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Optimization of a method for the cryopreservation of rabbit corneas: attempted application to human corneas
M Canals1, J Garcia, J M Potau
1Ocular Morphology Unit, Faculty of Medicine, University of Barcelona, Casanova 143, 08036 Barcelona, Spain; Author for correspondence (Tel.: 34-93-4021906; Fax:34-93-4035260;
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.
Insights
This study optimized a cryopreservation method for corneas using rabbit models. While effective in rabbits, the method yielded unsatisfactory results for human cornea cryopreservation and long-term storage.
Area of Science:
- Ophthalmology
- Tissue Engineering
- Cryobiology
Background:
- Long-term storage of human corneas is crucial for transplantation.
- Current cryopreservation methods require optimization for improved viability.
Purpose of the Study:
- To develop and evaluate a cryopreservation method for long-term human cornea storage.
- To optimize cryoprotectant type, concentration, addition/dilution, and temperature using rabbit corneas.
Main Methods:
- Optimized freezing solutions and protocols in 264 rabbit corneas.
- Tested various cryoprotectants (dimethylsulfoxide, 1,2-propanediol), concentrations, and temperatures.
- Assessed endothelial cell viability using vital staining and scanning electron microscopy.
- Applied the optimized method to 8 human corneas, with 4 as controls.
Main Results:
- Optimal rabbit cornea cryopreservation involved 10% cryoprotectant, direct addition/dilution at room temperature, cooling at 1°C/min, and thawing at 37°C.
- Dimethylsulfoxide yielded higher rabbit endothelial cell survival (2003 ± 372 cells/mm²) compared to 1,2-propanediol (1357 ± 667 cells/mm²).
- Human cornea cryopreservation resulted in significantly lower viability: 753 ± 542 cells/mm² with dimethylsulfoxide and 56 ± 56 cells/mm² with 1,2-propanediol.
Conclusions:
- The developed cryopreservation method is effective and easy to handle for rabbit corneas, meeting transplant criteria.
- The method's performance in human corneas is currently below expectations and requires further improvement for clinical application in eye banking.
