Hucpg

Cryopreserved primary human hepatocytes (Lonza #HUCPG) were cultured according to the supplier’s instructions. Hepatocytes were thawed at 37°C and placed in warmed thawing medium (Lonza # MCHT50P), resuspended in Hepatocyte Plating Medium (Lonza #MP100), plated in collagen-coated 96 well plates at 50,000 cells/well, and cultured at 37°C and 5% CO₂. Culture wells were precoated with 140 μg/ml Collagen I solution (Roche #11179179001) for 2 hrs at 37°C. The initial plating medium was exchanged for fresh plating medium after 1h. Plating media was then exchanged again for Hepatocyte Culture Medium (Lonza #CC-4182) after 24 h, and then daily for the next 3 days. Once primary human hepatocyte monolayers were established, 10% pooled human serum from pregnant or non-pregnant donors was added together with EdU (Click-iT^™ EdU Proliferation Assay for Microplates, ThermoFisher Scientific) and cultured for one week. At takedown, EdU was detected following the manufacturer’s protocol and fluorescence was measured with a microplate reader (Molecular Dives Spectramax M5) at 568/585 nm.