Abstract
Main objective of this study was to improve the success rate of human corneal endothelial cell (hCEC) cultures from single donor corneas. We could show that the use of stabilization medium prior to cell isolation may have a positive effect on the success rate of hCEC cultures from single research-grade donor corneas by allowing growth of otherwise possibly not successful cultures and by improving their proliferative rate. hCEC were obtained from corneo-scleral rims of 7 discarded human research-grade cornea pairs. The Descemet membrane–endothelium (DM–EC) sheets of each pair were assigned to 2 experimental conditions: (1) immediate cell isolation after peeling, and (2) storage of the DM–EC sheet in a growth factor-depleted culture medium (i.e. stabilization medium) for up to 6 days prior to cell isolation. hCEC isolated by enzymatic digestion were then induced to proliferate on pre-coated culture plates. The success rate of primary cultures established from single donor corneas were higher for DM–EC sheets kept in stabilization medium before cell isolation. All cultures (7/7) initiated from stabilized DM–EC sheets were able to proliferate up to the third passage, while only 4 out of 7 cultures initiated from freshly peeled DM–EC sheets reached the third passage. In addition, for the 4 successful paired cultures we observed a faster growth rate if the DM–EC sheet was pre-stabilized prior to cell isolation (13.8 ± 1.8 vs 18.5 ± 1.5 days, P < 0.05). Expression of the phenotypical markers Na+/K+-ATPase and ZO-1 could be shown for the stabilized cultures that successfully proliferated up to the third passage.
Highlights
Human corneal endothelial cells are crucial for maintaining corneal transparency, since loss of their functionality owing to endothelial diseases or trauma, results in corneal swelling and loss of corneal clarity (Gagnon et al 1997; Joyce 2003)
Descemet membrane endothelial keratoplasty (DMEK) is the most selective of these techniques to date and replaces the recipient’s diseased endothelium and Descemet membrane (DM) by a healthy donor DM–endothelium (DM–EC) sheet (Melles and Dapena 2014; Melles et al 2006). This method has several advantages over traditional penetrating keratoplasty, one of its limitations is the shortage of high quality healthy donor tissue. This has led to considerable interest in the development of new strategies to increase the pool of available donor tissue, such as the introduction of hemi- and quarter-DMEK (Gerber-Hollbach et al 2016; Muller et al 2017), in which the donor DM–EC is divided in 2 and 4 pieces, respectively, allowing a much more efficient use of donor tissue
Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), Dulbecco’s phosphate-buffered saline (PBS), TrypLETM Express (TE), ascorbic acid 2-phosphate, collagenase from Clostridium histolyticum (Type A), paraformaldehyde (PFA), bovine serum albumin (BSA), 40,6-diamidino-2-phenylindole (DAPI), and Triton X-100 were purchased from Sigma-Aldrich Chemistry BV (Zwijndrecht, The Netherlands)
Summary
Human corneal endothelial cells (hCEC) are crucial for maintaining corneal transparency, since loss of their functionality owing to endothelial diseases or trauma, results in corneal swelling and loss of corneal clarity (Gagnon et al 1997; Joyce 2003). Descemet membrane endothelial keratoplasty (DMEK) is the most selective of these techniques to date and replaces the recipient’s diseased endothelium and Descemet membrane (DM) by a healthy donor DM–endothelium (DM–EC) sheet (Melles and Dapena 2014; Melles et al 2006). This method has several advantages over traditional penetrating keratoplasty (shortens the recovery time, reduces the risk of inflammation and graft rejection), one of its limitations is the shortage of high quality healthy donor tissue. This has led to considerable interest in the development of new strategies to increase the pool of available donor tissue, such as the introduction of hemi- and quarter-DMEK (Gerber-Hollbach et al 2016; Muller et al 2017), in which the donor DM–EC is divided in 2 and 4 pieces, respectively, allowing a much more efficient use of donor tissue
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