Abstract
Nanofibrous scaffolds provide high surface area for cell attachment, and resemble the structure of the collagen fibers which naturally occur in the basement membrane and extracellular matrix. A label free and non-destructive method of assessing the interaction of cell tissue and scaffolds aids in the ability to discern the effective quality and magnitude of any scaffold modifications. Impedance cell spectroscopy is a biosensing method that employs a functional approach to assessing the cell monolayer. The electrical impedance barrier function of a cell monolayer represents the level of restriction to diffusion of charged species between all adjacent cells across an entire contiguous cellular monolayer. The impedance signals from many individual paracellular pathways contribute to the bulk measurement of the whole monolayer barrier function. However, the scaffold substrate must be entirely porous in order to be used with electrochemical cell impedance spectroscopy (ECIS) and cells must be closely situated to the electrodes. For purposes of evaluating cell-scaffold constructs for tissue engineering, non-invasive evaluation of cell properties while seeded on scaffolds is critical. A Transwell-type assay makes a measurement across a semi-permeable membrane, using electrodes placed on opposing sides of the membrane immersed in fluid. It was found that by suspending a nanofiber scaffold across a Transwell aperture, it is possible to integrate a fully functional nanofiber tissue scaffold with the ECIS Transwell apparatus. Salivary epithelial cells were grown on the nanofiber scaffolds and tight junction formation was evaluated using ECIS measurements in parallel with immunostaining and confocal imaging. The trans-epithelial resistance increased coordinate with cell coverage, culminating with a cell monolayer, at which point the tight junction proteins assemble and strengthen, reaching the peak signal. These studies demonstrate that ECIS can be used to evaluate tight junction formation in cells grown on nanofiber scaffolds and on effects of scaffold conditions on cells, thus providing useful biological feedback to inform superior scaffold designs.
Highlights
This research investigated a continuous, non-destructive method of characterizing the function of the cell–cell junctions in continuous epithelial cell line monolayers
TEER measurement reduces the likelihood of misinterpreting scaffold characteristics because of non-representative point-of-interest selection, which can occur during microscopy, by generating an average measurement over many cell junctions throughout the cell monolayer
Eliminating the glass substrate leaves a very thin sheet of nanofibers, which is permeable to diffusion of all media components, providing for the supply of necessary media nutrients to the Transwell cell monolayer as well as facilitating voltage-imposed trans-membrane diffusion
Summary
This research investigated a continuous, non-destructive method of characterizing the function of the cell–cell junctions in continuous epithelial cell line monolayers. The lock-in amplifier circuitry in the ECIS system allows for precise signal deconvolution of the resistance from the complex impedance, separating it from the capacitive element. This enables the active, direct probing of the permeability of the pathway (barrier function) without disturbing the living cells. These assays are performed by seeding cells on a thin, porous membrane in a Transwell assembly and allowing a monolayer to form, which retains a free permeable pathway through the membrane and monolayer to take electrical impedance measurements [3]. This opens up more types of investigation, and can explore even transient effects of scaffolds on cell behavior, other than molecular expression levels and localization, that are possible with destructive endpoint assays
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