An Optimized Mechanical Peeling Protocol for Mouse Corneal Endothelium

Corneal endothelium is a single layer of polygonal cells responsible for maintaining stromal dehydration and corneal transparency. Isolating corneal endothelium from murine models is exceptionally challenging without an established protocol, which historically limits the utility of mouse models to study corneal endothelial disease. Herein, a detailed method is presented to obtain a highly pure population of corneal endothelial cells via mechanical peeling of the endothelial sheet from both young and aged mice. Under a dissecting microscope, the corneal dome, including the limbus, was dissected. Following the removal of the iris and trabecular meshwork, the corneal dome was rinsed in phosphate-buffered saline (PBS). Fine, blunt forceps were utilized to stabilize the edge of the corneal dome, while ultra-fine forceps were employed to grasp the edge of Descemet’s membrane (DM). The edge of the DM was identified by performing a gentle micro-scratch along the limbal margin until a small flap emerged. Subsequently, the entire endothelial sheet along with the DM was carefully peeled from the periphery toward the corneal center. The isolated endothelial sheet can be dried flat on a microscope slide to investigate spatial gene expression, cell morphology, and cellular interactions. To obtain a single-cell suspension, the endothelial sheet was enzymatically digested in 0.25% Trypsin-EDTA for 8 min at 37 °C, yielding a highly purified population of corneal endothelial cells (95.7%). This protocol demonstrated high reproducibility for both young (approximately 1 month old) and aged (approximately 1 year old) mouse corneas. The total cell yields collected from young and old mice were 3634 and 4560 cells per eye, respectively, exhibiting high cell viability (young: 98.3%, old: 95.8%) and a high percentage of single cells (young: 90.6%, old: 90.1%). This detailed isolation protocol will facilitate advanced downstream research on the corneal endothelium using murine models.