Liver cells isolated from newborn rats and seeded on a non-adherent plastic substratum were found to spontaneously re-aggregate and to form, within a few days, spheroidal aggregates that eventually reached a plateaued diameter of 150-175 ~,m. Analyses on frozen sections from these spheroids by immunofluorescence microscopy using antibodies to various cytoskeletal elements and extracellular matrix components revealed a sorting out and a histotypic reorganization of three major cell types. A first type consisted of cells that segragated out on the aggregate surface forming a monolayer cell lining; a second type was identified as hepatocytes that regrouped in small islands often defining a central lumen; and a third group of cells reorganized into bileduct-like structures. This intercellular organization in the aggregates was paralleled by the accumulation of extracellular matrix components (laminin, fibronectin, and collagen) and their deposition following a specific pattern around each cell population structure. Determinations of albumin secretion and tyrosine aminotransferase induction by dexamethasone and glucagon at various times after the initiation of the cultures revealed a maintenance of the hepatocyte-differentiated functions for at least up to 2 mo at the levels measured at 3-5 d. It is concluded that cells dispersed as single cells from newborn rat liver conserve in part the necessary information to reconstruct a proper threedimensional cyto-architecture and that the microenvironment so generated most likely represents a basic requirement for the optimal functioning of these differentiated cells.

The cell of origin of the nonparenchymal epithelioid cells that emerge in liver cell cultures is unknown. Cultures of rat hepatocytes and several types of nonparenchymal cells obtained by selective tissue dispersion procedures were typed with monoclonal antibodies to rat liver cytokeratin and vimentin, polyvalent antibodies to cow hoof cytokeratins and porcine lens vimentin, and monoclonal antibodies to surface membrane components of ductular oval cells and hepatocytes. Immunoblot analysis revealed that, in cultured rat liver nonparenchymal epithelial cells, the anti-rat hepatocyte cytokeratin antibody recognized a cytokeratin of relative mass (Mr) 55,000 and the anti-cow hoof cytokeratin antibody reacted with a cytokeratin of Mr 52,000, while the anti-vimentin antibodies detected vimentin in both cultured rat fibroblasts and nonparenchymal epithelial cells. Analyses on the specificity of anti-cytokeratin and anti-vimentin antibodies toward the various cellular structures of liver by double immunofluorescence staining of frozen tissue sections revealed unique reactivity patterns. For example, hepatocytes were only stained with anti-Mr 55,000 cytokeratin antibody, while the sinusoidal cells reacted only with the anti-vimentin antibodies. In contrast, epithelial cells of the bile ductular structures and mesothelial cells of the Glisson capsula reacted with all the anti-cytokeratin and anti-vimentin antibodies. It should be stressed, however, that the reaction of the anti-vimentin antibodies on bile ductular cells was weak. The same analysis on tissue sections using the anti-ductular oval cell antibody revealed that it reacted with bile duct structures but not with the Glisson capsula. The anti-hepatocyte antibody reacted only with the parenchymal cells. The differential reactivity of the anti-cytokeratin and anti-vimentin antibodies with the various liver cell compartments was confirmed in primary cultures of hepatocytes, sinusoidal cells, and bile ductular cells, indicating that the present panel of antibodies to intermediate filament constituants allowed a clear-cut distinction between cultured nonparenchymal epithelial cells, hepatocytes, and sinusoidal cells. Indirect immunofluorescence microscopy on nonfixed and paraformaldehyde-fixed cultured hepatocytes and bile ductular cells further confirmed that both anti-hepatocyte and anti-ductular oval cell antibodies recognized surface-exposed components on the respective cell types.(ABSTRACT TRUNCATED AT 400 WORDS)

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