| Objective To develop a method of corneal organ culture preservation. To investigate an easier method of dehydration on the basis of the intrinsical formula. To compare the results of the two methods, and to discuss the effects to organ-cultured porcine corneas of two different dehydrated method from the view of cell apoptosis.Methods Prepare the medium of preservation: Prepare the preservation medium A according to the formula,which contains MEM,25mmol/L HEPES,2% calf serum,100U/ml penicillin +50U/ml streptomycin,NaHCO3. Prepare the transport medium A by add 5%dextran T500 to the above-mentioned. Prepare the preservation medium B by add 1.35% chondroitin sulfate to the above-mentioned.Preserve corneas and evaluate them:24 fresh Porcine corneas were divided into two groups by random,12 for group A and 12 for group B.They were correspondingly floated in preservation medium A and preservation medium B in DIN bottles which were put in 37℃calorstat. In group A, every 4 corneas were preserved in preservation medium A for 4 ,11 ,18 days respectively. They were shifted to transport medium A with 5 % daxtran 500 and kept for 3 days before use. In group B every 4 corneas were just preserved in preservation medium B for 7 , 14, 21 days respectively. After preservation, every corneas were cut into two pieces,which a half piece was measured thickness, stained with trypan blue- alizarin bordeaux, observed the appearance and shape of CECs, counted CECs and trypan blue positive CECs,and the other piece was ripped away the posterior elastic membrane to detect the apoptosis CECs after fixed by 4% paraformaldehyde by TUNEL technique.By the way above-mentioned, porcine corneas'thickness ,the lost rate of CEC, trypan blue positive CECs and condition of apoptosis were determined and compared respectively before preservation and after 7,14,21 days' preservation. The of media was checked before preservation at random.We observed the color and clarity of medium everyday, and discarded the contaminant corneas in time.We checked the sterility of overall preserved medium.Results The procine corneas which were preserved by the two ways for 3 weeks kept transparent. The longer the corneas were preserved, the shape of endothelial cell not more regular,and the lost rate of endothelial cell was higher,and the number of typan-blue positive ECs was increased. and the number of TUNEL positive cell was raised. Corneas in group B were thicker than group A when they were preserved after 7d,14d and 21d,the differences were significant(P<0.01). The differences of lost rate of endothelial cell and apoptosis rate in group A and B were not significant when they were preserved after 7d and 14d(P>0.05). Lost rate of endothelial cell, apoptosis rate, and typan-blue positive rate in group A are lower than in group B, and the differences were significant(P<0.05). Contamination rate of the two groups were 8.3%.Conclusion The two methods of corneal preservation were feasible. The effect of classic dehydration with dextran was much more obvious, so it was good for transplant operation. The method of add chondroitin sulfate to medium directly was convenient, but the effect of dehydration was not as good as the former. The results of the two methods were more or less identical; When time of preservation last for 3 weeks, the corneal activity which preserved in former medium has an advantage than the latter one. |
