原代大鼠肝细胞分离及培养鉴定

目的探讨原代大鼠肝细胞分离及培养的简易方法,并对其纯度及生物活性进行鉴定。方法采用改良原位两步非循环灌流法及多次过滤低速离心法分离原代大鼠肝细胞,常规DMEM高糖培养基培养原代大鼠肝细胞,锥虫蓝拒染法检测接种时肝细胞的存活率,倒置显微镜动态观察肝细胞形态变化。过碘酸-Schiff反应检测肝细胞糖原合成能力,并利用免疫细胞化学方法检测肝细胞角蛋白18(CK-18)的表达联合判断肝细胞纯度。结果每只大鼠可获取(1.38～1.74)×108个肝细胞,接种瞬时肝细胞活性>90%。倒置显微镜下观察肝细胞在接种后4 h基本完成贴壁,贴壁的细胞胞体变大变平,随后细胞相互靠拢呈岛状或条索状连接。糖原染色发现肝细胞内糖原被染成红色颗粒或片状。抗CK-18免疫细胞化学染色发现CK-18在肝细胞内均匀分布,被染成棕黄色。糖原染色及细胞CK-18联合鉴定肝细胞纯度达95%以上。结论用改良原位两步非循环灌注法及多次过滤低速离心法成功分离并培养原代大鼠肝细胞,可操作性强,所培养肝细胞数量、活力和纯度高,可在具备基本细胞培养条件的实验室推广。

Isolation, Culture and Identification of Hepatocytes of Primary Rat

Objective To investigate a simple and feasible method for primary rat hepatocytes isolation and culture,and to identify its purity and biological activity. Methods Primary SD rat hepatocytes were isolated by using a modified two-step in-situ non-circulating perfusion method followed by multiple filtration and low-speed centrifugation.The hepatocytes were cultured in Dulbecco′s modified Eagle′s medium with high level of glucose.The viabilities of cells evaluated by Trypan blue exclusion before cells were seeded.Cellular morphological changes were observed using an inverted microscope.Periodic acid Schiff(PAS) staining was performed to evaluate the status of hepatic glycogen storage,combining with the immunocytochemical staining targeting cytokeratin 18(CK-18) to identify the purity of hepatocytes. Results A total of(1.38-1.74)×108 hepatocytes per rat were obtained.The viabilities of hepatocytes were more than 90% when seeded.The hepatocytes with enlarged and flattened round shape were attached to the surface of collagen-coated dishes 4 hours after seeding and then connected with surrounding cells forming islands or cords.Glycogen preserved in hepatocytes was dyed purple using periodic-acid-Schiff staining.Through the PAS and anti-CK-18 immunocytochemical staining of hepatocytes,the purity of hepatocytes was more than 95%. Conclusions Primary rat hepatocytes can be successfully isolated and cultured using a modified two-step in-situ non-circulating perfusion method,which is highly feasible.The quantity,viability and purity of cultured hepatocytes were high.This method is available to perform in labs with basic cell culture conditions.