Morphology of sites of adhesion between hepatic stellate cells (vitamin A-storing cells) and a three-dimensional extracellular matrix.

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Hepatic stellate cells lie in the perisinusoidal space in a three-dimensionally distributed extracellular matrix (ECM). This three-dimensional structure of the ECM regulates the proliferation, morphology, and functions of the stellate cell. To investigate how the three-dimensional structure of ECM regulates behavior of the cells, we cultured stellate cells two- or three-dimensionally and examined the morphology of the cells in both cases as well as the localization of cell-surface adhesion molecules specific for the ECM. Isolated rat stellate cells and human stellate cells were cultured in Dulbecco's modified Eagle's medium. Rat stellate cells were cultured in non-coated polystyrene culture dishes, or on or in type I collagen gels. The morphology of cell-ECM adhesion was examined under transmission and scanning electron microscopes. Localization of integrin alpha2 and integrin beta1 in human stellate cells was examined by immunoelectron microscopy. Immunostaining was performed with a mouse monoclonal anti-human integrin alpha2 or integrin beta1 antibody and goat anti-mouse IgG coupled with 10-nm immunogold. Hepatic stellate cells cultured in polystyrene dishes spread well. However, the cells cultured on or in the type I collagen gel became slender. The cells extended long cellular processes onto or into the gel. The cellular processes were entangled three-dimensionally with the type I collagen fibers and directly adhered to these fibers. The cells inoculated in type I collagen gels formed a large number of adhesive structures that resembled focal adhesions. These adhesive structures were distributed not only on the lower side but also on the upper side of both the cell bodies and cellular processes. Moreover, each adhesive area formed a face but not a point. Integrin alpha2 and integrin beta1 were detected on the surfaces of cell bodies, cellular processes, and microprojections. The cells cultured in type I collagen gel develop a three-dimensional adhesive structure.