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

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

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

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 100-300 µm

Preparation of precision-cut liver slices Well plates => well validated system extra slide De Graaf et al, Nature Protoc. 2010, 5(9), 1540-1551.

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

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

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), 184-94.

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

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), 2778-2786.

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), 2778-2786.

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

Interorgan interactions on chip P.M. van Midwoud, M.T. Merema, E. Verpoorte, G.M.M. Groothuis, Lab Chip. 2010, 10(20), 2778-2786.

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), 2778-2786.

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

Metabolism in human liver slices (n=3)

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

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

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

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

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

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

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

On-line metabolism studies Pump Waste Waste Waste Peristaltic pump

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), 84-91. 25

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

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), 84-91.

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

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

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

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

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

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