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J. M. Simonis, N. Capito, V. Copeland, J. Prine, B. K. Ambati, J. Moore, Y. Song; Trehalose Enhanced Lyophilization of Corneal Tissue: Promise in Long-Term Storage and Corneal Transplantation. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5762. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Current methods for corneal transplant tissue preservation are limited by a brief period of donor tissue viability. Lyophilization of transplant tissue extends tissue preservation up to three months, but is limited by loss of endothelial viability. In this study we sought to develop a lyophilization procedure conferring endothelial protection and structural viability in the rabbit cornea. We also sought to demonstrate proof-of-principle in rabbit corneal transplantation.
Female corneas (n=10) were harvested and stored in Optisol with or without trehalose+cryoprotectant solution at 4ºC for 72 hours. All samples were cryogenically frozen to -80ºC, stored for one day at -140ºC, placed into liquid nitrogen for 1 hour, and then lyophilized for 24 hours. Dried samples were stored at room temperature for two months, whereupon tissue prehydration and rehydration with BSS was completed. Cell morphology was was assessed using PAS/H (periodic acid Schiff/hematoxylin) histological staining and scanning electron microscopy. Female donor corneas, after rehydration, were transplanted into male corneas, observed for 2 weeks with photomicrography, and then examined for Barr body presence using transmission electron microscopy.
Corneal endothelial layer structural viability of specimens stored in Optisol with trehalose + cryoprotectant was greater than specimens preserved without enhanced buffer. Immunohistochemistry confirmed the presence of an intact endothelial monolayer in the enhanced buffer, while no such structure was noted for corneas stored in the unenhanced buffer. Scanning electron microscopy substantiated these results, with preservation of a reticular hexagonal pattern and fewer microperforations in corneas preserved with the the enhanced solution. Successful transplantation was confirmed through the presence of a clear cornea two weeks post operation and through presence of barr bodies confirmed by TEM.
Upon rehydration of rabbit corneas, we found that structural integrity of the endothelial layer can be preserved using trehalose-enhanced buffer with controlled-rate temperature cryopreservation in concert with lyophilization. Furthermore, transplantation of a rehydrated cornea has shown functionality and viability of endothelial cells. Specific factors conferring protection to the endothelium need to be further examined, and functional restoration of the corneas needs to be replicated in future studies.
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