Method for Extracellular Electrochemical Impedance Spectroscopy on Epithelia

Epithelial tissues form barriers to the flow of ions, nutrients, waste products, bacteria, and viruses. The conventional electrophysiology measurement of transepithelial resistance (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 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 techniques, and error quantification. The measurement can be performed in approximately one minute using instrumentation capable of galvanostatic sinusoidal signal processing ( $4\mu \text{A}$ amplitude, 2 Hz-50 kHz). All fits to the model have less than $10\Omega$ mean absolute error, revealing repeatable values distinct for each cell type. On representative retinal pigment (n=3) and bronchiolar epithelial samples (n=4), we measured TER $500-667\Omega .{\text{cm}}^2$ and $955-1034\Omega .{\text{cm}}^2$ , within the expected range, TEC $3.65-4.10\mu \text{F}/{\text{cm}}^2$ and $1.07-1.10\mu \text{F}/{\text{cm}}^2$ , and membrane ratios 18-22 and 1.9-2.2, respectively.