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S.–H. Chung, E.K. Kim, K.Y. Seo; Morphological and Functional Characteristics of Primary–Cultured Human Conjunctival Epithelium . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4941.
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© ARVO (1962-2015); The Authors (2016-present)
To describe the establishment of a primary culture system of human conjunctival epithelial cells using a serum–free conditioned medium and the characterization of these cells using mucin gene expression as markers of differentiation
Small pieces of normal conjunctiva were biopsied from patients undergoing cataract surgery and subcultured in BEGM (bronchial epithelial growth medium) under serum–free condition. Passage 2 cells were differentiated in air–Liquid Interface (ALI) culture for 2 days, 1, 2, and 3 weeks after confluence. Cellular morphology was analyzed by light and scanning electron microscopy (SEM), and histology. The expression of cytokeratin 19 (CK 19) was evaluated by immunocytochemistry. The amount of apical secreted mucin was measured by dot blotting using monoclonal anti–MUC5AC and anti–mucin antibody (17Q2) on each culture period. The expression of mucin gene 1 (MUC1), MUC4, MUC16, and MUC5AC were determined on each culture periods by reverse transcription (RT)–polymerase chain reaction.
The primary culture of human conjunctival epithelium expressed CK 19, conjunctival differentiation marker. An increase in stratification of up to six to seven layers was noted on 2–week ALI culture and there was no significant increase in stratification compared with the 3– week ALI culture. Goblet cells stained with AB/PAS (Fig. 1) and microvili on the cell surface was noted in SEM on 2–week ALI culture. The expression of MUC1, MUC4, MUC16, MUC5AC and the secretion of mucin increased with culture time and showed a peak on the 2 weeks after confluence, and then decreased.
A primary–cultured conjunctival epithelium demonstrated well defined multi–layered epithelial features and exhibited the mucin gene expression repertoire of their native epithelium. These cells may be useful experimental tool in the field of ocular surface cell biology and determining regulation of ocular surface mucin gene expression, and goblet cell differentiation.
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