Isolation, identification and cultivation of primary rat hepatic stellate cells
- VernacularTitle:大鼠原代肝星状细胞分离、鉴定及培养方法的研究
- Author:
Yu ZHANG
1
;
Xi ZHOU
;
Long YI
;
Xin MA
;
Qian-Yong ZHANG
;
Man-Tian MI
Author Information
- Keywords: hepatic stellate cell; cell isolation; primary culture; cell identification
- From: Journal of Regional Anatomy and Operative Surgery 2018;27(1):5-11
- CountryChina
- Language:Chinese
- Abstract: Objeetive To improve the method for the isolation and purification of rat hepatic stellate(HSC) cells and to provide a stable cell source for the research on liver-related diseases.Methods Rat liver was digested in situ by a two-step infusion assay under a strict control of the infusion temperature,flow rate and time with a combined utilization of Pronase E and Collagenase Ⅳ.And then,the HSC cells were separated by Percoll density gradient centrifugation.The cell growth curve and survival rate were measured by CCK-8 and trypan blue staining,respectively.The HSC cells were identified by flow cytometry and immunofluorescence cytochemistry.Results With the improved methods,there were (2.1 ± 0.2) × 107 HSC cells isolated from one rat and the survival rate was (96.2 ± 0.8) ％.The percentage of HSC cells with a spontaneous fluorescent characteristic from the isolated cells was 96.3％.The immunofluorescence cytochemistry was used to detect the expressions of the surface antigens α-SMA and Desmin in the isolated HSC cells.Conclusion By strict control of infusion temperature,flow rate and perfusion time as well as the combined application of Pronase E and Collagenase Ⅳ,there is an increased harvest of HSC cells with improved cell viability and purity,which is helpful for further research on HSC cells.
