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

Abstract

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

“…Implementation of ECIS electrodes on porous membrane cell culture inserts has recently been performed by Rajasekaran et al 237 and by Chebotarev et al 247,248 The latter studies introduced a novel, cost-effective fabrication method for integrating gold electrodes onto porous membrane in Transwell-like inserts, enabling sensitive detection of barrier formation and disruption, as well as simultaneous validation against conventional TEER and molecular permeability in the same devices. 247,248 This electrode configuration lends itself well to OOC barrier modes, facilitating direct measurements of monolayers in complex OOC systems that contain multiple cell types, as demonstrated in models of the placenta 74 and BBB 73 (Fig. 5F).…”

“…270 Similarly, heat-bonded gold leaf provides a flat, continuous electrode surface with lower resistivity compared to sputtered gold. 247 Interestingly, inkjet-printed gold electrodes can have as much as 10-fold greater sensitivity compared to sputtered gold. 258 Meanwhile, the surface morphology – and therefore sensitivity – of nanoporous gold may vary depending on fabrication method ( e.g.…”

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

“…Each insert comprised gold electrodes patterned onto a porous transparent PETE membrane filter (3.0 μm, 6 × 10 5 pores/cm 2 ; Sterlitech, 1300032) on which endothelial cells were cultured. Device fabrication was performed based on published protocols . Briefly, this process involves thermal bonding of gold leaf (Gold Leaf Supplies, 2300/RUXX) to the membrane, followed by UV lithography to pattern the electrodes and laser cutting of the electrode-patterned membrane to size and shape for mounting in the custom insert (Figure A).…”

“…Briefly, this process involves thermal bonding of gold leaf (Gold Leaf Supplies, 2300/RUXX) to the membrane, followed by UV lithography to pattern the electrodes and laser cutting of the electrode-patterned membrane to size and shape for mounting in the custom insert (Figure A). We previously showed that heat-bonded gold leaf PM-ECIS electrodes have more uniform coverage and thickness than electrodes etched on sputter-coated membranes, resulting in similar variance but lower overall resistivity vs sputtered electrodes …”

“…Furthermore, solid substrates do not enable direct comparison against TEER, the gold-standard methods for electrical sensing of barrier integrity . Recently, ECIS electrodes have been incorporated into porous membranes configured as traditional Transwell-like cell culture inserts, , and use of such electrodes has also been explored in OOC format . While these initial studies demonstrated the potential of porous membrane ECIS (PM-ECIS) to monitor the formation, disruption, and permeability of cell barriers, they did not systematically assess the effect of working electrode size, which is known to impact the dynamic range of ECIS measurements in traditional solid-substrate systems. − Furthermore, direct comparisons between PM-ECIS and TEER measurements and their electrode size dependency have not been made.…”