Preparation of Keratinocyte Culture Medium for the Clinical Applications of Regenerative Medicine

Keratinocyte culture medium (KCM) has been used for the in vitro culture of keratinocytes and other types of epithelial cells, and the medium includes various ingredients. In this study, two modified KCMs were prepared. In the first, insulin, hydrocortisone and antibiotics that are normally included in KCM were replaced with clinically approved pharmaceutical agents, except transferrin and selenium; in the second, cholera toxin (CT) was replaced by L-isoproterenol (ISO). The modified KCMs were then compared to conventional KCM containing laboratory-grade reagents. Induced cell colony formations of canine oral mucosal epithelial cells cultured in both modified KCMs were found to be nearly equivalent to that in the control KCM, and there was no significant difference between the effect of CT and ISO. Canine oral mucosal cells proliferated to confluence in all three KCM formulations, with or without the use of 3T3 feeder layers. Cultured epithelial cells were harvested from temperature-responsive culture surfaces as an intact cell sheet, and the immunohistochemical analysis of the sheets showed that p63 and cytokeratin were expressed in the epithelial cell sheets cultured in all KCMs. Eventually, in the modified KCM formula, fetal bovine serum was replaced by autologous human serum, and the formula was found to be able to fabricate human oral mucosal epithelial cell sheets. These results indicated that the modified KCM was equally efficient as conventional KCM in the fabrication of transplantable stratified epithelial cell sheets.