Ex vivo development of a composite human oral mucosal equivalent.
OBJECTIVE
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.


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-cm2 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/cm2 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/cm2.


CONCLUSIONS
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.
D002118:Calcium / Q000008:administration & dosage; D002452:Cell Count; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D046508:Culture Techniques; D004847:Epithelial Cells / Q000166:cytology; D004848:Epithelium / Q000033:anatomy & histology; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000502:physiology; D009061:Mouth Mucosa / Q000033:anatomy & histology* / Q000166:cytology / Q000254:growth & development; D012867:Skin