Isolation and Culture of Hepatic Stellate Cells

C57BL/6 mice 8-12 week old were from Charles River. All animals received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals”. Hepatic stellate cells (HSC) were isolated from C57BL/6 mice by perfusion with collagenase-pronase as described (29 (link)) with small modifications. HSC were separated from parenchymal cells by 60×g centrifugation, collecting the supernatant for centrifugation at 450×g for 10min. Pellet or non-parenchymal cells were resuspended and purified over a 17.2% Hystodenz density gradient by centrifugation. The cloudy strip was collected and the HSC were cleaned with Krebs-Henseleit buffer by centrifugation of 450×g for 10 minutes. Cells were cultured in DMEM complemented with 10% FBS, and antibiotics at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Culture purity was assessed by retinoid autofluorescence. Mouse HSC were not passaged and were used from day-2 to day-10.