Establishment of a three-dimensional co-culture system by porcine hepatocytes and bone marrow mesenchymal stem cells in vitro

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.