Ex vivo produced oral mucosa equivalent preliminary report: a technical note

Aim: The aim of this study was to produce of ex vivo oral mucosal equivalent. The continuous stratified layer of human oral keratinocytes was grown on a cadaveric human dermal matrix in a defined culture medium without a feeder layer. Materials and methods: Human oral keratinocytes from a keratinized oral mucosa biopsy specimen were enzymatically dissociated and cultured in a serum-free defined culture medium, "EpiLife" (Cascade Biologics, Portland, OR, USA), containing a low calcium concentration of 0.06 mM. Oral keratinocytes were expanded one passage. Once a sufficient population of keratinocytes was reached, they were seeded onto the type IV collagen coated acellular nonimmunogenic cadaveric human dermis (AlloDerm), at cell densities of 1.8 x 10(5). To form a continuous epithelial monolayer, and to enhance keratinocyte differentiation, oral keratinocyte-AlloDerm composites were cultured while submerged in a high calcium concentration of 1.2 mM medium for 4 days. After 4 days, AlloDerm-keratinocyte composites were raised to an air-liquid interface to encourage stratification of the epithelial monolayer. After additional 7 days, they were taken for histologic examination at 11 days postseeding of the keratinocytes on the AlloDerms. Results: Histologically, on day 11, EVPOME's development showed multilayered epithelium comprising basal, suprabasal, and parakeratinized layers. Conclusion: Histologically, an ex vivo produced oral mucosa equivalent (EVPOME) that consisted of a stratified epidermis on a dermal matrix was successfully developed by the method used.