A new defined and serum‐free technique to obtain stratified oral mucosal epithelium for corneal surface reconstruction

Abstract

Abstract Purpose Corneal epithelial reconstruction by using cultivated oral mucosal epithelial transplantation (COMET) technique is a viable treatment option for severe ocular surface disorders. So far this has been based on utilization of xenogenic/allogenic components such as 3T3 mouse feeder cells, serum or amniotic membrane. In this study, we present a new defined and serum‐free method to obtain oral mucosal epithelium for COMET. Methods Epithelial cells of oral mucosal biopsies were isolated and seeded on collagen IV inserts in a defined progenitor cell targeted (PCT) medium. After 90% confluency the medium was switched to a defined non‐PCT medium, and differentiation was induced by increased calcium concentration and airlifting for 6 days. No serum was added to the cultures. The integrity of the epithelium was measured by transepithelial electrical resistance (TEER) before embedding in paraffin. 5 μm sections were studied for histology and immunohistochemical markers for epithelial keratins (K), cell proliferation and cell adhesion. Results The cultivated oral epithelial cells formed a stratified tissue of 4 to 15 cell layers. The cell sheets showed normal keratin expression profiles and were positive for K15 and p63, indicating the presence of putative progenitor cells. The cultivated epithelium also exhibited excellent TEER values, high integrity and ZO1 expression. Conclusion We have developed a novel method to produce tight multi‐layered epithelium with proliferative potential for COMET under defined, serum‐free conditions, potentially suitable for clinical applications.