Maintenance medium

Primary hepatocytes were purchased from Lonza (Basel, Switzerland). Hepatocyte culture was performed in Bio-Coat collagen I 24-well plates (Corning, Corning, NY, USA), and thawing medium, plating medium, and maintenance medium (Lonza, Basel, Switzerland) were used for hepatocyte cell culture. Cryopreserved hepatocytes were thawed in a water bath set to 37°C for less than 2 min. Once hepatocytes were almost completely thawed, the hepatocyte vial was wiped with 70% alcohol in the biosafety cabinet, and the cells were placed into the thawing medium using a wide-bore pipette tip to transfer. The thawing medium with the hepatocytes was inverted by hand prior to centrifugation. Human hepatocytes were centrifuged at 100 × g for 10 min, NHP cells at 100 × g for 6 min, and mouse hepatocytes at 70 × g for 5 min. Following centrifugation, the supernatant was aspirated, and cells were resuspended in 1 mL plating medium with a wide-bore pipette to achieve single-cell suspension. The suspension was mixed with 5-mL plating medium, and hepatocytes were diluted for ideal concentration for seeding (target concentration was approximately 1 million ± 20% cells/mL for humans and NHPs, 1 million ± 10% cells/well in a 24-well plate for dogs, and 0.45 million ± 10% cells/well in a 24-well plate for mice). Cells were evenly dispersed in the wells and then incubated.