Abstract
Abstract: :
Purpose: To characterize a spontaneous cell line derived from normal human conjunctiva (NHC). The final goal is to have an in vitro system to study the physiopathology of the human conjunctiva. Method: Cells were cultured in DMEM/F-12 supplemented with antibiotics, insulin, EGF, cholera toxin, fetal bovine serum and hydrocortisone. Cell viability was frequently checked. Plating efficiency, colony-forming efficiency and colony size were calculated around passage 20. Different techniques were used to characterize NHC cells in several passages: chromosome analysis; Giemsa staining to observe general morphology; Alcian blue/PAS and lectin (AIA, DSA, GNA, HPA, LFA, MAA, PNA, UEA-I, and VVA) stainings to identify secretory cells and secretion products; transmission and scanning electron microscopy to confirm epithelial characteristics; immunofluorescence with monoclonal antibodies against KI67, citokeratins, desmoplakins, vimentin, EGF receptor, FVIII, CD1 and AS02 to rate proliferative capability, to detect typical epithelial markers and to exclude contamination by other cell types; and RT-PCR analysis of mRNA expression for MUC1, MUC2, MUC4, MUC5AC and MUC7 mucin genes. Results: Summarized, results showed: 1) cells have been continuously proliferating until passage 100 always showing viability higher than 95%, 2) contaminating cell types were absent, 3) epithelial characteristics were maintained in vitro, 4) PAS+ cells were observed, 5) lectin-binding sites were detected for all lectins except LFA and UEA-I, 6) cytokeratins 3 and 7 and vimentin were immunodetected, and 7) MUC1, MUC2 and MUC4 mucin genes were expressed in vitro. Also, microorganism contamination was excluded. Conclusion: NHC cell line retain many of the morphologic and functional characteristics of conjunctival epithelial cells in vivo. Therefore, we propose this cell line as a new in vitro system to study the physiopathology of the human conjunctiva.
Keywords: 365 conjunctiva • 375 cornea: surface mucins • 376 cornea: tears/tear film/dry eye