Sensitivity and Validation of Porous Membrane Electrical Cell Substrate Impedance Spectroscopy (PM-ECIS) for Measuring Endothelial Barrier Properties.

Measurement of endothelial and epithelial barrier integrity is important for a variety of in vitro models, including Transwell assays, cocultures, and organ-on-chip platforms. Barrier resistance is typically measured by trans-endothelial electrical resistance (TEER), but TEER is invasive and cannot accurately measure isolated monolayer resistance in coculture or most organ-on-chip devices. 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 advanced the design and utility of PM-ECIS by investigating its sensitivity to working electrode size and correlation with TEER. Gold electrodes were fabricated on porous membrane inserts using hot embossing and UV lithography, with working electrode diameters of 250, 500, and 750 µm within the same insert. Sensitivity to resistance changes (4 kHz) during endothelial barrier formation was inversely proportional to electrode size, with the smallest 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 both EGTA and thrombin was detectable by all electrode sizes. 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 µm electrodes for endothelial monolayers (r = 0.71; p = 0.058). These data inform the design and selection of PM-ECIS electrodes for specific applications and support PM-ECIS as a promising alternative to conventional TEER for direct, noninvasive, real-time assessment of cells cultured on porous membranes in conventional and organ-on-chip barrier models.

Porous Membrane Electrical Cell-Substrate Impedance Spectroscopy for Versatile Assessment of Biological Barriers In Vitro.

Cell culture models of endothelial and epithelial barriers typically use porous membrane inserts (e.g., Transwell inserts) as a permeable substrate on which barrier cells are grown, often in coculture with other cell types on the opposite side of the membrane. Current methods to characterize barrier function in porous membrane inserts can disrupt the barrier or provide bulk measurements that cannot isolate barrier cell resistance alone. Electrical cell-substrate impedance sensing (ECIS) addresses these limitations, but its implementation on porous membrane inserts has been limited by costly manufacturing, low sensitivity, and lack of validation for barrier assessment. Here, we present porous membrane ECIS (PM-ECIS), a cost-effective method to adapt ECIS technology to porous substrate-based in vitro models. We demonstrate high fidelity patterning of electrodes on porous membranes that can be incorporated into well plates of a variety of sizes with excellent cell biocompatibility with mono- and coculture set ups. PM-ECIS provided sensitive, real-time measurement of isolated changes in endothelial cell barrier impedance with cell growth and barrier disruption. Barrier function characterized by PM-ECIS resistance correlated well with permeability coefficients obtained from simultaneous molecular tracer permeability assays performed on the same cultures, validating the device. Integration of ECIS into conventional porous cell culture inserts provides a versatile, sensitive, and automated alternative to current methods to measure barrier function in vitro, including molecular tracer assays and transepithelial/endothelial electrical resistance.

Bridging barriers: advances and challenges in modeling biological barriers and measuring barrier integrity in organ-on-chip systems.

Biological barriers such as the blood-brain barrier, skin, and intestinal mucosal barrier play key roles in homeostasis, disease physiology, and drug delivery - as such, it is important to create representative in vitro models to improve understanding of barrier biology and serve as tools for therapeutic development. Microfluidic cell culture and organ-on-a-chip (OOC) systems enable barrier modelling with greater physiological fidelity than conventional platforms by mimicking key environmental aspects such as fluid shear, accurate microscale dimensions, mechanical cues, extracellular matrix, and geometrically defined co-culture. As the prevalence of barrier-on-chip models increases, so does the importance of tools that can accurately assess barrier integrity and function without disturbing the carefully engineered microenvironment. In this review, we first provide a background on biological barriers and the physiological features that are emulated through in vitro barrier models. Then, we outline molecular permeability and electrical sensing barrier integrity assessment methods, and the related challenges specific to barrier-on-chip implementation. Finally, we discuss future directions in the field, as well important priorities to consider such as fabrication costs, standardization, and bridging gaps between disciplines and stakeholders.