Abstract
Purpose :
Central corneal endothelial cell (EC) count is one of most important parameters in eye bank evaluation of donor corneas. About one third of donor corneas have to be discarded during cultivation - one main reason are too low central EC counts. This work aims to better evaluate donor EC by imaging the EC reservoir in the endothelial periphery using different microscopy techniques. This would allow more precise charaterization of donor cornea quality beyond the current state-of-art.
Methods :
Donor corneas (n = 46) from 41 donors was obtained from the Cornea Eye Bank (DGFG) (Ethic Committee No. A2020-0108). Human corneas were examined using a special Heidelberg Retina Tomograph (HRTII/RCM). The different cell populations were also analyzed by scanning electron microscope (FE-SEM; Merlin VP compact, Carl Zeiss). For this purpose, corneas were fixed in 1% paraformaldehyde and 2% glutaraldehyde, as well as, afterwards, with 1% osmium, followed by dehydration through an ascending ethanol series (30, 50, 70, 90, and two times 100%), drying by a critical point dryer (Emitech850), and final sputtering with a thin gold layer. Furthermore, corneas were fixed with 2% paraformaldehyde (1h, Sigma) to analyze the cells with a confocal laser scanning microscope (LSM780, Carl Zeiss) for their function and properties using markers (such as ABCG2, LRG5, SOX2, ZO1, NaK-ATPase).
Results :
Our multimodal analyses using the different microscopic techniques of the ex vivo cornea highlight the need for advanced and high-resolution imaging approaches to assess corneal quality. These techniques allow the visualization and characterization of other cell populations, f.ex. in the endothelial transition zone. Using HRTII/RCM, ex vivo corneas could be analyzed without fixation. Using cLSM and FE-SEM, it was possible to characterize corresponding layers and cells after fixation. With cLSM, we could identify cells with stem cell markers in the TC (multicellular clusters), representing a niche of EC progenitor cells.
Conclusions :
Our analyses with the different microscopy techniques of ex vivo corneas highlight the need to use excellent and high-resolution imaging techniques to assess the cornea's quality. Examination of the endothelial transition zone in ex vivo donor corneas in daily eye bank routine could lead to better characterization of donor corneas - with the potential to decrease the amount of discarded donor tissue.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.