Human Corneal Epithelial Equivalents for Ocular Surface Reconstruction in a Complete Serum-Free Culture System without Unknown Factors

Abstract

To establish a culture technique for human corneal epithelial equivalents that do not require fetal bovine serum (FBS), feeder cells, or bovine pituitary extracts and compare this system with conventional culture medium with FBS and mouse 3T3 fibroblasts. Human corneal limbal tissue from donor corneas was dissociated on denuded amniotic membranes and then cultured for 3 weeks in feeder-cell- and serum-free medium containing epidermal growth factor and B-27. Then, the cell sheet was evaluated by light microscopy, immunohistochemistry, and electron microscopy. The epithelial proliferative capacity was compared between serum- and feeder-cell-free medium and conventional medium. The cultured cell sheets were transplanted onto the denuded rabbit ocular surface to cover the resected area. A stratified cell sheet expressing cytokeratin-3 and -12 was grown in serum- and feeder-cell-free medium without unknown growth factors. The epithelial proliferative capacity in feeder-cell- and serum-free medium determined by WST-1 and colony-forming efficiency was significantly higher than that in conventional medium. Scanning and transmission electron microscopy showed well-formed stratified epithelium with clear cell boundaries, microvilli, and hemidesmosomal/desmosomal junctions. The transplanted cell sheets remained transparent without epithelial defects during the follow-up period. This method using serum- and feeder-cell-free medium not containing unknown growth factors allows the highly proliferative culture of human corneal epithelium. It avoids exposure of the corneal epithelial equivalent to FBS and animal feeder cells, thus minimizing the risk of contamination by pathogens that could transmit diseases to recipients.