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 37C 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 widebore
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.

In vitro AAV transduction Twenty hours after seeding, plates were removed from the incubator, and medium was aspirated and re-plated with 600 mL fresh hepatocyte
basal medium (HBM). The concentration of AAV virus was calculated based on MOI, and the AAV virus was prepared in the HBM at 2 times the target concentration. Following this, 450 mL HBM was removed, and 150 mL AAV containing HBM was added to each well (volume was adjusted for mouse cells to achieve comparable
MOIs). After 24 h, medium was removed, and cells were washed with HBM twice before 500 mL HBM with supplements (Lonza, Basel, Switzerland) were added per well. Cell cultures were maintained by changing medium each day, and 6 days following transduction, cells were collected for measurements.