Impedance Spectroscopy for Accurate TEER Measurement

Impedance spectroscopy when combined with a fitting algorithm provides a more accurate representation of TEER values than traditional DC/single frequency AC measurement systems.^{34 (link)} Impedance spectroscopy is performed by applying a small amplitude AC excitation signal with a frequency sweep and measuring the amplitude and phase response of the resulting current. Figure 2 (a) indicates a schematic which illustrates the concept of impedance measurement.
Electrical impedance (Z) is the ratio of the voltage-time function V (t) and the resulting current-time function I (t):
where Vo and Io are the peak voltage and current, f is the frequency, t is the time, Φ is the phase shift between the voltage-time and current-time functions, and Y is the complex conductance or admittance. Z is a complex function and can be described by the modulus |Z| and the phase shift Φ or by the real part Z_R and the imaginary part Z_I, as illustrated in the Figure 2(b). An in depth analysis of impedance spectroscopy has been published.³⁵ Impedance measurement across a wide spectrum of frequencies instead of a DC/single frequency AC TEER measurement can provide additional information about the capacitance of the cell layer. An automated measurement system (cellZscope^®, nanoAnalytics GmbH, Germany) has been developed for measuring the transendothelial/epithelial impedance of various barrier-forming cells cultured on permeable membranes of standard cell culture inserts. An equivalent circuit analysis of the measured impedance spectrum is performed to obtain the electrical parameters that can be applied to characterize the cellular barrier properties. Figure 3 (a) (adapted from Benson et al.^{26 (link)}) shows a typical equivalent circuit diagram that can be applied to analyze the impedance spectrum of cellular systems.^{26 (link)} In this circuit, the current can flow through the junctions between cells (paracellular route) or through the cell membrane of the cells (transcellular route). The tight junction proteins in the paracellular route contribute to an ohmic resistance (R_TEER) in the equivalent circuit. Each lipid bilayer in the transcellular route contributes to a parallel circuit^{26 (link)} consisting of ohmic resistance (R_membrane) and an electrical capacitance (C_C). In addition to these elements, the resistance of the cell culture medium (R_medium) and the capacitance of the measurement electrodes (C_E) also have to be considered. The high values of R_membrane causes the current to mostly flow across the capacitor and allows an approximation where R_membrane can be ignored^{26 (link)} and the lipid bilayers can be represented with just C_C. Based on this approximation, the equivalent circuit diagram can be further simplified as shown in Figure 3 (b) (adapted from Benson et al.^{26 (link)}) and the impedance spectrum observed will have a non-linear frequency dependency as shown in Figure 3 (c) (adapted from Benson et al.^{26 (link)}). Typically, there are three distinct frequency regions in the impedance spectrum where the impedance is dominated by certain equivalent circuit elements. In the low frequency range, the impedance signal is dominated by C_E. In the mid frequency range, the impedance signal is dominated by circuit elements related to the cells, namely R_TEER and C_C. In the high frequency range, C_C and C_E provide a more conductive path and the impedance signal is dominated by R_medium. These equivalent circuit parameters can be estimated by fitting the experimental impedance spectrum data to the equivalent circuit model using non-linear least squares fitting techniques to obtain the best fit parameters.
The following sections describe the advantages of organs-on-chips for TEER measurement, in vitro models of some widely studied cellular barriers, TEER measurements with in vitro models and some microfluidic implementations, and the various factors affecting TEER values.