Barrier Function of Cultivated Limbal and Oral Mucosal Epithelial Cell Sheets

Abstract

Purpose.: To investigate barrier function of cultivated limbal and oral mucosal epithelial sheets. Methods.: Human and rabbit cultivated limbal epithelial sheets (CLES) and cultivated oral mucosal epithelial sheets (COMES) were subjected to immunohistochemistry for keratin K3, K4, K13, ZO-1, and occludin. Results were compared with those obtained from normal cornea and oral mucosa. Prepared under various culture conditions, the in vitro barrier function of these sheets was evaluated by biotin assay and transepithelial electrical resistance measurement. The CLES and COMES were transplanted to rabbit eyes with limbal deficiency, and fluorophotometry was performed before and after surgery. Transplanted corneas were subjected to immunohistochemistry after surgery. Results.: Both normal corneal epithelium and CLES were positive for K3 but negative for K13. Both CLES and COMES showed positive expression of ZO-1 and occludin in the apical epithelium. Both CLES and COMES demonstrated tight junction-like structures in the apical portion of the superficial epithelium by electron microscopy. Although rabbit COMES showed better barrier function than CLES, as evaluated by biotin assay and transepithelial electrical resistance, no such differences were noted in human sheets. Whereas epithelial barrier function was significantly improved after transplantation of CLES in the rabbit limbal deficiency models, recovery was not remarkable after COMES transplantation. Immunohistochemical study revealed that transplanted COMES had an irregular epithelial thickness with decreased expression of ZO-1 in some areas. Conclusions.: Epithelial barrier function was influenced by various factors such as culture condition and local environment after transplantation. CLES appears to offer better barrier function than COMES.