Construction of large area organotypical cultures of oral mucosa and skin.

Abstract

Skin and oral mucosal keratinocytes grown in vitro usually lose their normal patterns of differentiation, unless grown as organotypical cultures that are constructed by allowing collagen gels containing fibroblasts to contract before they are plated with keratinocytes and raised to the air/medium interface. However, the contraction process tends to produce small irregular cultures. To generate uniformly differentiating large cultures, we have investigated several aspects of the factors involved in the culture construction. By adjusting the number of fibroblasts used and by plating the matrices with keratinocytes prior to contraction, cultures of up to 72 cm2 were constructed. The cultures retained almost the full surface areas of the original matrices and showed uniform patterns of epithelial plating and differentiation. Immunostaining for cytokeratins and integrins indicated restoration of in vitro phenotypes similar to those of the epithelial tissues of origin. These methods successfully generate cultures required for certain types of investigations and tissues that are suitable for clinical use as grafts.