Impedance Spectroscopy of Cell Monolayers

The evaluation of the biological activity of pharmacological relevant substances is based on the application of cultured cells as sensor elements. Among suitable cell culture models competitive analytical methods are needed to detect cellular reactions as a function of pharmacological substances. The cell shape is a well-known criterion to analyze the cell fitness due to the fact that cells react very sensitive on changes of their environment. The method of electrical cell-substrate impedance sensing (ECIS) is a new electrochemical application to analyze changes in the cell shape as a function of biological, chemical, or physical stimuli without invasion of the cells.11'12 Cell-cell and cell-substrate interactions can be characterized independently. Thus, the change of the cell shape is interpreted by electrical parameters.

Figure 11.3. Electrical cell-substrate impedance sensing. (A) Scheme of the different impedance elements in cultured adherent cells. Cm represents the ion flux across the cell walls of the monolayer, a is the resistance between the cell monolayer and the substrate, and Rb is the resistance of the cell-cell contacts (for details see Ref. 13). (B) Impedance spectra of a gold electrode without (o) and with (•) a cell monolayer (for details, see Ref. 14).

Figure 11.3. Electrical cell-substrate impedance sensing. (A) Scheme of the different impedance elements in cultured adherent cells. Cm represents the ion flux across the cell walls of the monolayer, a is the resistance between the cell monolayer and the substrate, and Rb is the resistance of the cell-cell contacts (for details see Ref. 13). (B) Impedance spectra of a gold electrode without (o) and with (•) a cell monolayer (for details, see Ref. 14).

11.2.4.1. Electrical Cell-Substrate Impedance Sensing

Confluent cell monolayers are cultured on gold electrodes, and the impedance be-tween a working electrode (several square millimeters) and a counter electrode (1000 times larger) is analyzed mathematically. There are three different ways for the electrical current from the working to the counter electrode (Fig. 11.3A).

(1) When leaving the working electrode the current has to pass the cleft between the lower side of cells and the surface of the electrode, which is characterized by the electrical parameter a. This cleft is often smaller than 0.1 |xm, which makes it to the main part of the total impedance. Due to cellular adhesion and movement processes cells alter the distance between their lower side and the substrate, resulting in a change of a.

(2) Cell-cell contacts are also a kind of eye of a needle for the current, because the cleft between adjacent cells is also in the range of 0.1 |xm. By stimulating cell-cell contacts the cleft can be opened or closed resulting in a change of the electrical parameter b.

(3) Using alternating current with a high frequency (several kilo- or megahertz) the cells can be charged as a kind of capacitor. The capacity of the cell membrane Cm is a function of the cell shape, and changes of it are detected very sensitively. By applying a broad range of frequencies of the alternating current all three parameters, and corresponding changes in cell-cell and cell-substrate interactions or in the cell shape can be determined in a single experiment (Fig. 11.3B). Due to an electronic automatization and the parallele arrangement of eight electrodes, a set of experiments is possible as a function of time, simultaneously.

The expression of electrically tight intercellular junctions was proven by Arndt et al. who developed an apoptosis assay for endothelial cells from cerebral microvessels.14 The authors used cycloheximide to cause apoptosis, which leads to a disassembly of barrier-forming tight junctions. The changes in cell-substrate contacts was then measured as a decrease in total impedance resulting from a decrease of both, a and Rb. Keese et al. developed a wound-healing assay for confluent cell monolayers after current treatment.15 Cell monolayers on ECIS electrodes were subjected to currents resulting in severe electroporation and subsequent cell death. The authors were able to monitor cell migration into and ultimate healing of the wound as an increase in resistance and a decrease in capacitance of the cell monolayer.

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