An Experimental Study On Limbal Tissues With Cryopreservation

Objective To evaluate the vitality of limbal tissues with Cryopreservation.Methods Limbal grafts include 2mm of peripheralcornea and 2mm of sclera of 3 rabbits were preserved by simplified two-step Cryopreservation method for 90 days. The human corneal tissues were cryopreserved by the same method for 92 days and 726 days, and were made into limbal graft include 2-3mm of peripheralcornea and 2-3mm of sclera. Light and transmission electron microscopies were used to study the structure of limbal tissue cells. Enzyme histochemistry methods with image analysis system were used to evaluate the metabolic enzyme activity, lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH).Results (1) From light microscopic study, the cryopreserved limbal epithelial cells remained intact and lined up tightly. Partial epithelium arrayed radiately and shaped into Vogt bars, which projected downward over the limbus every 1 -2mm from the subepithelial papilla. And a lot of pigment granule was deposited. There were blood vessel tissue in Hypothallus. (2) From transmission electron microscopic study, the cryopreserved limbal epithelial cytomembrane remained intact and zonula occludens was as excellent as before. Partial mitochondria were swollen with no nuclei change in limbal tissue cells preserved by simplified two-step Cryopreservation method. (3) The activity of LDH and SDH in the epithelial cells of limbal tissue remained the same as in the fresh control group, which preserved by simplified two-step Cryopreservation method (P>0.05).Conclusions Simplified two-step Cryopreservation method can maintain the vitality of limbal tissue cells and provide viable limbal grafts at any time. And can provide an experimental evidence for cryopreserved limbal stem cells to treat ocular surface disorders.Postgraduated: Luo Lin(Ophthalmology) Directed by: Professor Wang Chuanfu...