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Published on: April 24, 2020
Analysis of Cryopreservation Protocols and Their Harmful Effects on the Endothelial Integrity of Human Corneas
Silvia Rodríguez-Fernández1,2,3, Marcelino Álvarez-Portela4, Esther Rendal-Vázquez5
1Grupo de Investigación en Terapia Celular e Medicina Rexenerativa, Departamento de Fisioterapia, Medicina e Ciencias Biomédicas, Facultade de Ciencias da Saúde, Universidade da Coruña (UDC), Campus de Oza, 15006 A Coruña, Spain.
Abstract:
Corneal cryopreservation can partially solve the worldwide concern regarding donor cornea shortage for keratoplasties. In this study, human corneas were cryopreserved using two standard cryopreservation protocols that are employed in the Tissue Bank of the Teresa Herrera Hospital (Spain) to store corneas for tectonic keratoplasties (TK protocol) and aortic valves (AV protocol), and two vitrification protocols, VS55 and DP6. Endothelial viability and general corneal state were evaluated to determine the protocol that provides the best results. The potential corneal cryopreservation protocol was studied in detail taking into consideration some cryopreservation-related variables and the endothelial integrity and stroma arrangement of the resulting cryopreserved corneas. TK corneas showed mostly viable endothelial cells, while the others showed few (AV) or none (DP6 and VS55). The corneal structure was well maintained in TK and AV corneas. TK corneas showed endothelial acellular areas surrounded by injured cells and a normal-like stromal fiber arrangement. Cryoprotectant solutions of the TK protocol presented an increasing osmolality and a physiological pH value. Cooling temperature rate of TK protocol was of 1 °C/min to -40 °C and 3 °C/min to -120 °C, and almost all of dimethyl sulfoxide left the tissue after washing. Future studies should be done changing cryopreservation-related variables of the TK protocol to store corneas of optical grade.
Insights
The tectonic keratoplasty (TK) protocol best preserved endothelial cell viability and corneal structure among tested methods for cryopreserving donor corneas. Further research on TK protocol variables could enable optical-grade corneal storage.
Area of Science:
- Ophthalmology
- Tissue Engineering
- Cryobiology
Background:
- Donor cornea shortage is a global challenge for keratoplasty.
- Cryopreservation offers a solution for storing corneas for transplantation.
Purpose of the Study:
- To evaluate and compare standard cryopreservation and vitrification protocols for human corneas.
- To identify the optimal protocol for preserving corneal integrity and endothelial cell viability.
Main Methods:
- Human corneas were cryopreserved using tectonic keratoplasty (TK), aortic valve (AV), VS55, and DP6 protocols.
- Endothelial viability and overall corneal structure were assessed post-preservation.
- Cryoprotectant solutions, cooling rates, and cryoprotectant removal were analyzed for the TK protocol.
Main Results:
- The TK protocol demonstrated the highest endothelial cell viability.
- Corneal structure was well-maintained in TK and AV protocols.
- TK protocol cryoprotectant solutions had increasing osmolality and physiological pH; cooling rates were 1°C/min to -40°C and 3°C/min to -120°C.
Conclusions:
- The TK protocol is the most promising for cryopreserving corneas, showing good endothelial viability and structural integrity.
- Further optimization of TK protocol variables is needed to achieve optical-grade corneal preservation.
