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Y. Yoshida, Y. Ban, S. Kinoshita; Evaluation of Barrier Function in hTERT Immortalized Corneal and Conjunctival Epithelium by Measuring Transepithelial Electrical Resistance. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2551. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the barrier function in hTERT immortalized conjunctival and corneal epithelium by measuring ion flux.
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. Cultures were maintained in serum-free medium during the first 6 days, then maintained in stratification medium. The morphology of the cultures was assessed by labeling actin filaments with fluorescently tagged phalloidin or tight junction-related proteins by indirect immunofluorescence. The TER (transepithelial electrical resistance) was measured every other day using endohm electrodes and an EVOM voltohmmeter (World Precision Instruments, LTD., Hertfordshire, UK). TER measures resistance to ion flow across the cell layer. The change of TER that occurred with Ca2+ elimination by adding 3mM EDTA (the chelate of Ca ions) was also investigated before and after the cell stratification.
Before the medium change to stratification medium, culture epithelium was only partially stratified to two layers. Stratification developed gradually, and finally became 3 layers. After 10 days in culture, the TER had reached a plateau; 200Ω ·cm3 for corneal cells and 50Ω ·cm3 for conjunctival cells. As expected for tight junctions, the TER was reversibly reduced by a15-min incubation in medium containing 3mM EDTA. Thirty minutes were required for recovery to the original TER. Before the stratification, TER was decreased to 10% but was decreased to just 70% after the stratification.
Conjunctival and corneal epithelium tight junctions are present between the most superficial cells, and form a barrier that isolates the eye from the outside environment and regulate the passive movement of fluid, electrolytes, macromolecules, and cells through the paracellular pathway. hTERT immortalized corneal epithelium had a tighter barrier than hTERT immortalized conjunctival epithelium, such as in the in vivo situation. In stratified epithelium, TER consist of two factors: 1) resistance made up from the tight junction and 2) that which comes from cell tiers. TER measurement was useful because it was technically easy and stable.
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