Study Of The Biologic Viability Of Rabbit's Limbal Corneal Epithelial Cells Cultured On Rabbit Superficial Layer Corneal Stroma After Cryopreservation

Objective To detect the structure and the activity of thawed rabbit's limbal corneal epithelial cells after Cryopreservation cultured on rabbit superficial layer corneal stroma .To observe the effect of superficial layer corneal stroma containing cultivated limbal epithelial cells were transplanted to allogeneic limbal stem cell damaged rabbit cornea. In order to obtain experiment evidence for clinical utilization of superficial layer corneal stroma containing cultivated limbal epithelial cells.Methods The laboratory stage: (1)Limbal corneal epithelial cells primary culture:Small tissue speciments of superficial layer limbus corneae were excised from the limbal regions in rabbits;each of them was cut into small tissue(1mm×1mm). We cultivated them in cell culture bottle with two kind of cell culture fluid containing 20% fetal bovine serum (FBS) ; (2) Limbal corneal epithelial cells serial subcultivation on Rabbit superficial layer corneal stroma: Limbal corneal epithelial cells from primary culture were digested into suspending liquid with VTP(0.25% pancreatina and 0.02% EDTA 1:1) and culture them onto corneal stroma ; (3) The cryopresevation of limbal corneal epithelial cells cultured on rabbit superficial layer corneal stroma: Limbal corneal epithelial cells stored in preservative fluid were frozen in nitrogen canister from sequent hypothermy and the preservative fluid is composed of 90% FBS and 10% DMSO (dimethyl sulphoxide); (4)The evaluation of limbal corneal epithelial cells from primary culture:The cultivated limbal epithelial cells were identified by inverted microscope and electron microscope and Immunocytochemical stain ; (5) Study on the biologic viability of frozen and non-frozen cells cultured on rabbit superficial layer corneal stroma :the structure and activity of frozen and non-frozen cells were compared by inverted microscope and electron microscope and Immunocyto- chemical stain.The animal experiment stage:30 rabbits undergone corneal epithelium and limbal ablation to induce limbal stem cell deficiency were randomly divided into three groups, corneal stroma group,non-frozen cells group and frozen cells group(n=10 for each group). To observe the activity of limbal corneal epithelial cells and transparency of the cornea of each group by transplantation experiment.Results (1) Limbal corneal epithelial cells from primary culture can culture on superficial layer corneal stroma to the future generation and cohere on the carrying agent well. The cultivated limbal epithelial cells on corneal stroma formed 2～3 layers after 14 days in culture .The cultured cells on corneal stroma were stained positive in antibody anti-proliferating cell nuclear antigen(anti-PCNA) . (2) The structure and the survival condition of frozen and non-frozen limbal corneal epithelial cells is different: The ultrastructure of frozen cells is diversity from non-frozen cells by transmission electron microscope examination . (3) The allogeneic transplantation of subculturing cells on corneal stroma indicated: 5 weeks after corneal limbal stem cell damage, rabbits eyes developed features of limbal stem cell deficiency including vascularization and conjunctival epithelial ingrowth .2 weeks after the allogeneic transplantation of frozen and non-frozen subculturing cells on corneal stroma , rabbit cornea were all successfully epithelialied and corneal new vessels gradually disappear while 2 weeks after transplanting corneal stroma, corneal epithelia remained defected and corneal new vessels remained invariable .Conclusion . The cultivated limbal epithelial cells can form multi-layered epithelium on superficial layer corneal stroma and they still remained character of epithelial cells and reproductive activity through refrigeration for 6 weeks .These cells growing on the carrier may be used to cure keratopathy of limbal stem cell deficiency by allogeneic transplantation of subculturing cells on corneal stroma.