MORPHOLOGICAL AND BIOCHEMICAL ANALYSES ON FIBROBLASTS AND SELF-PRODUCED COLLAGENS IN A NOVEL 3-DIMENSIONAL CULTURE

The addition of L-ascorbic acid 2-phosphate (Asc 2-P), which is active and stable under a conventional culture condition, could render derma l fibroblasts to the organization of a dermis-like structure on a plas tic dish without any prior treatment. The cell layer was composed of m ultilayered fibroblasts surrounded by dense extracellular matrices. Co nfocal microscopic examination disclosed that the fibroblasts in the u pper layer were spindle-shaped and those in the lower layer were polyg onal. Electron microscopic examination revealed the accumulation of ma ture collagen fibrils in the intercellular space. These morphological observations suggest that the cell layer may resemble the dermis-like structure. Biochemical analyses revealed that the hydroxyproline conte nt of the cell layer increased in a time-dependent manner, while the m onolayer culture system without Asc 2-P yielded no measurable amount o f hydroxyproline. On sodium dodecylsulphate-polyacrylamide gel electro phoresis, neutral insoluble collagens extracted from the cell layer-sh owed the identical electrophoretic pattern to those from the human der mis. in addition, these bands were completely digested by bacterial co llagenase. This novel culture system could provide a simple tool with which to investigate the collagen metabolism by fibroblasts under more physiological conditions.