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V. T. Copeland, C. Jenkins, Y. C. Song, J. Moore, K. Smith, P. Roon, B. K. Ambati; Long-Term Room Temperature Storage of Rabbit Corneas After Trehalose-Enhanced Lyophilization. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2691.
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Current methods of tissue preservation for corneal transplant are limited by the brief interval for which donor tissue is viable. Lyophilization of transplant tissues can lengthen tissue preservation time up to three months, but is currently limited by loss of endothelial viability in the freeze-drying process. In this study, we sought to develop a lyophilization procedure that conferred endothelial protection and structural viability in the rabbit cornea.
Corneas (n=5) were harvested and stored in Optisol with or without trehalose+cryoprotectant solution prior to being incubated at 4ºC for 72 hours. All samples were then cryogenically frozen to -80ºC at the set-rate of -0.3ºC/min. Samples were stored for one hour at -80ºC, and then were lyophilized for a period of at least 72 hours. Dried samples were then stored at room temperature for a period of two months, whereupon tissue prehydration and rehydration with BSS (balanced salt solution) was completed. Structural integrity of rehydrated corneas was determined using PAS/H (periodic acid Schiff/hematoxylin) histological staining, as well as scanning electron microscopy.
Corneal endothelial layer structural viability of those specimens stored in Optisol with trehalose + cryoprotectant was greater than that of 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 prervation of a reticular hexagonal pattern and with fewer microperforations in corneas preserved with the the enhanced solution_features that are usually associated with normal, intact corneal endothelium
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. Specific factors conferring protection to the endothelium need to be further examined, and functional restoration of the corneas needs to be assessed in future studies.
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