Culture of Cryopreserved Human Hepatocytes.

Various lots of human cryopreserved hepatocytes (Supplemental Table 4) were obtained from different commercial vendors, including CellzDirect (Durham, NC), Bioreclamations In Vitro Technologies (Baltimore, MD), and XenoTech LLC, (Kansas City, KS). As detailed in previous publications (Fahmi et al., 2010; Ramsden et al., 2015), cryopreserved human hepatocytes (Supplemental Tables 1 and 4) were thawed in hepatocyte thawing medium and were seeded in collagen I–coated 24- or 96-well plates at cell densities of 0.5 to 1 × 10⁶ viable cells per well in hepatocyte plating medium. Viability, as determined by trypan blue exclusion or other methods, was 85% or better when cells were plated. The cells were initially maintained at 37°C in a humidified incubator with 95% atmospheric air and 5% CO₂ overnight in hepatocyte incubation media. After overnight incubation, the cells were treated with various compounds. Compounds were dissolved in dimethylsulfoxide (DMSO) and added to the culture medium at various concentrations (final DMSO concentration, 0.1%). After 2 days of daily treatment, the medium was removed and the cells were washed with saline. The cells were lysed in lysis buffer and prepared for RNA isolation. Cell viability was assessed by visual inspection of the monolayer, checking for confluence and morphology. Different companies used different plating conditions and a representation of the conditions is shown in Supplemental Table 1.