Pre-dissected DMEK stored in bioreactor: feasibility study.

Abstract

Purpose : The number of endothelial grafts precut by corneal banks increases. Their shelf life is limited to a few days. We previously demonstrated the superiority of an active storage with the corneal bioreactor (BR) over organ culture (passive) for whole corneas. Aims: Measure endothelial viability of precut DMEK after 3 and 10 days of storage in our BR in a preclinical study.

Methods : Human pairs of corneas were included. The endothelial cell density (ECD in cells/mm²), thickness and transparency of corneas were measured before graft preparation. Descemet's membrane (DM) was peeled using the no-touch technique leaving the graft attached to the center of the cornea (on approx. 1/mm²). After randomization, one cornea was kept in organ culture (OC) and the other in the BR (21 mmHg, 2.6 µL/min) in the same medium (CorneaMax, Eurobio). The final viable ECD was determined using the triple staining with Hoechst-Ethidium-Calcein-AM. In addition, the expression of CD166 and NCAM (lateral membranes), ZO-1 (apical junctions), Na⁺/K⁺ATPase (endothelial pump function) and COX-IV (mitochondrial content) was studied by immunostaining to characterize endothelial cells after the storage.

Results : Initial ECDs were comparable: 2185±232 cells/mm² in BR versus 2276±328 cells/mm² in OC for the 3-day period and 2680±416 cells/mm² in BR versus 2644±420 cells/mm² in OC for the 10-day period. The DMs did not fold back in either BR or OC. Viable ECD did not differ significantly between BR and OC for either storage period: 2378±501 cell/mm² in BR versus 2342±503 cell/mm² in OC for the 3-day period (n=8 pairs and p=0.6238) and 2482±288 cell/mm² in BR versus 2579±315 cell/mm² in OC for the 10-day period (n=5 pairs and p=0.1756). Corneas were more transparent and thinner in BR than in OC after 3 days (916±86 versus 1193±136µm with p=0.0001) and 10 days (957±128 versus 1220±105µm with p=0.0625). The functional and structural markers studied were expressed in both groups after 3 and 10 days, some better preserved in BR.

Conclusions : The storage of precut DMEKs seems to be possible in BR and OC for at least 10 days. Interestingly, a precut endothelium continues to partially exert its pump function in the BR. In practice, this could allow the anterior part to be used for DALK. In addition to improving the storage of whole grafts, the BR allows the storage of precut DMEKs for up to 10 days with excellent endothelial survival.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.