Hepatic stellate cells are one of the central cell types responsible for liver fibrosis following their activation into fibrogenic myofibroblast-like cells. When cultured on uncoated plastic, stellate cells undergo spontaneous activation that closely correlates with their response in vivo.
Bouyant and lipid-rich stellate cells were obtained from normal liver tissue by collagenase/pronase treatment followed by fractionation on a Nycodenz density gradient. The primary culture isolate was plated onto an uncoated plastic dish, trypsinised after three days, and subcultured. The cells were then passaged 3 more times in order to remove potential contaminating hepatocytes, endothelial cells, and Kupffer cells, which typically do not survive trypsinisation and subculturing.
Isolated stellate cells were cultured in M199 medium containing fetal bovine serum (10%) 24 hours later and every 3–4 days thereafter. When the cultures reached confluence, they were trypsinised (0.05% trypsin/0.53 mM EDTA) and passaged at a ratio of 1:3. Subsequent passages were performed every 7–10 days.