Improving the success rate of human corneal endothelial cell cultures from single donor corneas with stabilization...

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.