Comparative observation of freeze-thaw-induced damage in pig, rabbit, and human corneal stroma.
OBJECTIVE
Although variations exist between species with respect to outcomes after cryopreservation, little is known about the differences in the susceptibility of the corneal stroma to cryoinjury. We performed this study to investigate freeze-thaw-induced damage in keratocytes and collagen in rabbit, pig, and human corneas.


METHODS
Rabbit, pig, and human.


METHODS
We prepared 250-microm-thick anterior stroma from rabbit, pig, and human corneas after scraping off the epithelium and endothelium. Each 250-microm-thick corneal stroma without epithelium was placed in a 50-mL tube, frozen with liquid N2 for 15 min and taken out to thaw rapidly at 37 degrees C. This procedure of rapid freezing and thawing was repeated three times. Differences between the species with respect to cells and collagen structures were examined using hematoxylin-eosin (H&E) staining, terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay, and transmission electron microscopy (TEM). We orthotopically transplanted the pig and rabbit corneal transplants after the triple freeze-thaw cycle into rabbit eyes and evaluated graft survival.


RESULTS
On gross examination, rabbit corneas became opaque after the triple freeze-thaw procedure, while pig and human corneas remained transparent. Histologically, keratocytes were apoptotic on TUNEL assay and TEM in rabbit, pig, and human corneas. Collagen fibrils were fragmented and the arrangement of collagen fibrils was severely disturbed in rabbit corneas on H&E staining and TEM; collagen was well preserved in pig and human corneas. Rabbit corneal stroma underwent autolysis after transplantation, whereas the pig corneal stroma remained clear for 1 month.


CONCLUSIONS
Our study showed that rabbit corneal stroma was more susceptible to freeze-thaw injury than pig and human corneas.
Oh
Joo Youn
JY
Seoul Artificial Eye Center, Seoul National University Hospital Clinical Research Institute, Seoul, Korea.
Kim
Mee Kum
MK
Lee
Hyun Ju
HJ
Ko
Jung Hwa
JH
Wee
Won Ryang
WR
Lee
Jin Hak
JH
D000818:Animals; D003319:Corneal Stroma / Q000502:physiology*; D015925:Cryopreservation; D005615:Freezing; D006801:Humans; D011817:Rabbits; D013048:Specimen Handling / Q000662:veterinary*; D013552:Swine; D013997:Time Factors