Hepatocytes, prepared from normal adult rat liver, were seeded onto a collagen substratum and cultured alone or in the presence of rat liver endothelial cells. When hepatocytes were cultured alone in a hormonally defined serum-free medium, decreased albumin production and rapid morphological deterioration of bile canaliculi structures and gap junctions occurred within 4 to 5 days. In contrast, hepatocytes cocultured with liver mesenchymal cells remained morphologically intact and biochemically functional for at least 4 weeks. They reorganized into small islands, continued to secrete high levels of albumin, did not express alpha-fetoprotein (a fetal marker), and remained strongly dye coupled. All of the hepatocytes synthesized albumin and retained their gap junctional channels. No junctional communication was observed between hepatocytes and endothelial cells. Long fibers containing fibronectin, Type I collagen and laminin distributed over the hepatocytes were induced in coculture but never appeared in hepatocytes cultured alone. Moreover, supplementation of the hormonally defined medium with phenobarbital and dimethyl sulfoxide, both of which improve the life span and functional activities of cultured hepatocytes, failed to induce reticulin fiber formation in pure culture of hepatocytes. The modulation of albumin secretion, biomatrix deposition and junctional communication observed in hepatocytes cultured with sinusoidal liver cells was also obtained when hepatocytes were in association with various epithelial or mesenchymal cells [rat liver epithelial cells (T51B), mouse embryonic fibroblasts (NIH 3T3), human or rat dermal fibroblasts and bovine aorta endothelial cells (AG 4762)].
Sinusoidal cells isolated from adult rat liver have been established in primary culture and in cell line. The presence of factor VIII R:Ag and peroxidatic/phagocytosis activities were the criteria used to distinguish in freshly isolated cells the endothelial cells from the Kupffer cells and suggested the endothelial origin of the cell line. Using a co-culture system, the effect of sinusoidal liver cells on hepatocyte functional activity was characterized. A plateau in which the state of differentiation was stabilized could be generated for co-cultured hepatocytes isolated from adult rat and a disappearance of the initial expression of alpha 1-fetoprotein (AFP) and the increase and/or maintenance of albumin secretion were measured with co-cultured hepatocytes isolated from suckling rat. The presence of dexamethasone was required for such beneficial effect. The hepatocyte-stabilizing activity was also produced by a pulmonary endothelial cell line.
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