Engineering of Multilayered Polarized Epithelial Grafts From Explant Culture Is Independent of Limbal Explant Orientation

Abstract

Purpose: : The engineering of multilayered epithelial sheets ex vivo for limbal stem cell transplantation depends on apicobasal polarization of the limbal epithelial cells. With our novel culture technique, we positioned limbal explants with either the epithelium or the stromal side facing the amniotic membrane in order to investigate polarization and surface morphology of the cultured epithelium.

Methods: : In previously reported methods where the amniotic membrane is fastened to the lower side of a bottomless culture plate insert, the culture medium is obliged to pass through the amniotic membrane by passive diffusion. To enhance the provision of nutrients to the cultures, we stitched human amniotic membranes onto permeable polyester membranes of culture plate inserts. Human limbal explants were positioned with either the epithelium or the stromal side facing the amniotic membrane. After two weeks of culturing, the morphology of the epithelial outgrowth and control human corneal epithelium was examined by the means of transmission electron microscopy, light microscopy and scanning electron microscopy.

Results: : Confluent epithelial outgrowth was observed in all the cultures independent of explant orientation. The morphological findings did not show any major difference between the two groups of explant orientations and were similar to the control samples. Transmission electron microscopy demonstrated a polarized well–differentiated epithelium with desmosomes, hemidesmosomes and deep basal interdigitations. Light microscopy revealed a squamous epithelium which was up to six cells in thickness. A surface consisting of closely apposed, flattened epithelial cells covered with microplicae and microvilli was uncovered by the means of scanning electron microscopy.

Conclusions: : Multilayered polarized epithelium may be engineered ex vivo independent of limbal explant orientation.

Keywords: cornea: basic science • cornea: epithelium • microscopy: electron microscopy