Mp100

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.

Mouse primary hepatocytes were prepared as previously described (12 (link)). Cells were seeded in 12- or 24-well plates (Corning BioCoat Cellware, Collagen Type I, 62405-607) and maintained in a complete culture medium (Williams’ Medium [GIBCO, 12551, contains 11 mM glucose] supplemented with 5% fetal bovine serum, 10 mM Hepes buffer [GIBCO, 15630-080], 2 mM L-glutamine [GIBCO, 25030-081], 1% antibiotic-antimycotic [GIBCO, 15240-062], 4 mg/L insulin [GIBCO, 12585-014], and 1 μM dexamethasone [Sigma, D4902]). Metformin and GSSG treatments were carried out in the complete culture medium. Cryoplateable primary human hepatocytes (MTOXH1002, lot 1914850-01, Caucasian female, 53 y old, BMI 38; MTOXH1000, lot 1912392-02, Caucasian male, 37 y old, BMI 39) were purchased from Sigma-Aldrich. Cells were thawed in thawing media (MCHT50, Lonza) and seeded in hepatocyte plating media (MP100, Lonza) in 12-well plates (Corning BioCoat Cellware, Collagen Type I, 62405-607). Metformin treatment and let-7a transfection were performed the next day and followed the same protocol for the mouse primary hepatocytes.

Cryopreserved human hepatocytes were recovered and cultured according to the protocol provided by Lonza. Briefly, cells were thawed and washed once with thawing medium (MCHT50, Lonza, Basel, Switzerland). Then the cells were resuspended in plating medium (MP100, Lonza, Basel, Switzerland), counted, and plated in 6-well collagen coated plates. The cells were cultured in a 37 °C/5% CO2 incubator. Four hours later, plating medium was replaced with maintenance medium, containing William’s E media supplemented with penicillin/streptomycin/fungizone (100 U/100 µg/0.25 µg per mL), 100 nM dexamethasone, 2 mM L-glutamine, 15 mM HEPES, and ITS (0.55 mg/mL human transferrin, 1 mg/mL bovine insulin and 0.5 µg/mL sodium selenite, from Sigma Aldrich, St. Louis, MO, USA). Eighteen hours after in culture, cells were treated with DMSO or 17-beta-estradiol (E2, 1 µM) for 8 h, this relatively high dosage was chosen because a previous report demonstrated that E2 is rapidly metabolized in hepatocytes and that they require a higher dosage than other cell lines [20 (link)]. The cells were then cross-linked for 15 min with 1% formaldehyde. Cells from the two donors were pooled and used for ChIP-Seq.