Culturing Discarded Peripheral Human Corneal Endothelial Cells From the Tissues Deemed for Preloaded DMEK Transplants

To investigate if the peripheral corneal endothelium that is discarded after the preparation of preloaded Descemet membrane endothelial keratoplasty (DMEK) grafts for transplantation could be successfully used for corneal endothelial cell culture.

Complete Descemet membrane-endothelial complex (11.00 mm) was peeled from research-grade tissues (n = 15). The periphery (2.75 mm) of clinical-grade tissues (n = 15) deemed for preloaded DMEK transplants was gently peeled and preserved for 48 hours in tissue culture media, followed by centrifugation at 1000 rpm for 5 minutes. After enzymatic digestion, the cells from each group were plated in 2 different wells of an 8-well chamber slide. Media were refreshed and the confluence rate was monitored every alternate day. Live/dead staining and the expression of ZO-1, Tag1A3, Tag2A12, and Ki-67 markers were used to assess the viability, morphology, tight-junctions, cell area, and number of proliferative cells. The Wilcoxon and Student's t test were applied, where P < 0.05 was deemed statistically significant.

Average endothelial cell density at confluence was 2,352 cells/mm2 from complete endothelium and 2,510 cells/mm from peripheral endothelium (P = 0.0351). The confluence rate (%), hexagonality (%), polymorphism (%), cell area (μm), and Ki-67 positivity (%) did not differ between both groups (P > 0.05). All the antibodies were expressed in both groups at confluence.

The discarded peripheral endothelial cells obtained after preparing a preloaded DMEK graft for clinical application has a huge reservoir of healthy endothelial cells having proliferative potential. Using these discarded tissue pieces from donor tissues will significantly increase the primary source of healthy donor endothelial cells for regenerative treatments, which are otherwise difficult to obtain.