Protocol for serial cultivation of epithelial cells without enzymes or chemical compounds

Deriving keratinocytes from epidermis or oral mucosa is a critical first step in the construction of cell-based tissue engineering and regenerative medicine applications. It would be advantageous to develop a methodology to grow adult somatic cells with maximum plasticity in a rapid fashion and in large numbers with minimal manipulation. With routine methods, keratinocytes are cultured in standard amount of medium and passaged with enzymes, and the confluence of the monolayer induces differentiation and eventual cell death. A protocol to expand keratinocytes in culture by growing keratinocyte in large numbers using a technique in which keratinocytes are released into the overlaying medium, effectively "popping-up" into suspension from the cell monolayer, is described in this chapter. This technique does not require the use of enzymes or chemical compounds for serial cultivation. The cells possess the ability of active cell proliferation at 100 % confluence over 1-2 months' time. Based on previous characterization reports, these are untransformed, normal keratinocytes that appear to be highly suitable for clinical applications.