Cellular Impedance Explained

Positioned between reductionistic biochemical assays and whole organism in vivo experimentation, cell-based assays serve as an indispensable tool for basic and applied biological research. However, the utility of many cell-based assays is diminished by: (1) the need to use labels, (2) incompatibility with continuous monitoring (i.e. only end point data is produced), (3) incompatibility with orthogonal assays, and (4) the inability to provide an objective/quantitative readout. Each of these shortcomings is, however, overcome by the non-invasive, label-free, and real-time cellular impedance assay.

Functional Unit of Cellular Impedance Assay

The functional unit of a cellular impedance assay is a set of gold microelectrodes fused to the bottom surface of a microtiter plate well (Figure 1). When submersed in an electrically conductive solution (such as buffer or standard tissue culture medium), the application of an electric potential across these electrodes causes electrons to exit the negative terminal, pass through bulk solution, and then deposit onto the positive terminal to complete the circuit. Because this phenomenon is dependent upon the electrodes interacting with bulk solution, the presence of adherent cells at the electrode-solution interface impedes electron flow. The magnitude of this impedance is dependent on the number of cells, the size and shape of the cells, and the cell-substrate attachment quality. Importantly, neither the gold microelectrode surfaces nor the applied electric potential (22 mV) have an effect on cell health or behavior.

 

Impedance Electrodes

The gold microelectrode biosensors in each well of Agient’s electronic microtiter plates (E-Plates) cover 70-80% of the surface area (depending if a view area is present). Rather than the simplified electrode pair depicted in Figure 1, the electrodes in each well of an E-Plate are linked into “strands” that form an interdigitating array (Figure 2). This arrangement enables populations of cells to be monitored simultaneously and thereby provides exquisite sensitivity to: the number of cells attached to the plate, the size/morphology of the cells, and the cell-substrate attachment quality.

Real-Time Impedance Traces Explained

The impedance of electron flow caused by adherent cells is reported using a unitless parameter called Cell Index (CI), where CI = (impedance at time point n – impedance in the absence of cells)/nominal impedance value. Figure 3 provides a generic example of a real-time impedance trace throughout the course of setting up and running an apoptosis experiment. For the first few hours after cells have been added to a well there is a rapid increase in impedance. This is caused by cells falling out of suspension, depositing onto the electrodes, and forming focal adhesions. If the initial number of added cells is low and there is empty space on the well bottom cells will proliferate, causing a gradual yet steady increase in CI. When cells reach confluence the CI value plateaus, reflecting the fact that the electrode surface area that is accessible to bulk media is no longer changing. The addition of an apoptosis inducer at this point causes a decrease in CI back down to zero. This is the result of cells rounding and then detaching from the well bottom. While this generic example involves drug addition when cells are confluent, impedance-based assays are extremely flexible and can also evaluate the rate and extent of initial cell adhesion to the electrodes, or the rate and extent of cell proliferation.

 

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