一种小鼠原代肝细胞的分离与培养方法

Methodological Studies on the Isolation and Culture of Primary Mouse Liver Cells

目的:探索一种高效、稳定的小鼠原代肝细胞分离方法,并延长其体外培养的时间。方法:取成年雄性C57BL/6小鼠,以含双抗的D-Hanks灌流液经肝门静脉充分灌注肝脏,然后以0.2 mg/mL的Ⅳ型胶原酶灌注消化,接着过滤、离心、洗涤,获得小鼠原代肝细胞。通过台盼蓝染色检验小鼠原代肝细胞活率,利用倒置显微镜观察肝细胞的形态变化,糖原染色鉴定细胞纯度,添加ITS-X和HGF细胞因子延长小鼠原代肝细胞的体外培养时间。结果:平均每只成年小鼠肝脏可提取获得原代肝细胞约2×107个,台盼蓝染色显示细胞活率均超过95%,糖原染色结果表明此方法获得的原代肝细胞纯度高于95%,通过添加HGF和ITS-X两种细胞因子能够有效地延长肝细胞形态的体外维持时间。结论:本实验在原有小鼠原代肝细胞两步灌流分离法和培养的基础上,经过优化,成功建立了一种高效、稳定的小鼠原代肝细胞分离和培养方法,为肝脏的体外研究提供了技术保障。

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 identified 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 established,and providing technical support for in vitro liver research.