1. Microfluidics Enables Small-Scale Tissue-Based Metabolism Studies with Scarce Human Tissue
Paul van Midwoud, Marjolijn T. Merema,
Geny M.M. Groothuis, Elisabeth Verpoorte
Pharmacokinetics, Toxicology and Targeting & Pharmaceutical Analysis
Department of Pharmacy

2. Prediction of metabolism and toxicity in man:
in vitro - in vivo extrapolation
animal in vivo
man in vivo
animal in vitro
man in vitro
interspecies differences

3. Introduction Intrinsic problems with current in vitro systems
No cell-cell interactions
Single organ: no interorgan interactions
Accumulation of waste products in static environment
In vitro systems are needed which better approach the in vivo situation

4. Approach Precision-cut organ slices
Intact architecture: all cell types
and enzymes are still present in a
physiological matrix
Combine slices with microfluidic techniques
Slices integrated in microchambers
Microchambers continuously perfused
Sequentially connect microchambers containing slices from different organs for interorgan studies
3-8mm µm

5. Preparation of precision-cut liver slices
Well plates => well validated system extra slide
De Graaf et al, Nature Protoc. 2010, 5(9),

6. Well-validated system
Volume: 1.3 mL
Medium exchange once every 24h
Accumulation of metabolites and
waste products
Well-validated system

7. Well-validated system Microfluidic system
Volume: 1.3 mL
Medium exchange once every 24h
Accumulation of metabolites and
waste products
Volume: 25 µL
Flow-through device
Continuous supply of nutrients
and oxygen
Continuous removal of waste
products
Possibilitiy to connect chambers
containing slices from different/same
organ(s)
Well-validated system
Microfluidic system

8. Liver slices in a microfluidic device
12/6/2018
| 8
Volume chamber 25 µL
P.M. van Midwoud, G.M.M. Groothuis, M.T. Merema, E. Verpoorte, Biotechnol Bioeng. 2010, 105(1),

9. Characterization of the Biochip
In-line measurements demonstrated constant pH and dissolved oxygen concentrations over time
Leakage of the enzyme, lactate dehydrogenase (LDH), was low, which indicated high viability up to 72 h
Phase I and II metabolism in rat liver slices were measured using 7-ethoxycoumarin
Constant substrate delivery
Constant metabolite production

10. 1 or 2 rat liver slices sequentially perifused
7-ethoxycoumarin (500 µM)
7-hydroxycoumarin (HC)
7-hydroxycoumarin glucuronide (HC-G)
(HC)
7-hydroxycoumarin sulfate (HC-S)
Mean ± SEM (n=3-4)
P.M. van Midwoud, M.T. Merema, E. Verpoorte, G.M.M. Groothuis, Lab Chip. 2010, 10(20),

11. Rat intestinal metabolism
Same metabolic rate in well plates and biochip
7-ethoxycoumarin
7-hydroxycoumarin
Mean ± SEM (n=3)
Mean ± SEM (n=3)
P.M. van Midwoud, M.T. Merema, E. Verpoorte, G.M.M. Groothuis, Lab Chip. 2010, 10(20),

12. Interorgan effects: intestine and liver
Well plates: co-incubation of two slices in the same well
Liver metabolites can influence intestinal metabolism and vice versa
Biochip: Sequential perifusion of intestinal slice and liver slice
Intestinal metabolites can influence liver metabolism, but liver metabolites cannot influence intestinal metabolism

13. Interorgan interactions on chip
P.M. van Midwoud, M.T. Merema, E. Verpoorte, G.M.M. Groothuis, Lab Chip. 2010, 10(20),

14. Intestinal-liver biochip
FGF15
protein
Chenodeoxycholic acid
(CDCA)
Activation FXR
mRNA FGF15
CYP7A1
CDCA
Ileum
Liver
P.M. van Midwoud, M.T. Merema, E. Verpoorte, G.M.M. Groothuis, Lab Chip. 2010, 10(20),

15. Best model for man is man precision-cut liver slices prepared from human tissue are a good model to represent human metabolism

16. Metabolism in human liver slices (n=3)

17. On-line analysis system for metabolism studies
Three chambers
Medium outflow directly to HPLC :
on-line measurement of metabolite formation
Advantages:
Direct result / high speed
Information about time-dependent changes in slice viability and function
Ability to detect unstable metabolites
Possibility for on-line induction/inhibition studies

18. On-line metabolism studies
Pump
control
Waste
Pump (100 nL/min)
Pump (100 nL/min)
drug
Pump (medium 10 µL/min)

19. On-line metabolism studies
Pump
control
Waste
Pump (100 nL/min)
Pump (100 nL/min)
drug
Pump (medium 10 µL/min)

20. On-line metabolism studies
Pump
control
Waste
Pump (100 nL/min)
Pump (100 nL/min)
drug
Pump (medium 10 µL/min)

21. On-line metabolism studies
Pump
control
Waste
Pump (100 nL/min)
Pump (100 nL/min)
drug
Pump (medium 10 µL/min)

22. On-line metabolism studies
Pump
control
Waste
Pump (100 nL/min)
Pump (100 nL/min)
drug
Pump (medium 10 µL/min)

23. On-line metabolism studies
Pump
control
Waste
Pump (100 nL/min)
Pump (100 nL/min)
drug
Pump (medium 10 µL/min)

24. On-line metabolism studies
Pump
Waste
Waste
Waste
Peristaltic pump

25. Liver metabolism measured on-line (500 µM 7-HC)
7-HC-S
7-HC-G
mean±std
3 slices measured simultaneously
P.M. van Midwoud et al, Anal. Chem. 2011, 83(1), 25

26. On-line analysis of diclofenac and its metabolites
Chemically
unstable
Reactive
metabolite

27. On-line analysis of unstable metabolites
Diclofenac acyl glucuronide is not stable
Well plates
4’OH-Dic
5OH-Dic
Acyl-G-Dic
Biochip
4’OH-Dic
5OH-Dic
Acyl-G-Dic
P.M. van Midwoud et al, Anal. Chem. 2011, 83(1),

28. Conclusions
Biochip designed allowing constant flow, O2 delivery, pH and temperature
Use of microfluidics makes more sophisticated experimentation possible
Interorgan effects can be measured
First on-line analysis system for tissue slices
Monitoring of time-dependent effects
Detection of unstable metabolites
On-line drug-drug interaction experiments
Reduction of the use of animals
Slices of human origin can be incorporated

29. Conclusions
Biochip designed allowing constant flow, O2 delivery, pH and temperature
Use of microfluidics makes more sophisticated experimentation possible
Interorgan effects can be measured
First on-line analysis system for tissue slices
Monitoring of time-dependent effects
Detection of unstable metabolites
On-line drug-drug interaction experiments
Reduction of the use of animals
Slices of human origin can be incorporated

30. Conclusions
Biochip designed allowing constant flow, O2 delivery, pH and temperature
Use of microfluidics makes more sophisticated experimentation possible
Interorgan effects can be measured
First on-line analysis system for tissue slices
Monitoring of time-dependent effects
Detection of unstable metabolites
On-line drug-drug interaction experiments
Reduction of the use of animals
Slices of human origin can be incorporated

31. Conclusions
Biochip designed allowing constant flow, O2 delivery, pH and temperature
Use of microfluidics makes more sophisticated experimentation possible
Interorgan effects can be measured
First on-line analysis system for tissue slices
Monitoring of time-dependent effects
Detection of unstable metabolites
On-line drug-drug interaction experiments
Reduction of the use of animals
Slices of human origin can be incorporated to better predict metabolism in man

32. Acknowledgement: Joost Janssen Funded by: Niek Verweij Arnout Janse
Department of Surgery
at the UMCG

33. Thank you for your attention