Long-term preservation of donor corneas in glycerol for keratoplasty: exploring new protocols.

Aim: To evaluate the role of temperature and adjunctive dehydration in better long-term preservation of human corneas when preserved and stored in glycerol.
Methods: Different preservation temperatures and effects of adding silica gel in glycerol-preserved corneal tissues were evaluated. Human corneal tissues not suitable for optical keratoplasty initially preserved in McCarey-Kaufman medium were transferred to glycerol and stored at four different temperatures for 3 months as follows: tissues in anhydrous glycerol with and without silica gel at -80°C, -20°C, 4°C and at room temperature (RT). Parameters evaluated included microbial sterility, thickness (Digimatic micrometer), transparency (slit lamp examination, UV-Vis spectrophotometer), mechanical strength (Instron 5848 Microtester), tissue integrity (H&E staining), antigenicity (immunohistochemistry) and ultrastructure of collagen (transmission electron microscopy, TEM).
Results: Microbial test after 3 months of glycerol preservation confirmed sterility of the tissues. The thickness increased in corneas preserved at RT with and without silica gel (p<0.001). RT corneas had the lowest transparency and tensile strength. Tissues in anhydrous glycerol stored with and without silica gel at -80°C were the most transparent (p<0.001) and had the highest tensile strength (p<0.001). Tissue integrity was maintained and expression of Human Leukocyte Antigen D related (HLA-DR) was less in glycerol-preserved corneas at -80°C. TEM studies indicated that parallel alignment of stromal collagen was disrupted at RT-preserved corneas.
Conclusions: Corneal tissue preserved at -80°C was the best method for preservation as it maintained the sterility, thickness, optical transparency, mechanical strength and ultrastructural features.
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