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Y. Ban, Y. Yoshida, S. Kinoshita; Evaluation of Barrier Function in hTERT Immortalized Corneal and Conjunctival Epithelium by Fluorescence-Based Cellular Permeability Assay. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2553. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To create a reproducible and accurate fluorescence-based cell culture assay and evaluate the barrier function in hTERT immortalized conjunctival and corneal epithelium.
Conjunctival and corneal epithelial cells immortalized by preventing telomere shortening by transduction with hTERT were cultured on 12-mm Transwell filters at a density of 4 x 105 cells/cm2. The morphology of the cultures was assessed by labeling actin filaments or tight junction-related proteins by indirect immunofluorescence. Permeability assays were performed after 12 days in culture, when the TER (transepithelial electrical resistance) had reached a plateau. Four mg/ml fluorescein isothiocyanate (FITC) Dextran 4000-conjugate (MW4000) was added to the apical medium chamber. Aliquots of 30µl were taken from cell cultures at 30, 60, and 90 min. The fluorescence of each sample was determined in a fluorescence spectrofluorometer at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. A standard curve was created based upon fixed concentrations of fluorescence. The TER was measured using endohm electrodes and an EVOM voltohmmeter (World Precision Instruments, LTD., Hertfordshire, UK).
After 12 days in culture, the permeability of FITC-Dextran was 0.0188µl/cm3/min for corneal cells and 0.0398µl/cm3/min for conjunctival cells.FITC-Dextran permeability assay and TER had moderate negative correlation (correlation coefficient: -0.44).
Corneal and conjunctival epithelium forms a barrier that isolates the eye from the outside environment and regulates passive movement through the paracellular pathway. Tight junctions create this barrier. By fluorescence-based cell culture assay, hTERT immortalized corneal epithelium had a tighter barrier than hTERT immortalized conjunctival epithelium, such as in the in vivo situation. This assay had moderate correlation with the TER, another measure of permeability which measures resistance to ion flow across the cell layer.
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