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  • Title: Ultrastructural and functional differentiation of hepatocytes under long-term culture conditions.
    Author: Knop E, Bader A, Böker K, Pichlmayr R, Sewing KF.
    Journal: Anat Rec; 1995 Jul; 242(3):337-49. PubMed ID: 7573981.
    Abstract:
    BACKGROUND: Studies on hepatocytes grown in different culture systems have shown that these cells rapidly dedifferentiate on a single support with liquid medium on top (single gel technique). However, in systems sandwiching them between two layers of extracellular matrix (double gel technique), the cells are able to regain and maintain typical light microscopical appearance and function. Their ultrastructural morphology is as yet unknown. METHODS: Isolated, adult rat hepatocytes were grown in both systems, and their fine structure (thin section electron microscopy) and the functional ability of albumin production (immunoassay) were studied and compared in both culture systems after 2, 7, and 14 days. RESULTS: The hepatocytes in conventional single gel culture did not completely regain their normal morphology and rapidly underwent progressive dedifferentiation. This was characterized by loss of cell polarization in terms of obliteration of the bile canaliculi-like intercellular expansions, loss of cell membrane differentiations, and reduction of organelles. Cytoskeletal components gradually increased, building up large filamentous zones underneath the plasma membrane. In double gel culture, the hepatocytes reachieved and maintained intact morphology and polarity over at least 14 days. The bile canaliculi were formed, preserved, or even enlarged and were associated with dense peribiliary bodies and Golgi fields. The plasma membrane facing both collagen layers bore numerous cytoplasmic microprojections like the sinusoidal surfaces of the hepatocytes in situ. Cell organelles, glycogen particles, and lipid droplets were always present. CONCLUSIONS: The hepatocyte is a cell type in which ultrastructural and functional differentiation are strongly interdependent. For these cells, the morphological microenvironment (i.e. the bipolar position of the extracellular matrix) may be as important or even more decisive for maintenance of normal cell differentiation than modifications of the composition of the matrix itself or addition of other cell types, as focused in other studies.
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