Method for Extracellular Electrochemical Impedance Spectroscopy on Epithelial Cell Monolayers

Epithelial tissues form barriers to the flow of ions, nutrients, waste products, bacteria, and viruses. The conventional electrophysiology measurement of transepithelial resistance (TEER/TER) can quantify epithelial barrier integrity, but does not capture all the electrical behavior of the tissue or provide insight into membrane-specific properties. Electrochemical impedance spectroscopy, in addition to measurement of TER, enables measurement of transepithelial capacitance (TEC) and a ratio of electrical time constants for the tissue, which we term the membrane ratio. This protocol describes how to perform galvanostatic electrochemical impedance spectroscopy on epithelia using commercially available cell culture inserts and chambers, detailing the apparatus, electrical signal, fitting technique, and error quantification. The measurement can be performed in under 1 min on commercially available cell culture inserts and electrophysiology chambers using instrumentation capable of galvanostatic sinusoidal signal processing (4 μA amplitude, 2 Hz to 50 kHz). All fits to the model have less than 10 Ω mean absolute error, revealing repeatable values distinct for each cell type. On representative retinal pigment (n = 3) and bronchiolar epithelial samples (n = 4), TER measurements were 500-667 Ω·cm² and 955-1,034 Ω·cm² (within the expected range), TEC measurements were 3.65-4.10 μF/cm² and 1.07-1.10 μF/cm², and membrane ratio measurements were 18-22 and 1.9-2.2, respectively. Key features • This protocol requires preexisting experience with culturing epithelial cells (such as Caco-2, RPE, and 16HBE) for a successful outcome. • Builds upon methods by Lewallen et al. [1] and Linz et al. [2], integrating commercial chambers and providing a quantitative estimate of error. • Provides code to run measurement, process data, and report error; requires access to MATLAB software, but no coding experience is necessary. • Allows for repeated measurements on the same sample. Graphical overview Electrochemical impedance spectroscopy measurement involves sending a galvanostatic signal through the electrophysiology chamber and across the epithelial cell monolayer (left) and results in complex impedance data at each frequency. This data is then fit to an electrical circuit model to output transepithelial resistance (TER), transepithelial capacitance (TEC), and membrane ratio (α) (right).