Ex vivo development of a composite human oral mucosal equivalent

Purpose: The aim of this study was the ex vivo development of a composite oral mucosal equivalent composed of a continuous stratified layer of human oral keratinocytes grown on a cadaveric human dermal matrix in a defined medium without a feeder layer. Materials and Methods: Enzymatically dissociated human oral keratinocytes from keratinized oral mucosa were cultured, submerged in a serum-free, low-calcium (0.15 mmol/L) supplemented medium, and expanded through several passages. Once a sufficient population of keratinocytes was reached, they were seeded on 1-cm(2) pieces of AlloDerm (LifeCell Co, Woodlands, TX), an acellular nonimmunogenic cadaveric human dermis, at cell densities of 2.5 x 10(4), 5.0 x 10(4), 1.25 x 10(5), or 2.5 x 10(5). The oral keratinocyte-AlloDerm composites were cultured while submerged in a high-calcium (1.8 mmol/L) medium for 4 days. After 4 days, the composites were raised to an air-liquid interface. Samples of the composites were taken for histologic examination at 4, 11, and 18 days postseeding of the keratinocytes on the AlloDerm. Results: At day 4, only the seeded cell density of 2.5 x 10(5) cells/cm(2) formed a continuous monolayer on the AlloDerm. At day 11, a continuous stratified epithelium was seen, and at day 18 a well-differentiated, confluent parakeratotic epithelial layer was developed at cell densities of 5.0 x 10(4), 1.25 x 10(5), and 2.5 x 10(5) cells/cm(2). Conclusion: With the method used, it was possible to successfully develop an ex vivo composite oral mucosal equivalent that consisted of a stratified epidermis on a dermal matrix.