To investigate the possibility of transplantation into rabbits of collagenase-induced cultured human corneal endothelial cell plate-rabbit corneal stromal complexes.

Human corneal endothelial cells were isolated from residual corneal limbus samples and treated with collagenase to create corneal endothelial cell sheets. Cultured sheets were transplanted into the rabbit stroma after the rabbit corneal endothelial cells and Descemet's membrane were removed. Hematoxylin-and-eosin staining and immunofluorescence staining for collagen VIIIa2 (COL8A2) and zonula occludens-1 (ZO-1) were performed. The cultured human corneal endothelial cell sheet-rabbit corneal stromal complex was transplanted into rabbits. On days 3, 5, and 7, the transplanted corneas were photographed and corneal opacity was measured. One week later, the rabbits were sacrificed. Hematoxylin-and-eosin staining and immunofluorescence staining for COL8A2 were performed.

The cultured cells were immunofluorescently stained for collagen VIIIa1 and ZO-1. Collagenase-treated cultured human corneal endothelial cells formed monolayers on day 7 after transplantation into the rabbit corneal stroma and immunofluorescently stained for COL8A2 and ZO-1. The cultured human corneal endothelial cell sheet-rabbit stroma complex transplanted into rabbits was transparent on days 3 and 5, but corneal opacity developed by day 7. Histologic examination revealed 3,3′-dioctadecyloxacarbocyanine perchlorate (DIO)-stained corneal endothelium (green) and hard-tissue lymphocytes had infiltrated the cultured corneal endothelial cell plate-rabbit corneal stromal complex graft group.

The cultured corneal endothelial cell sheet-rabbit corneal stromal complex prepared with the aid of collagenase showed a potential method for corneal endothelial cell transplantation.