小鼠肝细胞分离技术及无血清培养方法的介绍

An Introduction of the Techniques of Mouse Hepatocytes Isolation and Serum-free Culture

本工作改良了小鼠肝细胞的分离技术，并建立了小鼠肝细胞的无血清培养方法。采用二步原位灌流法分离的小鼠肝细胞，其活率及产量分别为８６．９±１．０％和２．３±０．３×１０７，能够满足实验的要求。肝细胞在无血清培养条件下，２４小时可完全贴壁，在继续培养的２４～４８小时期间，漏入培养液中的ＧＰＴ和ＧＯＴ可稳定于低水平，贴壁细胞数量（以贴壁细胞ＤＮＡ含量为指标）与培养前的活细胞数基本相同并保持稳定，肝细胞形态正常，糖原合成良好，表明在无血清培养２４～４８小时期间，肝细胞的存活状态和功能状态都保持良好，为进行实验研究的最佳时机。

e improved the technique of mouse hepatocytes isolation and established the technique of serum-free culture. We used two step in situ circulatory perfusion me- thod to isolate hepatocytes. The cell viability(86.9±1.0%)and(2.3±0.3×107)could meet the demand of our expe iments.Having been cultured for 24h in serum-free media, the mouse hepatocytes attached completely. During the following 24 to48h,le akages of GPT and GOT kept at a low level. The amount of attached cells-(took DNA contents of attached cells aS the index)equlled basically to that ofthe viable cells before culture and kept stable. The morphology of the hepatocytes presented s pherical cubic shapes and the function of glycogen synthesis was good. All indi cated that the viable and functional state of hepatocytes kept well duringthe 24 to 48 h of serum-free primary culture.