Cytoskeleton and pericellular matrix organization of pure adult human keratinocytes cultured from suction-blister roof epidermis.
Pure adult human keratinocyte cultures were raised from suction-blister roof epidermis and cultured in MCDB-151 medium. In primary culture the epidermal cells rapidly adhered, spread and began to proliferate on collagen-coated growth substrata but not on uncoated plastic or glass substrata. A fibrillar keratin-specific fluorescence, showing a typical cell-cell arrangement, was seen in all cells in indirect immunofluorescence microscopy, whereas only some cells also showed vimentin-specific staining. A fine fibrillar fibronectin-specific surface staining was seen at the margin of attaching cells and in marginal cells of spreading cell islands, whereas no fluorescence could be seen in epidermal cells, with antibodies against type IV collagen or laminin. Interestingly, the marginal cells also showed intracellular fibronectin. The synthesis of fibronectin in epidermal cell cultures could also be revealed by metabolic labelling experiments with [35S]methionine. In contrast to primary cultures, subcultivated keratinocytes also adhered to uncoated plastic and glass substrata. After subcultivation, keratin and surface fibronectin distribution remained unaltered but after some subcultivations, most of the cells also showed fibrillar vimentin and expressed fibronectin intracellularly. The results show that the suction-blister method provides an easy way to obtain pure epidermal cell cultures without contaminating mesenchymal cells. Our results also suggest a direct role for fibronectin but not for collagen type IV or laminin in adhesion and spreading of epidermal cells in vitro.
D000293:Adolescent; D000328:Adult; D001768:Blister; D002448:Cell Adhesion; D002455:Cell Division; D002478:Cells, Cultured; D000078404:Epidermal Cells; D005353:Fibronectins / Q000032:analysis; D006801:Humans; D007381:Intermediate Filament Proteins / Q000032:analysis; D007633:Keratins / Q000032:analysis; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D010452:Peptide Biosynthesis; D012867:Skin / Q000166:cytology*; D014746:Vimentin
D005353:Fibronectins; D007381:Intermediate Filament Proteins; D014746:Vimentin; D007633:Keratins