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Abstract

The purpose of the present study was to set up and test a cryopreservation method for long-term storage of human corneas. Therefore the freezing solution was optimized in 264 rabbit corneas by testing the type of cryoprotectant, its concentration, addition and dilution pattern and exposure temperature. Then rabbit corneas were frozen in the optimum solution at different cooling rates and thawed in a water bath at different temperatures. Eight human corneas were cryopreserved with the method showing optimum results in rabbit corneas and four additional corneas were used as controls. Endothelial viability was assessed after each step by vital staining and scanning electron microscopy. Best results after exposure of rabbit corneas to the freezing solution were achieved when using a 10% cryoprotectant concentration, with direct addition/dilution and exposure at room temperature (3512 +/-300 viable cells mm(2) when using dimethylsulfoxide; 3403 +/- 245 viable cells mm(2) when using 1,2-propanediol). Cryopreserved rabbit corneas had the highest endothelial cell survival when frozen at 1 degrees C/min and thawed at 37 degrees C (2003 +/- 372 viable cells/mm(2) when using dimethylsulfoxide and 1357 +/- 667 viable cells/mm(2) when using 1,2-propanediol). Cryopreserved human corneas had 753 +/- 542 viable cells/mm(2) when using dimethylsulfoxide and 56 +/- 56 viable cells/mm(2) when using 1,2-propanediol. We can conclude that the method developed is easy to handle and shows optimum results in rabbit corneas, with an endothelial cell survival that is consistent with transplant acceptability criteria. The results obtained in human corneas are below prediction and are still unsatisfactory for successful use in eye banking.

Analysis of Cryopreservation Protocols and Their Harmful Effects on the Endothelial Integrity of Human Corneas

“…Some CPAs, such as DMSO, proved to be low in cytotoxicity to ECs at low temperatures. Thus, DMSO was present in numerous standard cryopreservation and vitrification protocols from the beginning of corneal cryopreservation [3,4,18,27,28]. In TK and AV protocols, DMSO was added to corneas in cold CSs.…”

“…Some authors took this parameter into consideration, using ionic solutions that helped diminish the osmotic stress of EC in cold environments [10,13,[27][28][29]. These solutions provided better results than a commercial tissue organ medium, similar to those that we employed in TK and AV protocols or those that other authors used in their corneal cryopreservation protocols [3,5,6,18,29].…”

“…Despite all attempts, any cryopreservation protocol for human corneas has not provided a tissue with all the endothelial qualities required to ensure a valid cornea [1]. Normally, corneas after cryopreservation showed a disrupted endothelial integrity [3,18,22], low EC viability, or an impaired endothelial function [23,24]. Furthermore, although human cryopreserved corneas have been used in penetrant keratoplasties, the high variability of clinical results pushed the cryopreservation technique to the background.…”

“…The storage time limitation and the increasing worldwide scarcity of donor corneas should encourage us to find a long-term alternative for viable corneal storage. This alternative would be provided by cryopreservation [2,3].…”