The Experimental Studies For Limbal Epithelial Cells Culture And Allogeneic Transplantation

Objective To detect the effect of method which limbal epithelial cells were cultivated on amniotic membrane by tissue inoculation. To observe the effect of amniotic membrane containing cultivated limbal epithelial cells were transplanted to allogeneic limbal stem cell damaged rabbit cornea.Methods Small specimens (size, about 2.0mmx2.0mm; thickness, about 200um)of limbal epithelial cells were excised from the limbal regions in rabbits; each of them was cut into four tissues(lmm x 1mm). Making tissues loosen by enzyme digestion and cultivating them on acellular human amniotic membrane. The cultivated limbal epithelial cells on amniotic membrane were observed by inverted microscope every two or three days, and examined by histology, immunohistochemical and electron microscop. 26 rabbits underwent corneal epithelium curettement and limbal ablation to induce limbal stem cell deficiency and were randomly divided into two groups, experimental group and control group(n=12 for each group). Limbal epithelial cells were cultivated by tissue inoculation on amniotic membrane for about sixteen days, experimental group were transplanted with amniotic membrane that contained cultivated allogeneic limbal epithelial cells. Control group were transplanted with amniotic membrane transplantation only. Outcome(corneal opacity, vascularization,the process of corneal reepithelialization) was assessed by daily slit-lamp examination, and histopathologic examination was carried out regularly.Results A confluent primary culture of limbal epithelial cells was established on acellular human amniotic membrane on the 8th day.The cultivated limbal epithelial cellson the amniotic membrane formed multilayer after 16 days in culture, the cultivated limbal epithelial cells formed 3~4 layers on acellular human amniotic membrane and attached well to the underlying amniotic membrane. The cultured cells on amniotic membrane were stained positive in Pan-keratin antibody and anti-proliferating cell nuclear antigen(anti-PCNA). 6 weeks later, 26 rabbit eyes of corneal epithelial curettement and limbal ablation developed features of limbal stem cell deficiency including conjunctival epithelial ingrowth, vascularization and chronic inflammation. Experimental group entire corneal were all successfully epithelialied up to 5 days after surgery while control group rabbits eyes of corneal epithelia remained full defected. From the 16th day, experimental group rabbits eyes appeared to undergo rejection while control group not.Conclusions The cultivated limbal epithelial cells by tissue inoculation can form multi-layered on amniotic membrane, and some of them have ability in proliferation. Cultivated limbal epithelial cells on amniotic membrane by tissue inoculation can be transplantated to rabbit eyes of limbal stem cell deficiency. All experimental group corneas were all epithelialized in the earlier postoperative period and were undergone rejection in the later postoperative period.