Can Organ-on-a-Chip Technology Really Replace Animal Testing of Drug and Chemical Safety? J Malcolm Wilkinson, CEO, Kirkstall Ltd. Quasi Vivo - saving lives through better science

2 The drug development process DRUG DISCOVERY PRE-CLINICAL Human Cellular Assays Phase I CLINICAL TRIALS Phase II Phase III FDA review CLINIC GAP 10,000 Compounds 2D plastic culture 250 Compounds Animal Studies IND GAP 5 Compounds NDA 1 Approved drug 1.8 B$ Do not exactly represent Human physiology 6.5 years 6 years 1.5 years Time and Attrition Slide courtesy of Prof Martin Yarmush, Rutgers University & Harvard Medical School

3 The opportunity for in vitro assays DRUG DISCOVERY PRE-CLINICAL 2D plastic culture do not predict clinical response Phase I CLINICAL TRIALS Phase II Phase III FDA review CLINIC 10,000 Compounds Advanced in vitro assays which will provide good 250 Compounds IND predictions of clinical outcomes 5 Compounds NDA 1 Approved drug H 1.8 B$ Animal Studies Do not match Human physiology 6.5 years 6 years 1.5 years Time and Attrition

4 4 Problems with animals Expensive (breed, nurture, test, disposal) Different metabolism from humans Physiology different (eg rat brain) Licence needed for animal experimentation Ethical backlash Subject to variability (as are human volunteers) Sometimes they bite!..but despite ethical and scientific arguments it is still a regulatory requirement to use animal tests before clinical trials Quasi Vivo - saving lives through better science

5 5 How do animal tests complement existing HTS in vitro screening? Requirements list (not the same as HTS) Systemic models the whole organism Long term and repeat dose exposure Cultures that maintain homeostasis (before insult) Good disease models (the use of transgenic mice is growing rapidly but no amount of genetic manipulation will make them human) So this is our checklist of goals for organ on a chip

6 6 Advances in cell culture 2D cell culture 3D cell culture Perfusion of media through scaffolds and tissue slices Coculture and Systemic models Organ on a chip? 1960 s

7 The Multi-Organ-Chip concept human in vitro SKIN primary cells LIVER HepaRG INTESTINE CaCo 2 Molecular initiation subcellular cellular organ organism Pathway of Toxicity (PoT) Mode of Action (MoA) Adverse Outcome Pathway (AOP) 7 Edinburgh 2014 Materne

8 Next generation Multi-Organ-Chip 8 Edinburgh 2014 Materne

9 Benchtop bioreactor bioreactor design bioreactor in operation Features: Controlling up to 24 pneumatic actors Up to 4 chips per system Adjustable temperature and fluid flow Software control (e.g. WINDOWS, LINUX, MAC) 9 Telemonitoring Edinburgh 2014 Materne

10 10 Other Considerations Ease of use (run by technicians not postdocs) Reproducibility (no hidden uncontrolled variables) Robustness(same results across 6 or more labs) A predictive assay is much more than fancy cell culture equipment.it must have good sensitivity and specificity Good sensitivity reduces false negatives Good specificity reduces false positives Quasi Vivo - saving lives through better science

11 11 Micro scaling challenges Can a collection of micro organs predict the behaviour of 75Kg human? Can microchannels provide enough O 2 and nutrient and remove waste? Can long term viability and metabolic function be achieved? Can micro scale system provide 3D capability? Can practical problems like blockages and bubbles be solved? Can non-specific binding of drugs/proteins be avoided? Quasi Vivo - saving lives through better science

12 Smallest possible scale of organs a single liver lobule is of 1,3 µl in scale 1 million liver lobules constitute a human liver Molecule Cell Organoid Organ Individual Ten liver lobules the basis for a human-on-a-chip 12 Edinburgh 2014 Materne

13 13 Metabolic functionality Figure 1. Oxygen concentration profiles for micro- and milli-fluidic systems and the MCmB. Hypoxic regions are represented in white. Taken from Mattei, G., Giusti, S. & Ahluwalia, A. Design Criteria for Generating Physiologically Relevant In Vitro Models in Bioreactors. Processes 2, (2014). Quasi Vivo - saving lives through better science

14 Hepatocytes must be metabolically competent Phase I Phase II Phase III O 2 R (Eg: UDPG, GSH) X X Eg: CYP X-OH Eg: GST, UGT X-OR X-OR Eg: MRP Eg: MRP Therapeutic Effect Hepatocyte Elimination 14 General pathway of xenobiotic metabolism

15 Cell metabolism changes dependent on culture conditions and must be maintained long term Days 0 to 7 in Static Culture QVS flow conditions restore function Day Static conditions in 24 well plate destroy functionality Flow conditions in microfluidic devices can upregulate gene expression by factor of 2 or 3 relative to a static well plate (24 or 96 wells) Optimised flow in QVS upregulated activity by factor of 32 Drug Dose causing 50% cell death (LD 50 ) can be in error by same factor

16 LD 50 for Diclofenac underestimated by factor of 30 times in static culture Diclofenac IC50 assay 1.4 Fractional vitality Flow Static Flow fit Static fit Concentration ( M) Clinical trial result

17 17 Not just CYP 3A4 Significant down regulation in static condition compared to flow Phase I Phase II Xenobiotic transcription factor Transporter (P-gp) Quasi-Vivo.saving lives through better science

18 Depth of culture in microns No standards have emerged yet in 3D or scaffold technology 3D >10,000 >1000 >100 Not enough cells to do assays like western blot Liable to necrosis without flow Quasi Vivo System Spheroids 3D Printing Gels >10 1 Microfluidics Scaffolds 1 Cell , ,000 1M Number of Cells Cells become necrotic

19 19 Ease of Use example: Organ on a plate Equipment can be used by technicians Utilises existing capital investment Re-usable (sterilisable) elements Single use (disposable) elements Different tubing options available (teflon/silicone) Integrated organ on a plate system available now from Kirkstall and Parker Hannifin Quasi Vivo - saving lives through better science

20 Lego kit for cell biologists! Reservoir bottle provides enough media for 3 to 7 days Individual chambers for cell culture Recirculating flow allows cells to condition media Peristaltic pump provides controlled flow in 2 or more channels Quasi Vivo - saving lives through better science Adherent cells on scaffolds /in gels or coverslip Or even tissue slices

21 21 Prognosis Scale/Function Current well plate QVS organ on plate 3D? Oxygen Supply? Long Term Viability >28 days Multi-organ Metabolic competency Current organ on chip (2015) in development In development In development Future organ on chip (2020) Quasi Vivo - saving lives through better science

22 22 Thanks for your attention! J Malcolm Wilkinson jmw@kirkstall.org Web: Phone: Quasi Vivo - saving lives through better science