Effect of limbal explant orientation on the histology, phenotype, ultrastructure and barrier function of cultured limbal epithelial cells

Purpose: To compare the histology, phenotype, ultrastructure and barrier function of cultured limbal epithelial cells using two explant culture protocols. Methods: Epithelial cells were cultured for 16 days from limbal explants, positioned with either the stromal side (stromal group) or the epithelial side (epithelial group) on intact amniotic membranes. The cultured epithelium (n = 56) was examined using light microscopy, immunohistochemistry for K3, Cx43, ABCG2 and p63 expression, Western blot analysis of Delta Np63 alpha, transmission electron microscopy, a horseradish peroxidase (HRP) permeability assay and scanning electron microscopy. Results: The epithelial group demonstrated a significantly higher expression of p63-positive cells (85.7 +/- 4.2%) than the stromal group (75.3 +/- 8.9%), and Western blots showed a stronger band of Delta Np63 alpha. K3 and ABCG2 were not detected in either group, whereas Cx43 displayed moderate immunostaining in the suprabasal layer. The number of cell layers, the desmosome number and the undulation length in the epithelial group were not significantly different from those in the stromal group. In both groups, HRP accumulated on the apical surface of the superficial cells, and scanning electron microscopy demonstrated tightly apposed superficial cells. Conclusions: Our findings indicate that limbal explants positioned epithelial side down may give rise to cultured epithelia with higher expression of p63 and Delta Np63 alpha.