Ex Vivo Preservation And Expansion Of Human Limbal Epithelial Stem Cells On Amniotic Membrane Cultures By Subcutaneous Implantation For The Limbal Stem Cell Deficiency

Purpose:To study the method of culturing limbal stem cells (LSCs) of rabbit by subcutaneous implantation. To investigate the treatment of rabbit eyes with limbal stem cell deficiency (LSCD) by transplanting cultured limbal stem cells with the ammon.Methods:The LSCs tissues of left eye from the fifty-five rabbits (55eyes) rabbits were culture on human amniotic membrane (AM), then transplanted into the subcutaneous tissue of their abdomen. The follow-up time was once per week in the one month. Epithelial morphology was studied by histology, and phenotype was defined by immunostaining with monoclonal antibodies to P63and E-cadherin. The LSCD models in the right eye of all rabbits were established by moderate chemical burn. At last, fifty rabbits(50eyes) were divided into five groups (the AM+LSC1W group, the AM+LSC2W group, the AM+LSC3W group, the AM+LSC4W group, and the AM group) randomly, with10cases in each group. The AM+LSC1W group were performed the culture limbal stem cells (1W) with the amniotic membrane. The AM+LSC2W group were performed the culture limbal stem cells (2W) with the amniotic membrane transplantation. The AM+LSC3W group were performed the culture limbal stem cells (3W) with the amniotic membrane transplantation. The AM+LSC4W group were performed the culture limbal stem cells (4W) with the amniotic membrane transplantation. The AM group were performed the amniotic membrane transplantation. The follow-up time was once per day in one week, then once per week in the one month. The symblepharon were observed, and the corneal neovascularization and opacity were scored at postoperative1month.Result:The cultivated rabbit limbal stem cells on the amniotic membrane grew well after subcutaneous implantation. The limbal explants cultured on AM (1W-2W) were strongly positive for P63but negative for E-cadherin, after subcutaneous implantation in rabbits.3weeks, the explants were slightly positive in the basal epithelial cells for P63, while the suprabasal epithelial cells were slightly positive for E-cadherin.4weeks, the explants strongly positive in suprabasal cells for E-cadherin, and were slightly positive in the basal epithelial cells for P63. There were not statistical differences in the average score of corneal neovascularization and opacity in all groups, after the LSCD models were established. After the surgery, the average score of corneal neovascularization of the AM+LSC (1W、2W、3W) group were (0.80±0.630) points,(0.90±0.571) points, and (1.40±0.70) points; the average score of corneal opacity were (0.90±0.57) points,(0.90±0.32) points, and (1.30±0.48) points. There were statistical differences in score of corneal neovascularization and opacity before and after surgery (all P<0.05). The average score of corneal neovascularization of the AM+LSC4W group and AM group were (3.30±0.48) points and (3.20±0.63) points. The average score of corneal opacity were (2.40±0.52) points and (2.50±0.52) points. There were all not statistical differences before and after surgery (all P>0.05).Conclusion:The limbal stem cells of rabbit could be successfully cultured on AM by subcutaneous implantation. Using amniotic membrane as a carrier to transplant cultured limbal stem cells for the LSC, restoration of the epithelialization, inhibition of neovascularization could be achieved.