Morphological and biochemical analyses on fibroblasts and self-produced collagens in a novel three-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 dermal fibroblasts to the organization of a dermis-like structure on a plastic dish without any prior treatment. The cell layer was composed of multilayered fibroblasts surrounded by dense extracellular matrices. Confocal microscopic examination disclosed that the fibroblasts in the upper layer were spindle-shaped and those in the lower layer were polygonal. Electron microscopic examination revealed the accumulation of mature 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 content of the cell layer increased in a time-dependent manner, while the monolayer culture system without. Asc 2-P yielded no measurable amount of hydroxyproline. On sodium dodecylsulphate-polyacrylamide gel electrophoresis, neutral insoluble collagens extracted from the cell layer showed the identical electrophoretic pattern to those from the human dermis. In addition, these bands were completely digested by bacterial collagenase. This novel culture system could provide a simple tool with which to investigate the collagen metabolism by fibroblasts under more physiological conditions.