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Thomas Duncan, Koichi Baba, Yoshinori Oie, Kohji Nishida; A Novel Method for Testing Tight Junction Function in Cultured Epithelial Cell Sheets. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5512.
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© ARVO (1962-2015); The Authors (2016-present)
A cultured epithelial cell sheet should serve as a physical barrier to the passage of ions, molecules and other particles. Here, we develop a novel method for testing the barrier function of cultured epithelial cell sheets, specifically targeting tight junction complexes.
Epithelial cell sheets derived from oral or corneal stem cells were cultured until stratified and mature - as confirmed through immunohistochemistry and TEER (transepithelial electrical resistance). A calcium chelator (EGTA) was used to disrupt tight junction function and provide a model for an immature or defective cell sheet. Suspensions of varying fluorescent nanoparticles were applied to a range of cell sheets with properly functioning or affected tight junctions. This method was compared with routine TEER measurements. Confocal microscopy and 3D modelling was then used to obtain qualitative and quantitative data on the nanoparticles fluorescence wavelength, relative intensity and distribution throughout the cell sheet.
The nanoparticles fluorescence wavelength was dependent on their shape and size - smaller nanoparticles emitted 525nm wavelength light, whilst the larger nanoparticles fluoresced at 655nm. In epithelial cell sheets with properly functioning tight junctions, the intercellular spaces were restricted. Consequently only trace amounts of small sized nanoparticles were detected through low intensity fluorescence (1250I) at predominantly the 525nm wavelength. Following application of EGTA, TEER showed a 9-fold reduction in electrical resistance (28±2Ω) when compared to control samples (3±1Ω). In these EGTA treated cell sheets, a 2-3 fold increase in fluorescence intensity (3250I) was observed, with both the 525nm and 655nm wavelength nanoparticles present more ubiquitously throughout the intercellular spaces.
The predominant nanoparticle size, relative intensity and distribution were affected by tight junction function and subsequently how tightly bound together adjacent cells were. This novel method offers unique qualitative and quantitative data on surface morphology and cell sheet integrity that correlates specifically to tight junction functionality. As such, it may be used for quality testing of cultured cell sheets for use in tissue repair and regeneration.
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