Objective:To explore an efficient and stable method for isolating primary mouse liver cells and extend the time of in vitro culture.Methods:Adult male C57BL/6 mices were selected,and the liver was fully perfused with D-Hanks perfusion solution which containing dual antibodies through the hepatic portal vein.Then,0.2 mg/mL type Ⅳ collagenase was perfused to digest the liver,followed by filtration,centrifugation,and washing to obtain the primary liver cells of the mice.Trypan blue staining was used to test the viability of mouse primary liver cells.The morphological changes of liver cells were observed using an inverted microscope,cell purity was i-dentified using glycogen staining,and cytokines(ITS-X and HGF)were added to prolong the in vitro culture time of mouse primary liver cells.Results:Approximately 2*107 primary liver cells can be extracted from average per adult mouse liver.Trypan blue staining showed cell viability exceeding 95％,and glycogen staining results showed that the purity of primary liver cells obtained by this method was higher than 95％.The addition of HGF and ITS-X cytokines can effectively prolong the in vitro maintenance time of liver cell morphology.Conclusion:Based on the original two-step perfusion separation and culture method of mouse primary liver cells,an efficient and stable method for isolating and cultivating mouse primary liver cells was optimized and successfully estab-lished,and providing technical support for in vitro liver research.
维普
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