一种改良的胶原酶灌流高效分离肝细胞技术的建立

Establishment of an improved collagenase perfusion technique to isolate the hepatocyte effectively

目的探讨改良、简化肝细胞灌注、分离的方法,以提高细胞产量,降低制作成本。方法建立一种改良胶原酶灌注分离技术,改变灌注方式,分离的小鼠肝细胞采用Percoll离心,台盼蓝染色检测成活率,比较胶原酶剪切消化法和和改良方法在细胞产量、成活率的差异。结果采用改良的分离方法可获得大量的肝细胞,细胞产量为(1.74±0.35)×107/L,细胞成活率为90.1%±1.9%;而对照组胶原酶剪切消化法的细胞产量为(0.44±0.06)×107/L,细胞成活率为35.3%±1.4%,P<0.01,两组差异有统计学意义。结论该方法要求设备简单、容易操作、细胞产量及成活率较高。

Objective To establish an efficient,effective hepatocyte isolation technique in order to increase cell production and decrease the prime cost.Methods An improved collagenase perfusion isolation technique was established to isolate mouse hepatocytes by a two-step collagenase perfusion in situ.Hepatocytes were purified by centrifugation with Percoll,the hepatocytes yields and viabilities were measured.The difference of hepatocyte yields and viability were compared between the collagen slicing digestion group and the improved technique group.Results We establish an improved collagenase perfusion isolation technique.The isolation protocol used result in a high cell yield of（1.74±0.35）×107/L and the viability is 90.1%±1.9%.The yield of isolated hepatocyte using collagenase slicing digestion technique is（0.44±0.06） ×107/L and the viability is 35.3%±1.4%,P〈0.01.Conclusion The improved mouse hepatocyte isolation technique is simple and easy,the yield and viability of isolated hepatocyte is high.