猪肝细胞与骨髓间充质干细胞最适共培养体系的建立

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。

Establishment of an optimal co-cultivation of porcine primary hepatocytes and bone marrow mesenchymal stem cells in vitro

ObjectiveTo establish an optimal co-cultivation of porcine hepatocytes with bone marrow mesenchymal stem cells (MSCS) in vitro for finding the ideal cell source of bioartificial liver.MethodsMononuclear cells were isolated from bone marrow,which aspirated from the anterior superior iliac spine of swines (n=3) and isolated by density gradient centrifugation. A randomly distributed co-culture system composed of porcine hepatocytes harvested by a two-step in situ collagenase perfusion technique and MSCS of passage 3 generated. The morphological and functional character of varying group of co-cultured hepatocytes were observed.ResultsThe purity of primary hepatocytes was more than 99%. Hepatocyte viability was greater than 95%. A rapid attachment and self-organization of three-dimensional hepatocyte spheroids were encouraged in co-culture. Heterotypic junctions remained similar to that of hepatocytes in vivo. The maximal induction of albumin production, urea synthesis, and cytochrome P4503A1 activities was achieved at seeding ratio of 2∶1 (P<0.05). The best hepatic function levels were achieved on day 2 and moderately decreased in the following co-culture days (P<0.05).ConclusionsCo-cultivation of porcine hepatocytes and MSCS at a seeding ratio of 2∶1 can preserve hepatocyte morphology and functions, and could contribute highly potent cells to the functional bioartificial liver.