An improved method of isolating rat hepatic stellate cells

Objective To improve the method of isolating rat hepatic stellate cells(HSCs).Methods Rat hepatic stellate cells were isolated for several times with multiple density centrifugation within 1.040～1.060 g/mL after enzyme perfusion of liver.The cell viability was determined by Trypan blue exclution staining. The purity of HSCs was identified by retinoid antofluorescence and immunostaining by Desmin.The phenotype of HSCs was evaluated by characteristic immunofluorescence using antibodies specific for Desmin and α-SMA.Results About(3～5)×107 HSCs were harvested from each rat through multiple density centrifugation within 1.040～1.060 g/mL.The cell viability was more than 95% determined by the trypan blue exclusion method.The purity of freshly isolated HSCs and the activated HSCs were more than 90% and almost 100% respectively assessed by retinoid antofluorescence and immunostaining cells using Desmin antibody.Conclusion The method of multiple density centrifugation greatly increased the production of HSCs by reducing loss of cells due to error of density preparation and their relative heterogeneity of density during isolating HSCs.This method is more convenient,efficient and reliable.