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目的建立猪肝细胞与骨髓间充质干细胞(MSCS)最适宜共培养体系,为生物人工肝的构建提供理想的细胞来源。方法抽取中华实验猪(n=3)髂前上棘骨髓,采用密度梯度离心法分离单个核细胞,贴壁传代培养至第3代;采用原位两步胶原酶法分离猪肝细胞后与MSCS按1:1,2:1,5:1和10:1的比例混合培养,观察各组共培养肝细胞形态和功能的变化水平。结果所获肝细胞纯度>99%,存活率>95%。共培养组肝细胞迅速黏附于MSCS表面,在三维空间呈球形聚集生长;肝细胞与MSCS间出现细胞连接,超微结构与正常肝细胞接近。肝细胞/MSCS以2:1共培养组的肝细胞清蛋白分泌水平、尿素合成能力和细胞色素P450为各组中之最佳,自第1天培养起均显著高于对照组(P<0.05),并在第2天达到高峰,而且下降趋势较缓慢。结论猪肝细胞/MSCS按2:1组成的最适共培养体系可最大程度地维持肝细胞功能,为构建功能性生物人工肝提供高效细胞材料。
Objective To establish the most suitable co-culture system for porcine hepatocytes and mesenchymal stem cells (MSCs), and provide an ideal cell source for the construction of bioartificial liver. Methods Bone marrow from the anterior superior iliac spine of experimental pigs (n = 3) was collected. The mononuclear cells were isolated by density gradient centrifugation and subcultured to the third passage. The in situ two-step collagenase method was used to isolate porcine hepatocytes, According to the ratio of 1: 1, 2: 1, 5: 1 and 10: 1, the changes of morphology and function of hepatocytes in each group were observed. Results The purity of hepatocytes obtained was> 99% and the survival rate was> 95%. Co-culture group of liver cells adhered to the surface of MSCS rapidly in three-dimensional spherical accumulation of growth; hepatocytes and MSCS cell connection between the ultrastructure and normal liver cells close. The hepatocyte albumin level, urea synthesis ability and cytochrome P450 in hepatocyte / MSCS co-culture group at 2: 1 were the best among all groups, which were significantly higher than that of the control group (P <0.05 ) And peaked on the second day with a slower downward trend. Conclusion The optimized 2: 1 co-culture system of porcine hepatocytes / MSCS can maintain the function of hepatocytes to the maximum and provide a highly efficient cell material for the construction of functional bioartificial liver.