Isolation and Culture of Primary Human Hepatocytes

A published collagenase perfusion technique was employed for PHH isolation from liver wedges [13 (link)]. Briefly liver was digested and centrifuged to isolate PHH. These cells were resuspended and then plated on 24-well plates previously coated with rat tail collagen type 1 at a density of 3x10⁵ cells/well in Dulbecco’s Modified Eagle’s Medium (DMEM) (Catalogue number [CN]:41965–039; Gibco laboratories, Gaithersburg, MD, USA) supplemented with 10% fetal calf serum (FCS) (CN:10270106; Gibco) and 5% glutamine/penicillin/ streptomycin (GPS) (CN:10378016; Gibco). After 2 hours the cells were washed with Phosphate-buffered saline (PBS) (CN:10010023; Gibco) and the media changed to our standard medium for PHH culture, constituted of Arginine-/Glutamine-free Williams E (CN:12551032; Gibco) with 1% GPS, hydrocortisone (2μg/ml) (H4001; Sigma-Aldrich, St. Louis, MO., USA), insulin (0.124 U/ml) (I2643; Sigma-Aldrich) and L-ornithine (400μM) (O6503; Sigma-Aldrich), subsequently the cells were kept at 37°C in 95% air/ 5% CO₂.