Department of Hepatobiliary Surgery, DrumTower Clinical Medical College of Nanjing Medical University, Nanjing, China.
Hepatol Res. 2009 Apr;39(4):398-407. doi: 10.1111/j.1872-034X.2008.00472.x. Epub 2008 Dec 19.
The application of porcine hepatocytes in liver support systems has been hampered by the short-term survival. Co-cultivation of hepatocytes with non-parenchymal cells may be beneficial for optimizing cell functions via heterotypic interactions. In this study, we present a new cultivation system of porcine hepatocytes and mesenchymal stem cells (MSCs) in a randomly distributed co-culture manner.
Mononuclear cells were isolated from bone marrow aspirate of swines (n = 3) by density gradient centrifugation. MSCs were characterized by flow cytometry with CD29, CD44, CD45 and CD90, respectively. Then freshly isolated hepatocytes were simultaneously inoculated with MSCs in a hepatocyte dominant manner. The morphological and functional changes of heterotypic interactions were characterized.
Ninety percent MSCs of passage 3 were positive for CD29, CD44 and CD90, but negative for CD45. A rapid attachment and self-organization of three-dimensional hepatocyte aggregates were encouraged. The cell ultrastructure indicating heterotypic junctions remained similar to that of hepatocytes in vivo. Fluorescence microscopy further verified that MSCs served as a feeder layer for hepatocyte aggregates. Hepatocyte performance levels such as albumin secretion, urea synthesis and CYP3A1 induction were all significantly enhanced in co-culture group compared with hepatocyte homo-culture (P < 0.05). The best hepatic function levels were achieved on day 2 and moderately decreased in the following co-culture days. Moreover, the cell cycle of hepatocytes manifested the same trend in parallel to the enhancement of hepatocyte functionality.
A three-dimensional co-culture system by porcine hepatocytes and bone marrow MSCs was for the first time established in vitro. Enhanced liver-specific functions make such a co-culture system a promising tool for tissue engineering, cell biology, and bioartificial liver devices.
猪肝细胞在肝脏支持系统中的应用受到短期存活的阻碍。与非实质细胞共培养可能通过异型相互作用有益于优化细胞功能。在这项研究中，我们提出了一种新的猪肝细胞和间充质干细胞（MSCs）的培养系统，以随机分布的共培养方式进行。
通过密度梯度离心从猪骨髓抽吸物中分离单核细胞（n=3）。通过流式细胞术分别用 CD29、CD44、CD45 和 CD90 对 MSCs 进行特征描述。然后，以肝细胞为主的方式同时将新鲜分离的肝细胞接种到 MSCs 中。对异型相互作用的形态和功能变化进行了特征描述。
第 3 代的 90%MSCs 对 CD29、CD44 和 CD90 呈阳性，但对 CD45 呈阴性。三维肝细胞聚集体的快速附着和自组织受到鼓励。细胞超微结构表明异型连接与体内的肝细胞相似。荧光显微镜进一步证实了 MSCs 作为肝细胞聚集体的饲养层。与肝细胞同培养相比，共培养组的白蛋白分泌、尿素合成和 CYP3A1 诱导等肝细胞性能水平均显著提高（P<0.05）。最佳的肝功能水平在第 2 天达到，并在随后的共培养天数中适度下降。此外，肝细胞的细胞周期与肝细胞功能的增强呈相同趋势。
首次在体外建立了猪肝细胞和骨髓间充质干细胞的三维共培养系统。增强的肝脏特异性功能使这种共培养系统成为组织工程、细胞生物学和生物人工肝脏设备的有前途的工具。
