Epidermal cell culture using Sephadex beads coated with denatured collagen (cytodex 3)

Epidermal cell culture using microcarriers of Sephadex beads coated with denatured collagen (cytodex 3) was performed. Epidermal basal cells (above 95%) obtained from human skin by trypsinization were cultivated statically on the beads in 96-well culture plates. Proliferation was rapid and great in synchronous waves during 2-7 days after inoculation. The growth rate depended on the inoculation cell population densities. When cells were inoculated at 7.75 X 10(4)/well, the maximum increase was 3.6-fold and at 1.69 X 10(4)/well and 0.32 X 10(4)/well, 2.5-fold and 2.1-fold, respectively. Differentiation was assessed visually on a hemocytometer. The percentage of basal cells of the total cells present in each well was reduced from 98% (on inoculation) to approximately 25% in a week and thereafter. In 1 month after inoculation, cells with keratohyaline-like granules were observed at 12%. The attachment of cells to the beads was rather loose. Cells were supposed to be attached to denatured collagen on the beads via fibronectin contained in the serum of the medium, because denatured collagen had the property to bind strongly to fibronectin. Loose attachment made it possible to harvest intact cells without the use of trypsin. This cell culture system with such new characteristics will be a useful tool for studying epidermal cell biology and biochemistry.