Organ-on-a-chip: practical applications & challenges. Remko van Vught

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1 Organ-on-a-chip: practical applications & challenges Remko van Vught

2 A leap forward in physiological relevance The challenge of reductionism: Make things as simple as possible, but not simpler, Albert Einstein 2D culture 2D culture 3D culture Animal testing Human tissues Co-culture Perfused ECM embedded Tubes & vessels Mimetas BV Leiden,

3 The OrganoPlate Mimetas BV Leiden,

4 PhaseGuide Technology PhaseGuide Vulto et al 2011 Phaseguides: a paradigm shift in microfluidic priming and emptying. Lab Chip 11(9) Mimetas BV Leiden,

5 The OrganoPlate Watch full movie Mimetas BV Leiden,

6 The OrganoPlate Watch full movie Mimetas BV Leiden,

7 Pump-free continuous perfusion Mimetas BV Leiden,

8 Organ models in the OrganoPlate Liver Kidney clearance Kidney Brain Blood-brain barrier Vasculature Organoids Solid tissues Endothelium - Pericyte Co-culture Gut Barrier tissues Mimetas BV Leiden,

9 Tube seeding in OrganoPlates Boundary Perfusion lane Phaseguide Gel Mimetas BV Leiden,

10 Human Proximal Tubule Model basal basal apical apical apical Acetylated tubulin ZO-1 DNA basal apical perspective basal perspective Human Renal Proximal Tubular Epithelial Cells form a tube in the 3-lane OrganoPlate RPTECs are polarized (cilia on apical surface) and express tight junction markers Mimetas BV Leiden,

11 NephroScreen: toxicity screening with a proximal tubule-on-a-chip NephroScreen assay availability NephroScreen Validation Screen 2017 A range of nephrotoxicity read outs were implemented in the 3D NephroScreen and are undergoing validation using 4 benchmark compounds and 8 blinded compounds with known clinical effects supplied by the Sponsors. Partners: Funding: Sponsors: Mimetas BV Leiden,

12 Real-time Barrier Integrity Assay No cells (NC) Barrier integrity in time Leak tight tube (PC) Mimetas BV Leiden,

Real-time barrier integrity assay MDCK (canine distal tubule cell line) in 3-lane OrganoPlate Day 5: Staurosporine exposure (15h) Dose dependent loss of barrier integrity measured in real

13 Real-time barrier integrity assay MDCK (canine distal tubule cell line) in 3-lane OrganoPlate Day 5: Staurosporine exposure (15h) Dose dependent loss of barrier integrity measured in real time on tubular model Trietsch SJ et al.: Membrane-free culture and real-time barrier integrity assessment of perfused intestinal epithelium tubes. Nat Commun 2017, Mimetas BV Leiden,

14 OrganoTEER Barrier assessment 120 channels High data quality Realtime and long-term Non invasive Flow Incubator Launch Q Beta open now! Mimetas BV Leiden,

15 In tra c e llu la r flu o re s c e n c e (F IT C /D A P I) In tra c e llu la r flu o re s c e n c e (F IT C /D A P I) Efflux- and Influx Inhibition Efflux Inhibition of the P- gp (MDR1) Transporter Influx Inhibition of Glucose Analog 6-NBDG 2.5 **** 0.8 ** C a lc e in -AM µ M C yc lo s p o r in e -A Significant inhibition of calcein-am efflux by cyclosporine-a N B D G µ M p h lo r id z in Significant inhibition of 6-NBDG influx by phlorizin Mimetas BV Leiden,

16 Case study: Cisplatin induced toxicity Renal toxicity has been noted in 28% to 36% of patients treated with a single dose of 50 mg/m2 The dose is reduced when the patient's creatinine clearance (a measure of renal function) is reduced The maximum human plasma concentrations (Cmax) for free cisplatin is: 4.5 ± 1.6 μm (at dosing of 30 mg/m2) 14.3 ± 2.3 μm (at dosing of 100 mg/m2) Evaluation of a single, 48 hour exposure in Human Proximal Tubule Model using multiple end-points Mimetas BV Leiden,

Normalized Fluorescence (AU) R a tio Toxicity assessment: barrier integrity S ig m a R P T E C - P 3 4 8 h c is p la tin e x p o s u re F IT C -d e x tra n 2 0 k D a S ig m a R P T E C - P 3 4 8 h c

17 Normalized Fluorescence (AU) R a tio Toxicity assessment: barrier integrity S ig m a R P T E C - P h c is p la tin e x p o s u re F IT C -d e x tra n 2 0 k D a S ig m a R P T E C - P h c is p la tin e x p o s u re T R IT C -d e x tra n k D a u M C is p la tin u M C is p la tin u M C is p la tin u M C is p la tin 3 0 µ M c is p la tin 3 0 µ M c is p la tin u M C is p la tin u M C is p la tin.4 5 u M C is p la tin V e h ic le c o n tro l u M C is p la tin V e h ic le c o n tro l.2 M e d iu m o n ly N o c e lls 0.2 M e d iu m o n ly N o c e lls T im e (m in ) Time (min) Non leak-tight tubule T im e (m in ) Leak-tight tubule Dose-dependent decreased barrier integrity of proximal tubules can be measured after 48h exposure to cisplatin Mimetas BV Leiden,

18 Toxicity assessment: morphological Medium only 30 µm Cisplatin Loss of proximal tubule viability can be observed by phase contrast imaging Mimetas BV Leiden,

C e ll Cell v ia viability b ility (% (% of o f med m e only) d o n ly ) Toxicity assessment: Enzymatic activity S ig m a R P T E C - P 3 - V ia b ility 4 8 h C is p la t i n e x p o s u r e T o p c

19 C e ll Cell v ia viability b ility (% (% of o f med m e only) d o n ly ) Toxicity assessment: Enzymatic activity S ig m a R P T E C - P 3 - V ia b ility 4 8 h C is p la t i n e x p o s u r e T o p c h a n n e l a n a ly s is S ig m a R P T E C - P 3 - V ia b ility 4 8 h C is p la t i n e x p o s u r e B o t h c h a n n e ls a n a ly s is m o n ly V e h ic le c o n tr o l 5 µ M C is p la tin 1 5 µ M C is p la tin 3 0 µ M C is p la tin 9 0 µ M C is p la tin µ M C is p la tin -5 0 M e d iu m o n ly V e h ic le c o n tr o l 5 µ M C is p la tin 1 5 µ M C is p la tin 3 0 µ M C is p la tin 9 0 µ M C is p la tin µ M C is p la tin Dose-dependent decrease in WST-8 signal can be measured after 48h exposure to cisplatin Mimetas BV Leiden,

L D H a c tiv ity (% o f m e d iu m o n ly ) Toxicity assessment: Enzymatic activity S ig m a R P T E C - P 3 - L D H a c tiv ity 4 8 h C is p la t i n e x p o s u r e 1 4 0 0 1 2 0 0 1 0 0 0 8 0 0 6

20 L D H a c tiv ity (% o f m e d iu m o n ly ) Toxicity assessment: Enzymatic activity S ig m a R P T E C - P 3 - L D H a c tiv ity 4 8 h C is p la t i n e x p o s u r e N o c e lls M e d iu m o n ly V e h ic le c o n tr o l 5 µ M C is p la tin 1 5 µ M C is p la tin 3 0 µ M C is p la tin 9 0 µ M C is p la tin µ M C is p la tin Dose-dependent increase in LDH activity can be measured in the medium of proximal tubules after 48h exposure to cisplatin Mimetas BV Leiden,

21 Toxicity assessment: DNA damage Medium only Vehicle control 5 µm Cisplatin 15 µm Cisplatin 30 µm Cisplatin 90 µm Cisplatin 270 µm Cisplatin H2AX Dose-dependant increase in H2AX expression of proximal tubules can be measured after 48h exposure to cisplatin Mimetas BV Leiden,

22 Overview: Cisplatin induced toxicity Barrier function Morphology Enzymatic activity (WST-8) Enzymatic activity (LDH) DNA damage 5 µm 15 µm 30 µm 90 µm 270 µm All readouts detected cisplatin induced toxicity after a single, 48 hour exposure. The DNA damage and LDH assays are most sensitive for the detection of cisplatin induced renal toxicity. Mimetas BV Leiden,

23 Fluorescence Intensity (AU) F lu o re s c e n c e In te n s ity (A U ) Fluorescence Intensity (AU) F lu o re s c e n c e In te n s ity (A U ) Time dependent toxicity 015hrs cisplatin o v e r n ig h t exposure d a y 8 to n o c e lls V C 1 0 µ M µ M days 0 1after cisplatin d a y 1 2 B I exposure a s s a y 1.0 n o c e lls V C 1 0 µ M µ M Time (hr) tim e (h o u rs ) Time (min) tim e (m in u te s ) Cisplatin influences RPTEC barrier integrity after 4 day recovery after 24h exposure of 10 and 100 µm cisplatin Mimetas BV Leiden,

Repeated dosing: Modelling Treatment Effects Long term treatment can

resistance in the Organoplate Long term culture under low drug dose

points Day 0 Seeding of glioma cells Day 1 TMZ treatment Day 6

RealTime-Glo After Day 56 the cells weren t treated with TMZ until

24 Repeated dosing: Modelling Treatment Effects Long term treatment can select and/or induce resistance Modelling patient specific resistance in the Organoplate Long term culture under low drug dose Determine compound sensitivity of 3D cultures at different time points Day 0 Seeding of glioma cells Day 1 TMZ treatment Day 6 RealTime-Glo TMZ treatment Repetition of drug exposure and RealTime-Glo After Day 56 the cells weren t treated with TMZ until Day 63 when the exp. ended. Responder Non-Responder Recovery (Resistance) Mimetas BV Leiden,

phase contrast Kidney proximal tubule Bloodvessel Gel ECM gel van Duinen V et al: 96 perfusable

25 Next step: Renal clearance model Adding a blood vessel to the kidney tube allows creation of a functional kidney unit Applications: renal clearance, acute kidney injury model Top view in phase contrast Kidney proximal tubule Bloodvessel Gel ECM gel van Duinen V et al: 96 perfusable blood vessels to study vascular permeability in vitro. Sci Rep 2017, 7: Mimetas BV Leiden,

26 Next step: Vascularized tissues Nucblue Actingreen RFP VE-Cadherin Mimetas BV Leiden,

27 Conclusions Organ-on-a-chip is ready to use for safety assessment Kidney validation study has been completed, results will be published soon Assays for: Permeance, Transport and Toxicity assessment Long-term and repeated dosing is possible Low adsorption, non-pdms material Excellent inter-plate and inter-lab reproducibility Mimetas BV Leiden,

Organ-on-a-Chip Workshop 2-day hands-on experience Establish 3D cell cultures Take stunning high-content images Leiden (The Netherlands) or Gaithersburg, MD (USA) Free

28 Organ-on-a-Chip Workshop 2-day hands-on experience Establish 3D cell cultures Take stunning high-content images Leiden (The Netherlands) or Gaithersburg, MD (USA) Free lunch included Get 2 OrganoPlates for free! Sign-up: Perfectly organized and executed The hands-on sessions are great! Mimetas BV Leiden, 2018

29 Acknowledgements Mimetas BV Leiden,

30 Remko van Vught JH Oortweg CH Leiden The Netherlands

Genetically Modified Human Renal Proximal Tubule Epithelial Cells (RPTEC/TERT1) A New Model for Drug Toxicity Studies

Genetically Modified Human Renal Proximal Tubule Epithelial Cells (RPTEC/TERT1) A New Model for Drug Toxicity Studies Chaozhong Zou, Ph.D. Senior Scientist, ATCC About ATCC Founded in 1925, ATCC is a non-profit