Protocol for culturing healthy primary endothelial cells isolated from research-grade human corneoscleral donor tissue

Studying the function of healthy corneal endothelial cells has been challenging due to a variety of culturing strategies. Here, we present a standardized protocol for culturing primary human corneal endothelial cells, isolated from research-grade corneoscleral donor tissue, without the addition of mitotic enhancers. We describe the required enzymes, medium composition, cell density, and incubation times. Additionally, we use immunofluorescence microscopy to illustrate optimal outcomes and discuss troubleshooting procedures.