Culture of human keratinocytes in serum free medium

Keratinocytes are useful for cellular transplantation studies in order to improve functional outcome in burn patients and chronic wounds. Recently they have been used for generation of iPS cells with high efficiency. In this study, we described the details on separation, culture and proliferation of human keratinocytes from foreskin samples. In this experimental and qualitative study we obtained neonatal foreskin samples following newborn circumcision under sterile conditions. We used dispase enzyme to separate the epidermis from the dermis. Trypsin enzyme was used for isolation of keratinocyte cells from the epidermis layer. Isolated cells were cultured in type I human collagen-coated dishes and serum-free Epilife medium. We assessed morphological and immunocytochemical aspects of the isolated cells. Morphological and Immunocytochemical analyses revealed that isolated cells have typical keratinocyte morphology and they could express CK14 which is a specific marker of the keratinocytes. The cells were successfully sub-cultured at a split ratio of 1:2 every 4 to 5 days. After passage 10, a significant decrease in the proliferation of the human keratinocyte cells was observed. Morphologically the cells were flat and thin. In this study we improved the method of isolation and cultivation of human keratinocyte from foreskin. Using this method, we isolated human keratinocytes for more than 20 times. Thus, it can be concluded that this method is reproducible in other laboratories.