Exploration and Application of a Liver-on-Chip Device in Combination with Modelling and Simulation for Quantitative Metabolism Studies

Microphysiological systems (MPS) are complex and more physiologically realistic cellular in vitro tools that aim to provide more relevant human in vitro data for quantitative prediction of clinical pharmacokinetics while also reducing the need for animal testing. The PhysioMimix liver-on-a-chip integrates medium flow with hepatocyte culture and has the potential to be adopted for in vitro studies investigating the hepatic disposition characteristics of drug candidates. The current study focusses on liver-on-a-chip system exploration for multiple drug metabolism applications. Characterization of cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT) and aldehyde oxidase (AO) activities was performed using 15 drugs and in vitro to in vivo extrapolation (IVIVE) was assessed for 12 of them. Next, the utility of the liver-on-a-chip for estimation of the fraction metabolized (fm) via specific biotransformation pathways of quinidine and diclofenac was established. Finally, the metabolite identification opportunities were also explored using efavirenz as an example drug with complex primary and secondary metabolism involving a combination of CYP, UGT and sulfotransferase enzymes. A key aspect of these investigations was the application of mathematical modelling for improved parameter calculation. Such approaches will be required for quantitative assessment of metabolism and/or transporter processes in systems where medium flow and system compartments result in non-homogeneous drug concentrations. In particular, modelling was used to explore the effect of evaporation from the medium and it was found that the intrinsic clearance (CLint) might be underestimated by up to 40% for low clearance compounds if evaporation is not accounted for. Modelling of liver-on-a-chip in vitro data also enhanced the approach to fm estimation allowing objective assessment of metabolism models of different complexity. The resultant diclofenac fm,UGT of 0.64 was highly comparable with values reported previously in the literature. The current study demonstrates the integration of mathematical modelling with experimental liver-on-a-chip studies and illustrates how this approach supports generation of high quality of data from complex in vitro cellular systems.

At Day -5, the LC-12 plate was primed with plating medium to avoid dry spots in the channels and to acclimatize the media and the components of the system to the incubator temperature. The LC-12 plates and the PhysioMimix TM MPS Drivers were first wiped with 70% ethanol and afterwards combined. The plating medium was pre-warmed to 37°C and was added (500 µL) to the reservoir side of the reservoir chamber (see Figure S1). The drivers were slid into the PhysioMimix TM Docking Station to run the "Prime" Program, which induces an up-flow of 2.5 µL/s for 3 minutes to the medium in the wells. After this step, the wells were filled with 1100 µL to cover the whole surface of the well with medium. The plate was returned to the docking station to run the "Incubate'' program, which induces an up-flow of 2.5 µL/s to medium in the wells until the seeding at Day -4.

Media Exchange
The plates were removed from the incubator and the medium in the wells was aspirated until the remaining dead volume of 200 µL. Then, 400 µL of pre-warmed (37 °C) maintenance medium was added to the wells and the LC-12 plates were returned to the docking station to run the "Media Exchange" program, which induces a down-flow of 1.0 µL/s for 3 minutes. After the 3 minutes, the plates were removed from the docking station and the medium was again aspirated until the remaining dead volume of 200 µL. Then, 1400 µL of plating medium was added to each well and plates were returned to the docking station to run the "Incubate" program.

Seeding of the Cells
At Day -4, the hepatocytes were seeded on the scaffold of the wells in the LC-12 plate. For the process, the cryopreserved hepatocyte recovery medium (CHRM) and plating medium were pre-warmed to 37 °C in the water bath. Hepatocytes vial(s) were thawed, and the cells were transferred and suspended into 50 mL of CHRM. The cell suspension was centrifuged at room temperature at 100G for 10 minutes, and afterwards, the supernatant was carefully aspirated. Hepatocyte pellet was loosened by gently tapping the falcon tube and re-suspended in 3.0 mL (for two vials of hepatocytes) of plating medium. 50 µL of cell suspension was transferred to 0.1% Trypan Blue for the cell count with the hemocytometer.
The primed plates were removed from the incubator and the medium in the wells was removed as described in the media exchange program, with the difference that the medium added in the last step was not 1400 µL maintenance medium, but 300 µL plating medium in order to prepare the plates for the seeding process. The prepared hepatocyte suspension was now equally distributed to the different scaffolds (100 µL for each well). Note: Important to ensure a well-mixed hepatocyte suspension and to disperse the hepatocytes over the whole scaffold to avoid inter-well variability. Afterwards, the plates were returned to the docking station to run the "Seed" program, which induces a down-flow of 1.0 µL/s for 2 minutes. The plates were removed and 1000 µL of plating medium was very slowly added to the wells to cover the surface of the wells. Finally, plates were returned to the docking station to run the remaining "Seed" program that runs for another 7 hours and 58 minutes and automatically changes to the "Incubate" program afterwards.

Lysis of the Hepatocytes in the Scaffolds of Control Wells
The scaffolds in the control wells were removed, washed twice in 1000 µL PBS and subsequently placed into 500 µL PBS containing 1% Triton-X. The surface of the scaffold was then thoroughly scratched with a pipette tip to ensure maximal retrieval of contained cells, and the lysing process was continued for half an hour. This process was repeated twice to ensure complete detachment and lysis of the cells.   Area absolut The sampling volume was reduced for metabolically stable compound in order to reduce the impact of the volume depletion due to sampling. For the compounds incubated for 96 h (posaconazole, efavirenz, tolbutamide, irnotecan, and ketoprofen) the initial volume was 1818 µL, whereas 1800 µL was the initial volume for all other compounds.  As additional information about the media evaporation, the kev of ketoprofen was 0.12 µL/min for all three wells used as triplicates. Table S5. Compound with the respective source of data for the calculation of the unbound fraction in the medium. The fuinc,pred was also reported and it was calculated as described in the main manuscript.