Hepatocyte Isolation and Treatment Protocols

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.