Background: Failure of autologous keratinocyte culture from small split-thickness skin specimens or contamination of the keratinocyte culture by melanocytes represents practical problems in basic medical research and clinical studies. Purpose: To establish a simple and reliable method of harvesting pure autologous keratinocytes from a small split-thickness skin specimen. Methods: Split-thickness (0.3 mm) skin explants (1 × 2 mm) were firstly cultured in DMEM containing 10% FCS till formation of keratinocyte strips, then cultured in serum-free keratinocyte growth medium or cocultured with lethally irradiated 3T3 fibroblasts (J2) in a mixture of DMEM and Ham’s F12 (DF) medium. Results: Pure autologous keratinocyte culture is easily and reliably established by this organ culture technique. Conclusion: Culturing of skin explants in serum-free keratinocyte growth medium or coculturing of the skin explants with lethally irradiated 3T3 cells in DF medium is proved to be a useful, simple and reliable method of harvesting pure autologous keratinocytes from a small split-thickness skin biopsy.

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