Abstract: Objective To establish a method for simultaneous isolation and primary culture of rat hepatocytes, hepatic stellate
cells, Kupffer’s cells and hepatic sinus endothelial cells. Methods By combining in situ perfusion, in vitro perfusion, density
gradient centrifugation and differential adhension, primary rat hepatocytes, hepatic stellate cells, Kupffer’s cells and hepatic
sinus endothelial cells were obtained. The purity of these cells were assessed with morphological observation,
immunofluorescent staining and ink phagocytosis assay. Results We successfully obtained the 4 primary cells simultaneously
by combining in situ perfusion with in vitro perfusion, density gradient centrifugation, and differential attachment. The cell
yield rate, cell viability and purity all met requirements for the subsequent cell experiment. Conclusion The combined cell
isolation and culture method is feasible to isolate primary rat hepatocytes, hepatic stellate cells, Kupffer’s cells and hepatic
sinus endothelial cells simultaneously.