Advanced method for isolation and culture of hepatic stellate cells from mice with different degrees of liver fibrosis

Abstract

Objective To establish an advanced method for the isolation and culture of hepatic stellate cells (HSCs) from mice with different stages of liver fibrosis. Methods HSCs were isolatedby collagenase perfusion in combination with in vitro digestion and density gradient centrifugation. The cell purity was determined by morphological observation and HSCs’ autofluorescence. The viability of HSCs was identified by trypan-blue stain. Immunofluorescent stain was used to assess the activation state of HSCs. Results The yield, purity and viability of HSCs isolated from normal mice are 1.14×106, 95.7% and 92.8% respectively while they are 1.85×106, 90.6% and 97.3% from mice with different stages of liver fibrosis. Cells from both normal and fibrotic liver glowed well and activated gradually in vitro culture. Conclusion A simple and efficient method based on density gradient centrifugation for isolation of hepatic stellate cells from mice with different stages of liver fibrosis has been developed in this study. Key words: Mouse; Hepatic stellate cells; In situ perfusion; Density gradient centrifugation; Primary culture