CAI Weixiang,FANG Jianfeng,ZHUANG Wei, et al. Isolation, identification and cultivation of hepatocytes, hepatic stellate cells and Kupffer cells from rats[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2016, 46(9): 638-643,648.
Abstract：Objective: To establish a protocol for isolation, identification and cultivation of hepatocytes (HC), hepatic stellate cells (HSC) and Kupffer cells (KC) from rats. Methods: By combining in situ perfusion with collagenase, density gradient centrifugation and selective adhension, primary HC, HSC and KC were obtained. Cell yield and viability were assessed by automated cell counter. The purity of these cells were detected with immunofluorescent staining, special staining and phagocytosis assay. Results: The average yield, viability and purity of isolated HC were (21.0±8.2)×106/per gram liver, 92.1%±2.1%, and 96.5%±2.2%, respectively. The average yield, viability and purity of isolated HSC were (2.5±0.8)×106/per gram liver, 90.1%±3.8%, and 93.1%±1.5%, respectively. The average yield, viability and purity of isolated KC were (4.1±1.2)×106/per gram liver, 90.1%±3.8%, and 95.1%±1.4%, respectively. The isolated cells could be cultured in vitro for long-term. HSC underwent a gradual phenotypic transition to a myofibroblast-like phenotype with vitamin A losing in vitro culture. KC could proliferate, and had the ability of phagocytosis, and the expression of CD 68 at 14 d of culture. Conclusion: The simultaneous method is feasible to isolate and culture primary rat HC, HSC, and KC.
