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Preclinical testing of “micro” Descemet's membrane endothelial keratoplasty grafts to increase tissue availability

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First Author: A.Miron THE NETHERLANDS

Co Author(s):    S. Ni Dhubhghaill   J. Lie   I. Dapena   G. Melles   S. Oellerich        

Abstract Details

Purpose:

In this study we describe a process of preparing, surgically manipulating, and validating a novel 4 mm circular “micro” DMEK graft in vitro. Three of these “micro” 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 to place the grafts in explant culture to determine whether cells retained their capacity to migrate uniformly.

Setting:

Netherlands Institute for Innovative Ocular Surgery, Research & Development (NIIOS R&D) department, and Amnitrans EyeBank Rotterdam (AER).

Methods:

Thirty-six micro-DMEK grafts obtained from twelve corneas of ten donors, ineligible for transplantation, with intact and viable endothelial cells were included in this study. Micro grafts were tested by simulating DMEK surgery in vitro using two systems, i.e. a human donor cornea without DM mounted onto the artificial anterior chamber (AC) and human globes. Graft viability was assessed before and after in vitro surgeries by Calcein-AM staining and a regular DMEK-graft was used as a surgical reference model. In parallel, cell migration experiments were performed in a three-dimensional culture system using a temperature-reversible hydrogel and cultured over 2 weeks.

Results:

Endothelial cell density (ECD) determined on seventeen micro-DMEK grafts showed an average decrease from 2500 (±230) cells/mm2 before to 2240 (±413) cells/mm2 after preparation. Twenty-four micro grafts were successfully stained with 0.06% trypan blue, loaded into a straight DMEK injector, unfolded, positioned, and centered in the ~4 mm descemetorhexis area. The estimated % area populated by viable cells on the micro DMEK grafts decreased from ~92% before to 76% after in vitro surgery, a decline of 17% in viability. When grafts were embedded in the 3D hydrogel system, they displayed a capacity for uniform cell migration from all graft edges.

Conclusions:

Using an in vitro model of DMEK surgery it was possible to test the 4 mm “micro” DMEK grafts from eye bank preparation to surgical implantation. The cell loss after in vitro surgery was comparable with the in vivo ECD decline in the early postoperative phase after DMEK. The capacity of the cells to migrate to cover bare stroma highlights the potential of treating central endothelial disease.

Financial Disclosure:

... receives non-monetary benefits from a company producing, developing or supplying the product or procedure presented