Primary cell culture of canine corneal endothelial cells

Abstract

Objective To establish a primary cell culture and clarify the characteristics of canine corneal endothelial cells in vitro. Procedures The eyes were enucleated from dogs that were euthanized for reasons unrelated to this study. Enucleated canine eyes were dissected, and the intact corneas were isolated from the globes. Using enzymes, the corneal endothelial cells were dispersed from the cornea. The obtained canine corneal endothelial cells were cultured in a cell culture dish. Cultured corneal endothelial cells were morphologically evaluated using phase‐contrast microscopy. Immunohistochemical analysis of the cultured cells, particularly of the corneal endothelial cell marker, zonula occludens‐1 (ZO‐1), Na+/K+‐ATPase, and vimentin, was performed to clarify whether the cultured cells were actually corneal endothelial cells. Furthermore, the post‐passage morphology of cultured cells was evaluated. Results Canine primary cultured corneal endothelial cells showed morphologically small, cobblestone‐like structures. The isolated cells had proliferative ability in vitro and demonstrated positive expression of the corneal endothelial cell markers, ZO‐1, Na+/K+‐ATPase, and vimentin. However, repeated passages resulted in larger cell sizes as assessed by phase‐contrast microscopy. Repeated passages also resulted in lower cell density. Conclusions This study demonstrated the successful culture of canine corneal endothelial cells. This might enhance the understanding of corneal endothelial cell characteristics in dogs.