Clonal growth of mouse epidermal cells in medium with reduced calcium concentration

Mouse keratinocytes can be grown at clonal densities in dermal fibroblast conditioned medium with a calcium concentration of 0.02 MM. Colony forming efficiencies of approximately 1-3% can be achieved with primary and secondary cultures and up to 6% with selected subclones. A substrate of frozen-thawed dermal fibroblasts enhances colony formation over plastic. Colony size increases more rapidly in the presence of epidermal growth factor in conditioned medium. Conditioning of medium by fibroblasts is optimum after day 6 of culture and conditioned medium can be sotred at -20 degrees C for at least 4 mo.