Recovery of endothelial function after vitrification of cornea at -110 degrees C.
OBJECTIVE
To determine whether endothelial function is retained after ice-free cryopreservation of cornea by vitrification at -110 degrees C.


METHODS
Rabbit corneas, mounted on support rings, were exposed to a solution containing 6.8 M propane-1,2-diol (PROH) and cooled at approximately 7 degrees C/min to -110 degrees C, which was below the glass transition temperature (T(g)) of the solution. After rewarming at approximately 12 degrees C/min and removal of the PROH, endothelial function was assessed by monitoring corneal thickness during perfusion at 34 degrees C.


RESULTS
Addition and removal of 6.8 M PROH without cooling to -110 degrees C did not markedly impair endothelial function, although corneas were thicker than control samples. There was no visible crystallization of ice during cooling to -110 degrees C; but a few small, discrete sites of crystallization remote from the endothelium, were observed during warming. After removal of the PROH, corneas approximately doubled in thickness during the first 3 hours of perfusion, but they then started to thin, which suggested active control of stromal hydration by the endothelium. This was confirmed in a further set of experiments by removal of bicarbonate ions from the perfusate at this point, which resulted in further swelling at +58 +/- 2 microm/hour (SD; n = 4). Restoring bicarbonate to the perfusate halted this swelling, and the corneas then thinned at -13 +/- 2 microm/hour (n = 4). Morphologically, staining with trypan blue and alizarin red S showed an apparently intact endothelial monolayer.


CONCLUSIONS
Rabbit corneal endothelium tolerated exposure to 6.8 M PROH, and endothelial function was evident after vitrification at -110 degrees C. Preliminary morphologic results with vitrified human cornea also showed retention of endothelium.
Armitage
W John
WJ
Division of Ophthalmology, Bristol Eye Hospital, University of Bristol, Lower Maudlin Street, Bristol BS1 2LX, UK. w.j.armitage@bristol.ac.uk
Hall
Samantha C
SC
Routledge
Caroline
C
D000368:Aged; D000818:Animals; D027321:Anion Transport Proteins; D017920:Antiporters / Q000378:metabolism; D003080:Cold Temperature; D003315:Cornea; D015925:Cryopreservation / Q000379:methods*; D004728:Endothelium, Corneal / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D019946:Propylene Glycol / Q000494:pharmacology; D011817:Rabbits; D020127:Recovery of Function / Q000502:physiology; D014021:Tissue Preservation / Q000379:methods*
D027321:Anion Transport Proteins; D017920:Antiporters; C425809:SLC4A11 protein, human; D019946:Propylene Glycol