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目的探讨体外原代培养大鼠肝细胞的分离方法。方法以Wistar大鼠做为肝细胞供体 ,分别采用直接分离法与胶原酶消化法分离获取肝细胞并进行原代培养 ;以台盼蓝染色法测细胞存活率 ,在位相差倒置显微镜下观察细胞形态变化 ,MTT法测培养细胞活性 ,并检测不同时期培养上清液中白蛋白的含量。结果直接分离法获取的肝细胞活性及功能欠佳 ,而胶原酶消化法所获取的肝细胞形态完整、贴壁良好、活性高、功能强。结论经胶原酶消化分离获取肝细胞的方法优于直接分离法。
Objective To study the isolation method of primary cultured rat hepatocytes in vitro. Methods Wistar rats were used as donor of hepatocytes. The hepatocytes were harvested by direct separation and collagenase digestion respectively. Cell viability was measured by trypan blue staining and observed under inverted phase contrast microscope The morphological changes of cells were observed. The activity of cultured cells was measured by MTT assay. The content of albumin in culture supernatant was detected at different time points. Results The activity and function of hepatocytes obtained by direct isolation method were poor, while the morphology of hepatocytes obtained by collagenase digestion method was intact, well attached, high activity and strong function. Conclusion The method of obtaining hepatocytes by collagenase digestion is better than the direct separation method.