| Objective To search for the feasibility and the effect that cultural limbal corneal epithelial cells were cryopreserved by detecting the structure and the function of thawed cells.Methods The laboratory stage: (1)Limbal epithelial cells culture: Small specimens of limbal epithelial cells were excised from the limbal regions in rabbits; each of them was cut into small tissues(1mm 1mm). Making tissues loosen by enzyme digestion and cultivating them in twelve-well culture plate; (2) The cryopreservation and thawing of limbal epithelial cells: Some monolayer cells were digested into suspending liquid and were cryopreserved in two groups of cryoprotectant solutions respectively. Glycerol and dimethyl sulfoxide were chosed as cryoprotectant in group A and group B, while 32.5%F12 , 32.5%DMEM , 20%calf serum and 90%calf serum acted as cryoprotectant vehicles in group A and group B. Cryopreserved cells were frozen in liquid nitrogen according to the approach of stage freezing and were thawed after half a month,a month and two month differently; (3) The subculturing and evaluation of the frozen and non-frozen cells: thawed cells and some non-frozen cells were cultivated on acellular human amniotic membrane respectively. The cultivated limbal epithelial cells were identified by immunohistochemical and electron microscope; (4)Study on the biologic viability of frozen and non-frozen cells: the structure and activity of frozen and non-frozen cells were compared by inverted microscope, electron microscope,MTT colorimetry and trypan blue staining.The animal experiment stage: 40 rabbits underwent corneal epithelium curettement and limbal ablation to induce limbal stem cell deficiency and were randomly divided into four groups, simple amniotic membrane group, non-frozen cells group, glycerol group and dimethyl sulfoxide group(n=10 for each group). Searching for the activity of frozen and non-frozen cells more completely by transplanting subculturing cells and observing the accomplishment of cells function.Results (l)Subculturing cells were positive staining for monoclonal antibody cytokeratin AE1 and anti-proliferating cell nuclear antigen(anti-PCNA), were rarely positive for monoclonal antibody cytokeratin AE5. (2) Study on biologic viability of frozen and non-frozen cells: the adherence and growth of cryopreserved cells delayed 1-2 days compared with that of the fresh cells. After in vitro culrure,the time that the non-frozen cells group and the dimethyl sulfoxide group fromed confluent monolayer were 10 days and 12-14 days, while the glycerol group couldn't form confluent monolayer. Electron microscope revealed that cryopreserved cells appeared different extent dropsy and the recovery of the cell ultrastructure were in 7 days. Cryopreservation did severe harm to glycerol group cells. MTT colorimetry and trypan blue staining proved that the proliferation and the survival rate of frozen cells were lower than that of non-frozen cells after thawing(P <0.05) and after 5 days of subculturing(P >.05).There were no significant differences among the detective values of different frozen time groups(P >0.05), whereas the difference was statistically significant between that of the dimethyl sulfoxide group and the glycerol group(P <0.05). (3)The allogeneic transplantation of subculturing cells revealed: Corneas were positive staining for monoclonal antibody cytokeratin AE5 , were negative or weak positive for PAS in cells transplantation groups.Corneas were negative staining for monoclonal antibody cytokeratin AE5 ,were weak positive for PAS in the simple amniotic membrane group .The allogeneic transplantation experiment of subculturingcells showed that the keratopathy score of frozen cells were higher than that of non-frozen cells in the post-transplantation. Among cells transplanted groups, the score was not significantly different between non-frozen cells group and the dimethyl sulfoxide group(P >0.05),whereas the difference was statistically significant between non-frozen cells group and the...
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