Preclinical testing of small diameter Descemet membrane endothelial keratoplasty grafts to increase tissue availability

In this study, we describe a process of preparing, surgically manipulating, and validating a novel “small diameter” 4mm circular Descemet membrane endothelial keratoplasty (DMEK) graft in vitro. Three small diameter DMEK grafts can be prepared from a single donor endothelium and could, therefore, potentially expand the donor pool. Prior to clinical use, however, we aimed to examine each step of the process to determine the effect on the endothelial cell loss and whether or not cells retained their capacity to migrate uniformly. For this study, circular small diameter grafts, obtained from twelve corneas of ten donors deemed ineligible for transplantation, were included. Small diameter DMEK graft preparation was successful in all cases (n = 36). Endothelial cell density (ECD), determined in the eye bank on seventeen grafts, showed an average decrease from 2413 (±189) cells/mm(2) before to 2240 (±413) cells/mm(2) after preparation. Twenty-four grafts were used to simulate DMEK-surgery in vitro and were successfully stained with 0.06% trypan blue, loaded into a straight DMEK-injector, unfolded, positioned, and centered within the circular ~ 4mm descemetorhexis. The estimated % area populated by viable cells on the grafts decreased from on average 92 (±3) % before to 78 (±10) % (n = 4) after in vitro surgery. Cells displayed a capacity for uniform cell migration from all edges of the graft (n = 4) when embedded in the 3D hydrogel system. Our data show, that by using an in vitro model of DMEK-surgery it was possible to test the 4mm circular DMEK grafts from eye bank preparation to surgical implantation. The cell loss after in vitro surgery was comparable with the in vivo ECD decline early after DMEK and the capacity of the cells to migrate to potentially cover bare stroma indicates that these small diameter grafts may be a viable clinical option to treat central endothelial disease.