Preoperative Evaluation of Cultured Human Corneal Limbal Epithelium on Amniotic Membrane by Confocal Microscopy

Abstract

Although sheet transplantation with cultured corneal limbal epithelium has been widely performed as a strategy for ocular surface reconstruction, there has been no optimal method for evaluating the morphology of these sheets prior to transplantation. We propose the use of in vivo confocal microscopy as a novel method for the evaluation of limbal corneal epithelium cultured on amniotic membrane. Human limbal epithelial sheets were grown on amniotic membranes by following a standard protocol and were stained with hematoxylin and eosin. Morphology was studied using in vivo confocal microscopy for cultured corneal epithelium on amniotic membrane, human intact amniotic membranes, and epithelium-denuded human amniotic membranes. Histologic examination showed a stratified corneal epithelium sheet by the fourth week of culture. The surface and basal layers of the cultured limbal epithelium and amniotic membrane were clearly distinguished by in vivo confocal microscopy. A monolayer of amniotic epithelial cells was observed on the intact amniotic membrane, but not on the epithelium-denuded human amniotic membrane. Our findings support the use of in vivo confocal microscopy as a valid technique for the preoperative evaluation of cultured corneal limbal epithelial cell sheets on amniotic membrane.