Studies On The Establishment, Characterization And Cloning Of An Untransfected Human Corneal Epithelial Cell Line

Corneal blindness is a common eye disease, and there are more than4million blind patients in China caused by corneal blindness, especially by pathological changes in corneal epithelium. Corneal transplantation is the sole way to cure corneal blindness, however most of the patients could not accecpt corneal transplantation because of the serious shortage of donated corneas. In recent years, corneal tissue engineering has opened a new path for clinical corneal transplantation, and in vitro reconstruction of tissue-engineered human corneal epithelium as the alnertives of human corneal epithelium(HCEP), is the effective way to solve donated corneas shortage and cure HCEP diseases. The key issues of HCEP reconstruction are the source of good HCEP seed cells and suited carriers. To seed cells, human corneal cell lines as a powerful research tool can provide a available source of HCEPC, however the established HCEPC cell lines are transfected by oncygenes and unable use for transplatation. Unfortunately, there is no report on the establishment of a HCEP cell line without transfection. In order to solve the HCEP seed cells source problem, This study was conducted to establish an untransfected corneal epithelial cell line from donated corneal tissue, and to obtain a monoclonal cell line with normal morphology and characteristic HCEP functions on the basis of the HCEP cell line.To initiate the in vitro culture of HCEPC successfully, the corneal epithelium was digested with0.25%trpsin, and were sliced and inoculated into a0.01%gelatin-coated well of a24-well culture plate with the epithelial side down. After about12h, the medium was replaced with20%FBS-DMEM/F12medium (pH7.2) supplemented with fibroblast growth factor (bFGF), epidermal growth factor (EGF), chondroitin sulfate, carboxymethyl-chitosan and collagen IV, The primary culture was carried out at37℃with5%CO2with medium refreshed every4days. During primary culture, HCEPC migrated from cornea pieces in3d, growing into a confluent monolayer in22d. They were highly transparent and polygonal in shape. During subculture, the HCEPC maintained their polygonal shape, and grew and proliferated at a steady rate. After frozen reservation, their shape and proliferation are not affected. After being subcultured to over passage60, a novel continuous untransfected HCEP cell line was established. Morphological observation, growth properties, chromosome analysis, immuno-cytochemistry analysis and tumorigenesis assay were used to identify the properties, functions and latent risk of tumorigenicity of established HCEP cell line. According to the results of morphological observation (including optical microscopy and scanning electron microscopy), vitro-cultured HCEPC own higher transparency and plump conformation as stable epithelial-like cell morphology and the cell surface is rich in microvilli and locomotion. Proliferation time of the cells is40.56h indicated keep strong ability to cleavage. Chromosome analysis showed that the cell lines have their Characteristic chromosome number in46, although some cells are chromosomal aneuploidy. The cells express cytokeratin3/12/19positively, which is a specific marker of HCEPC. Besides, the cells had no tumorigenicity. Therefore, the untransfected HCEPC can be used as seed cells for in vitro reconstruction of TE-HCEP.In order to screen out the HCEPC with46chromosomes as seed cells, clonal culture was performed on the basis of the cell line. The cells were clonal amplificated using finite dilution method. A monoclonal cell line HCEP-5A1has chromosome number in46. According to the results of immunocytochemistry analysis, the HCEP-5A1cells positively express HCEP makers K3/12, connectional proteins Connecxin-43and Integrin131, and functional proteins PMCA1/4and14-3-3s. All these indicated that the HCEP-5A1cells were derived from HCEPC; and they maintain the characteristic HCEP functions like cell junctions, membrane transportations and forming stratified layers. Therefore, the HCEP-5A1cell line can be used as HCEP seed cells.In conclusion, current article successfully established an non-transfected, non-oncogenic HCEP cell line, and screened out a monoclonal cell lines HCEP-5A1with46chromosomes. Therefore, we successfully obtain the HCEP seed cells, and supply reference for large scale vitro-reconstruction, clinical application and vitro-reconstruction of tissue engineering of human whole cornea.