Ex Vivo Culture And Biological Characteristics Study Of Fetal Limbal Stem Cells

Objective To investigate ex vivo culture technique of fetal limbal stem cells and observe their biological characteristics in order to discover an excellent source of limbal stem cells. Methods Fetal and adult limbal epithelial tissues were cultured in vitro respectively by enzymatic digestion culture method, then the morphological characteristics and the colony-forming efficiency(CFE) of cultured cells were recorded. The expression of 64KD keratin(AE5), acidic keratin of low molecular weight(AE1), proliferating cell nuclear antigen(PCNA) and HLA-DR antigen in the limbus and cultured cells of fetus and adult were detected by enzyme immunocytochemistry staining. The surface characteristics of primary culture cells were observed by scanning electron microscope. Results Fetal limbal stem cells could develop and be passaged in this culture condition. They grew swiftly and reached growth peak at 8～10 days in primary culture when their CFE was (26.18±4.75)%. They could keep high proliferating rate in subculture. Adult limbal stem cells grew less swiftly than fetal cells and reached growth peak at 6～8 days in primary culture when their CFE was (15.45±3.88)%, moreover, their proliferating ability declined distinctly along with the passage times increasing. The difference of the CFE value of the two kinds of primary culture cells was statistically significant (t＝6.78, P＜0.001). In the results of enzyme immunocytochemistry staining, many cells in basal layer of fetal limbus were AE5 negative, AE1, PCNA and HLA-DR positive. A absolute majority of primary culture fetal cells were AE5 and HLA-DR negative, AE1 and PCNA positive, moreover, a majority of fetal cells in the third passage were AE5 negative. By comparison to the staining results of fetal limbus and cultured cells, the proportion of the cells with staining characteristics of above-mentioned stem cells was lower but the staining results of HLA-DR were similar in the staining results of adult limbus and cultured cells. The morphological characteristics of primary culture cells of fetus and adult were mainly spherical shape or short column shape and had plenty of processes and microvilli on surface by scanning electron microscope observation, but adult cells were bigger in diameter. Conclusion Fetal limbal stem cells possessing stem cells characteristics including high proliferating ability can be cultured successfully by enzymatic digestion culture method. Because the antigenicity of cultured fetal cells is lower than that of fetal limbus and the biological characteristics of fetal limbal stem cells are superior to that of adult limbal stem cells, fetal stem cells are an excellent source of limbal stem cells and may show broad applying prospect in clinical ocular surface reconstruction.