Cc 4182

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.

Cell culture plates were coated with 0.5 mg/ml rat tail tendon collagen type Ⅰ (Solarbio, C8062, Beijing, China) overnight at 4°C and washed with PBS for three times before cell seeding. Murine hepatocytes were isolated from C57BL/6 N mice by a retrograde perfusion of liver with 30–40 ml HBSS (Gibco, 14175–095) containing 0.5 mM EGTA (Sigma, 03777), followed with about 40 ml low glucose DMEM containing 100U/mL collagenase type IV (Gibco, 17104–019). Hepatocytes were cultured in DMEM with 10% fetal bovine serum for 4 h for attachment. Then the medium was replaced with hepatocyte culture medium (LONZA, CC-3199 and CC-4182). The hepatocytes were treated with APAP (5 and 10 mM) in the absence or presence of CSA (50 μM) or chloroquine (20 μM) for 24 h. Cells were double stained with calcein and propidium iodide (Beyotime Biotech, C2015). After incubation at 37°C for 30 min, the fluorescence intensity was detected by Tecan’s Spark multimode reader to assess the percentage of cell death. For representative images, hepatocytes were stained with propidium iodide and Hoechst 33258 (10 μg/ml) for 30 min followed by fluorescence microscopy.