The Research About Human Corneal Limbal Stem Cells In Primary Culture

Purpose: To approach cell culture and identifition method of person limbus of cornea stem cells. And human corneal limbal stem cells were observated in morphology. For clinical application of limbal stem cells lay the foundation for treatment.Methods : Limbal organizations were fetched from Ophthalmic surgery patients in 2007. Of these, under the microscope a small piece of limbal choice organizations were fetched from pterygium patients of six cases, some for the (1x1mm). Limbal week organizations were fetched from removable eye patients of 5 cases (three cases of absolute glaucoma, two cases of perforated corneal ulcer). The training, method was same:(1) Tissue culture(2) The same reagents and culture conditions. DMEM/F12 culture medium containing 20% FBS , 10ng/ml EGF, 100u/ml penicillin and 100μg/ml streptomycin 1:1 mixed solu, 37℃incubator greenhouse.Human limbal stem cells were cultured in vitro. The growth of cultured cells in vitro were observed under inverted microscope. To identifice the cells with AE5 that it was anti-keratin monoclonal antibody K3 .Results: First of all, none of cell growth appeared by grooming six cases of small organizations in vitro. In order to find the reasons for no growth of cells, limbal week tissues that we got were trained in vitro. We found all cell growth.We observed morphological of cells and identificed. After 2 days , cells growed from the edge of tissue, the cells are smaller and round. After 4 days, Cells were single-layer, the density were uniform. Fibroblasts cells were arranged at the surface of cells. After 6 days, cells were covered near all the bottom, cell volume increased, Cell morphology were round,polygonak etc. Nucleus were a single cell or multi-core.We identificed the cells that we cultured and found that only a small number of stem cells, and the majority of corneal epithelial cells. Conclusion: 1, Tissue culture: cell cann't growed by small piece of tissue cultured. Limbal stem cells growed by a week limbal tissue culture, but the small number of stem cells.2, AE5 was monoclonal antibody K3 keratin can be used as identification of limbal stem cells.