title: Comparison of three methods for human corneal cryopreservation that utilize dimethyl sulfoxide.
abstract: We compared endothelial cell survival in human corneas after cryopreservation by three methods that utilize dimethyl sulfoxide. Twenty-eight human cadaver corneas were cryopreserved by one of three methods, stored briefly over liquid nitrogen, thawed, cultured at 37 degrees C for 3 days, and fixed for scanning electron microscopy. Seventeen control corneas underwent identical cryoprotectant immersion and culture protocols but were not frozen. Endothelial photographs taken after 1 and 3 days of culture were analyzed. Endothelial cell losses in cryopreserved corneas by Methods 1, 2, and 3, respectively, were 36, 22, and 10% after 1 day of culture and 57, 36, and 27% after 3 days of culture. Cryopreservation by Method 3 had less cell loss than Methods 1 or 2 (P<0.02) but greater cell loss than the control corneas for Method 3 (P<0.001). No loss of cells occurred in the control corneas for Methods 1 and 3 but substantial cell loss (26%) occurred in the control corneas for Method 2. Polymegethism and pleomorphism of the endothelial cells were seen in the corneas that lost cells. The endothelial cell loss of 10% seen after 1 day of culture in human corneas cryopreserved by Method 3 is similar to the loss that occurs during organ culture storage as currently used clinically and therefore would be acceptable for clinical use. After 3 days of culture, however, the cell loss had increased significantly to 27%. This additional decrease in cell number that occurs in culture may represent latent cryodamage and must be understood and overcome in vivo before the technique can be used clinically.
authors.affiliation: Department of Ophthalmology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA. bourne.william@mayo.edu
authors.forename: W M
authors.initials: WM
authors.lastname: Bourne
authors.forename: L R
authors.initials: LR
authors.lastname: Nelson
authors.forename: D O
authors.initials: DO
authors.lastname: Hodge
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002102:Cadaver; D002470:Cell Survival; D003315:Cornea; D016039:Corneal Transplantation; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D004121:Dimethyl Sulfoxide; D004728:Endothelium, Corneal / Q000166:cytology; D005069:Evaluation Studies as Topic; D006801:Humans; D066298:In Vitro Techniques; D008855:Microscopy, Electron, Scanning; D008875:Middle Aged
chemical_list: D003451:Cryoprotective Agents; D004121:Dimethyl Sulfoxide