Studies on Differentiated Phenotypes of Rat Hepatocytes in Spheroid Culture

In order to examine the expression of differentiated phenotypes of hepatocytes in spheroid culture, we established a primary culture of rat hepatocytes on P-N-p-vinylbenzyl-Dlactonamide （PVLA） -coated dishes and observed changes in cytoskeletal organization and intercellular junctions with use of fluorescent microscope and con focal laser scanning microscope （LSM）. Rat hepatocytes on PVLA-coated dishes formed spheroids in serum-free medium containing insulin, transferrin, dexamethasone and epidermal growth factor （EGF） by the 48th hr after plating. Thus, the culture conditions resulted in the hepatocytes forming spacial organoids nearly identical to those in vivo. Fluorescent examination of spheroids by rhodamine-phalloidin revealed that the network of bile canalicular structures （BC） was reorganized in the spheroids. Alkaline phosphatase activity and bile canalicular membrane specific protein were demonstrated in the BC. It was also shown that the fluorescein Na added to the culture medium was secreted into the BC, indicating that the reconstructed BC is functional in the spheroids. Immunohistochemical examinations of the spheroids by desmoplakin antibody and α-tubulin antibody showed that desmosomes were present until 48 hrs after culture, and that the microtubules were localized mostly along the plasma membrane of each hepatocyte composing the spheroids. These results suggested that rat hepatocytes cultured on PVLA-coated dishes not only maintain their cuboidal shape, but also reorganize functional BC and maintain cell-cell junctions. Thus, this culture system, allowing cells to reorganize spacial cell-cell interaction, was shown to yield similar conditions to those of hepatocytes in vivo.departmental bulletin pape