Real-Time Electroportation


The ECIS Method

What is Electroporation?

By subjecting a cell to a short voltage pulse, the cellular plasma membrane is reversibly permeabilized allowing the introduction of polar, non membrane-permeable compounds (ie DNA) into the cell. This temporary disruption of membrane integrity is able to quickly repair itself so the cell stays intact and can then be studied by various methods.
Why use ECIS for Electroporation Experiments?

With ECIS, the cells can monitored before and after this electroporation process. Pulse application is integrated into the regular data acquisition so that the cell response to the introduction of any extracellular compounds can be followed in real time. In contrast to all existing electroporation devices - predominately using cells in suspension - the cells are in direct contact with the electrode surface and the same electrodes are used to detect the subsequent cell response immediately after pulse application. In addition ECIS makes use of AC instead of DC voltage pulses for electroporation in order to minimize electrochemical generation of reactive molecular species and perturbation of the electrical properties of the ECIS electrode (Wegener et al, 2000).
Electroporation Experiments using ECIS

Loading Efficiency: the optimum pulse parameters for cell loading can be easily determined from studies using fluorescent probes. The cells are bathed in a buffer supplemented with a fluorescent probe like Lucifer Yellow or a FITC-labled dextrane. After pulse application, the extracellular buffer is aspirated and replaced by a buffer with no probe. Microscopic inspection the reveals the loading of the cells as illustrated in figure 1. Pulse conditions can be adjusted with respect to amplitude, frequency and duration. In this example the amplitude was set to 4.0 V (corresponds to app. 1kV/cm field strength) and the frequency of the pulse 40 kHz. The duration was gradually increased from 50 msec to 500 msec. When the field is applied for 500 msec, there is a fraction of cells unstained (arrow) as their membrane has been irreversibly damaged and the dye has been washed out.
Cell Damage and Survival: Electroporation is an invasive operation that may do harm to the cells if the pulse parameters are not carefully adjusted. Cell recovery from the pulse is sensitively monitored by ECIS readings as shown in figure 2 for the same pulse conditions that have been used to study cell loading. Time-resolved ECIS readings show that the cells recover from the electroporation pulse within 60 min to pre-pulse values unless the duration of the high field is too long (500 msec). By combining loading (fig. 1) and recovery studies (fig. 2) it is possible to perform tailored electroporation experiments with maximal loading efficiency but minimal invasiveness such that the cell response. Under these conditions the cell response to the introduction of small organic molecules, peptides, antibodies, siRNA or even DNA can be studied.
Figure 1: Loading of confluent NRK cells with Lucifer Yellow by electroporation. The pulse conditions are: amplitude 4.0 V, frequency 40 kHz, duration is gradually increased.
Next, the response of the cells to Electroporation pulses was measured using 3 frequency ECIS measurements. Figure 4A and B show typical time courses of the impedance magnitude at a sampling frequency of 4 kHz when confluent NRK cells were exposed to a 100 msec voltage pulse of the specified frequency (A) and amplitude (B).
Figure 2: Response of confluent NRK to electroporation pulses of different specifications: amplitude 4.0 V, frequency 40 kHz, duration is gradually increased. The impedance readout reports on the invasiveness of the pulse.

Electroportation
Related ECIS Publications

Shikataa, Y., Riosa, A., Kawkitinaronga, K., DePaolab, N., Garciaa, J.G.N., Birukov, K.G., "Differential effects of shear stress and cyclic stretch on focal adhesion remodeling, site-specific FAK phosphorylation, and small GTPases in human lung endothelial cells." Experimental Cell Research Volume 304, Issue 1, 40-49 (2005).

DePaola, N., Phelps, J.E., Florez, L., Keese, C.R., Giaever,I., Minnear, F.L., and Vincent, P. "Electrical Impedance of Cultured Endothelium Under Fluid Flow." Annals of Biomedical Engineering 29, pp. 1-9 (2001)

Phelps, J.E., and DePaola, N. "Spatial Variation in Endothelial Barrier Function in Disturbed Flows inVitro." American Journal of Physiology: Heart and Circulatory Physiology, 278: H469-H476, (2000).

Wegener, J., Keese, C.R., Giaever, I., "Electroporation of adherent cells on a conductive substrate and electrical in situ monitoring of the cell response." Biotechniques 33: 348-357 (2000).