人结膜上皮细胞的培养鉴定及液氮冻存

目的：探索人眼结膜上皮细胞体外培养和细胞保存的最佳方法,建立人结膜上皮细胞,进一步研究人结膜的病理、生理特点及为毒理试验提供可靠的细胞模型。方法：分别用组织块培养法、机械分离法及混合消化液培养法体外培养正常成年人结膜上皮细胞,通过观察细胞形态、生长特性并用原位免疫组化方法鉴定培养细胞;收集第３代和第４代融合的细胞液氮冻存,保存３０天后复苏,观察复苏成功率。结果：３种取材方法中,混合消化液培养法细胞

The Culture and Cryopreservation of Human Conjunctival Epithelium in Vitro

Purposes: To detect the best method of culture and preservation of human conjunctivalepithelia.Methods: Human conjunctival epithelia were cultured by tissue inoculation, mechanicalseparation or enzyme digestion with 0. 25% trypsin. The cultured cells were identifiedthrough their morphology, growing features and immunohistochemical staining. The thirdand fourth passage confluent cells were frozen in liquid nitrogen, and resuscitated 30days later.Results: Cells from tissue digested with 0.25% Trypsin grew much better than thosefrom tissue inoculation. No cell outgrew in mechanical separating group. The culturedcells spread along the flask in polygonal shape and positive in pan-Keratin staining.Ninty percent of the cryopreserved cells were successfully resuscitated .Conclusion: Tissue digestion with trypsin is the best approach to culture humanconjunctival epithelium, and the cultured cells can be cryopreserved in liquid nitrogen.Eye Science 2000; 16: 135 - 138,