The Clinic Studies For Human Limbal Stem Cells Culture In Vitro And Autologous Transplantation

Objective To construct the model of human limbal stem cells culture in vitro and evaluate the effect of autologous limbal stem cells transplantation treating with unilateral ocular surface disease.Methods Small specimen (size, about 2.0×2.0 mm;thickness, about 0.2 mm) of limbal epithelial cells were extracted from the corneoscleral donor rims following penetrating keratoplasty, each of them were cut into four tissues and loosen by enzyme digestion. We cultivated them on acellulax human amniotic membrane with DMEM/F12 which containing 20%FBS (fetal bovine serum) for 14d in total. We observed the cells using inverted microscope every day. Then the cells were examined using methods of histology and immunohistochemistry on the 7th and the 14th day respectively. The limbal epithelial cells of same size were extracted from the patients' healthy fellow eye and were cultivated with the method mentioned above. Autologous transplantation was performed when the cells formed 3~4 layers. All observations were kept in records periodically after the operation for the patients' visual acuities, cornea re-epithelialization, neovascularity, and corneal opacification.Results A confluent primary culture of limbal epithelial cells was established on a cellular human amniotic membrane on the 7th day. The cultivated limbal epithelial cells on the amniotic membrane formed multi-layer after 14d 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...