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.