Expression of differentiation markers in human adult keratinocytes cultured in submerged conditions

A number of studies have shown that human keratinocytes cultured in submerged conditions with non-delipidized serum do not express the major differentiation markers, i.e. 67 kDa keratin, ceramides, and lanosterol. However, they were mostly performed with neonatal or juvenile keratinocytes after a few passages, and not all the markers were analyzed in parallel. In this study, we compared the expression of several differentiation markers in preconfluent and postconfluent adult breast keratinocytes in primary and secondary cultures before and after cryopreservation. When primary cultures reached confluence, the 67 kDa keratin was synthesized, transglutaminase activity was increased, and, although overall lipid synthesis dropped, both lanosterol and free fatty acids contents were augmented. The same pattern was observed in postconfluent subcultures at Passage 2; however decreased overall lipid synthesis was more pronounced. Cryopreservation of keratinocytes just after isolation or after a few days in culture did not result in the loss of expression of these specific epidermic markers. Thus, adult breast keratinocytes in postconfluent submerged cultures represent an in vitro model that possesses various features of the normal epidermis, even after cryopreservation.