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bioRxiv · 10.1101/2023.07.11.548631

Sensitivity and validation of porous membrane electrical cell substrate impedance spectroscopy (PM-ECIS) for measuring endothelial barrier properties

Ugodnikov, A.·Chebotarev, O.·Persson, H.·Simmons, C. A.
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Abstract

Conventional trans-endothelial electrical resistance (TEER) setups are invasive and cannot directly measure monolayer integrity in co-culture. These limitations are addressed by porous membrane electrical cell-substrate impedance sensing (PM-ECIS), which measures barrier integrity in cell monolayers grown directly on permeable membranes patterned with electrodes. Here we advance the design and utility of PM-ECIS by investigating its sensitivity to working electrode size and correlation to TEER. Gold electrodes were fabricated on porous membrane inserts using hot embossing and UV lithography, with working electrode diameters of 250, 500, and 750 {micro}m within the same insert. Frequency scans of confluent primary endothelial monolayers showed normalized resistance peaking at 4 kHz, consistent with traditional solid substrate ECIS. Sensitivity to resistance changes (4 kHz) during endothelial barrier formation was inversely proportional to electrode size, with the smallest electrodes being the most sensitive (p<0.001). Similarly, smaller electrodes were most sensitive to changes in impedance (40 kHz) corresponding to cell spreading and proliferation (p<0.001). Barrier disruption with EGTA was detectable by 250 and 750 {micro}m (p<0.01), and 500 {micro}m electrodes (p=0.058). Resistances measured by PM-ECIS vs. TEER for sodium chloride solutions were positively and significantly correlated for all electrode sizes (r>0.9; p<0.0001), but only with 750 {micro}m electrodes for endothelial monolayers (r=0.71; p=0.058). These data inform design and selection of PM-ECIS electrodes for specific applications, and support PM-ECIS as a promising alternative to conventional TEER due to its capacity for direct, non-invasive, and real-time assessment of cells cultured on porous membranes.

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Ugodnikov, A., Chebotarev, O., Persson, H., Simmons, C. A.. 2023-07-12. Sensitivity and validation of porous membrane electrical cell substrate impedance spectroscopy (PM-ECIS) for measuring endothelial barrier properties. https://doi.org/10.1101/2023.07.11.548631

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