Impedance Spectroscopy Screening the Cellular Parameters of Electrophysiological Inactive Cells

Impedance spectroscopy, which is also known as cellular dielectric spectroscopy (CDS) or electric impedance spectroscopy (EIS), can be used to measure frequency-dependent changes in the passive electrical properties of single cells or complex tissues by applying defined alternate currents. The bioimpedance of single cells or complex tissues, when combined with a working and counter electrode, is determined by different cellular parameters such as the electric resistance and capacitance of the (sub)cellular membranes, the resistance of the extracellular medium and cytoplasm (intracellular), and the contact between cells and cell-electrode system (Fig. 3.3).

For the measurement of impedance, an alternate voltage current is applied to a biological sample, whereby the current flows from an active working electrode through and beneath the cell or tissue to a counter electrode. Under these conditions the cell itself can act as a resistor and capacitor affecting the recorded impedance. Depending on the dielectric properties of subcellular structures, it is possible to classify three different frequency-dependent main dispersions (a-, |3-, and y-dispersion). It is assumed that the low-frequency dielectric behavior (up to 1 kHz) of current flow through ion channels mainly contributes to the a-dispersion

Fig. 3.3 Basic circuit model of a single cell positioned on an electrode. If an alternate current is applied to living cells or tissues, the bioimpedance is influenced by several physical parameters such as the resistances of the intracellular medium (Ri), extracellular medium (Re), membrane (Rm), and the membrane capacitance (Cm) [60].

Measuring Extracellular Impedance

Fig. 3.3 Basic circuit model of a single cell positioned on an electrode. If an alternate current is applied to living cells or tissues, the bioimpedance is influenced by several physical parameters such as the resistances of the intracellular medium (Ri), extracellular medium (Re), membrane (Rm), and the membrane capacitance (Cm) [60].

a-dispersion ß-dispersion 7-dispersion up to 1kHz 1 kHz-100 MHz 100 MHz-100 GHz

Classification of dispersions according to cellular structures counterions cytoplasm membrane free and bound water membrane ion channel glycocalyx cytoplasm membrane free and bound water proteins protein-protein interactions proteins protein-protein interactions

Fig. 3.4 Frequency-dependent classification of the three main dispersions according to cellular structures.

(Fig. 3.4), whereas the P-dispersion (1 kHz to 100 MHz) is associated with the dielectricity of the cytoplasm-membrane interface, intracellular membrane systems, and cytoplasm. The third dispersion range is termed y-dispersion (100 MHz to 100 GHz), and characterizes the dissociation/association relaxation of small charged groups, protein-protein interactions and bound, as well as free, water [17-19].

The increasing popularity of impedance recording is reflected by the large number of reports published during the past few decades. This is, in all probability, due to the availability of commercial hardware and software which allows feasible and reliable impedance measurements to be made. Additionally, the still-expanding computer capacity is now sufficient for adequate data acquisition, especially in terms of HTS. Moreover, the recording of cellular parameters under noninvasive and real-time conditions makes this technique attractive for both basic research and pharmaceutical screening.

One commonly used impedance recording method is the so-called electric cellsubstrate impedance sensing (ECIS), introduced by Giaever and Keese [20]. These authors were among the first to report that impedance recording is a suitable method for monitoring morphological changes of cells. In principle, for ECIS, cells must be grown on a small gold electrode implemented at the bottom of a culture dish. If an alternate current voltage is applied between a small working electrode and a large counter electrode, the impedance of a cell can be observed at a given time and on one single frequency. Over the years, ECIS has been further optimized for automated, noninvasive, real-time, and high-throughput analysis. For example, Applied Biophysics Inc. offers a complete ECIS system that provides an 8-well or 96-well format, and promises a wide range of biological screening applications such as analysis of cell attachment, signal transduction, cell-substrate interaction, barrier function, chemotaxis, toxicology, proliferation, and apoptosis (for ECIS literature, see under www.biophysics.com).

culture dish culture dish adherent cells

Adherent Cell Culture

adherent cells culture dish counter electrode adherent cells counter electrode adherent cells

Fig. 3.5 Principle of a biosensor for electric cell-substrate impedance sensing (ECIS). (Copyright © BBZ/Andrea Robitzki, Maik Schmidt).

glass substrate \ H

medium \

working electrode gold conductors

Fig. 3.5 Principle of a biosensor for electric cell-substrate impedance sensing (ECIS). (Copyright © BBZ/Andrea Robitzki, Maik Schmidt).

To illustrate the potential of ECIS, the details of a more recent study are provided below. Here, ECIS has been used to monitor the programmed cell death (apoptosis) of endothelial cells under real-time conditions [21]. In that approach, cells were cultured to confluence at thin gold-film electrodes (Fig. 3.5), whereby the impedance of the cell-electrode system was measured over an extended frequency range of 1 to 10 Hz.

The comparison of electric impedance of cell-covered and uncovered electrodes has shown that the contribution of cell bodies to the total impedance lies in a frequency range of 10 Hz to 100 kHz, with a maximal amplitude at 1 kHz. For that reason, the impedance of endothelial cells before and after induction of apoptosis by cycloheximide (CHX) was measured at a sampling frequency of 1 kHz. As cell bodies undergo dramatic structural changes during apoptosis, it was not surprising that the impedance of endothelial cells dramatically decreased within 12 h after CHX application. The authors of this study also pointed out that, in comparison to classical immunolabeling techniques, ECIS is apparently more sensitive to detecting apoptotic changes in real-time. If this technique is adapted to an automated work station, it probably represents a well-suited HTS system, especially in terms of drug-induced apoptosis or other adverse reactions.

Ciambrone and co-workers [11] utilized a 96-well microplate with interdigitated electrodes at the bottom of each well. Impedance measurements in a frequency range of 1 kHz to 100 MHz were carried out using adherent cell lines to detect subtype-specific activation of G-protein-coupled receptors (Gs, Gq, Gi) and protein tyrosine kinase receptors under noninvasive and labeling-free, high-throughput conditions. Sample application and buffer exchange was performed with a 96-head delivering system. As reported by the authors, the time for impedance measurements without consideration of liquid handling amounted to only 20 s for a complete 96-well plate.

Another chip-based approach for electronic impedance measurement has been developed and used by Robitzki and co-workers [10, 14, 22, 23]. These authors developed an impedance recording platform that enables the impedimetric measurement of3-D spherical in-vitro tissues (e.g., tumor spheroids, neurospheres or cardiomyocyte spheres), as well as single cells. The fabricated cell-based sensors consist of either multiple microcapillaries or microcavities, each with two or four implemented electrodes where cells or 3-D in-vitro tissues can be hydrodynamically positioned (Fig. 3.6), without requiring time-consuming procedures for precultivating cells on planar electrodes. This innovative system allows HTS and HCS of biological samples in which tested cells are released and new cells or tissues can be repeatedly positioned and monitored. A further advantage is that each single cell or tissue sample can be addressed in such arrays for a specific monitoring of cellular or physiological and molecular changes. Although single cells produce adequate impedimetric signals, the major advantage of using 3-D histotypic sphere-like in-vitro tissues is that they fit more precisely into the 3-D geometry of in-vivo tissues, such as intracellular and extracellular properties, representing a consolidated response of thousands of cells, and allowing a more reliable impedance analysis of cellular changes. This type of tissue-based sensor is an outstanding biohybrid system for functional drug discovery, and allows the screening of one substance per minute at, for example, 30 individual sites simultaneously.

Based on the time needed for sample application, liquid handling, and the time range of the expected drug effect (e.g., 2 min plus 1 min for data reading out), it is possible to perform an automated functional impedance screening of 600 substances per day. In this way, the use of this technique clearly accelerates the complex and cost-intensive process of identifying potential leads in secondary screening. However, the chip platform is also applicable as a screening tool to test the efficiency of cytostatic drugs on cancer cell lines, or directly on biopsies obtained from patients. In the latter case, biopsies are applied immediately or after generation of tumor spheroids on the chip, and subsequently the effects of cytostatic drugs - for example, on the proliferation or apoptosis of tumor cells - can be measured in real-time via impedance spectroscopy.

In this context it has been shown by Robitzki and colleagues that nontreated tumor spheroids derived from a mamma carcinoma cell line similar to other types oftissues (e.g., heart muscle and neural tissues) showed a cellular resistance in a range of 1 kHz to 100 kHz (Fig. 3.7a). If tumor spheroids were genetically

Measuring Extracellular Impedance

Fig. 3.6 Cell- or spheroid-based multielec-trode impedance sensors. Planar glass or silicon chips can be fabricated with small cavities (a) or capillaries (b) for an improved cell or tissue positioning and electric impedance spectroscopy via two or multiple electrode arrangements. In the case of capillary multiarrays, positioning of the cells or spheroids is facilitated by a medium permeable membrane (green). Cavities can be produced with different diameters ranging from 10 to 1000 |jm, whereas capillaries may have diameters of 100 |m, 200 |m, 300 |m, and 400 |m. (Copyright © BBZ/Andrea Robitzki, Maik Schmidt).

Fig. 3.6 Cell- or spheroid-based multielec-trode impedance sensors. Planar glass or silicon chips can be fabricated with small cavities (a) or capillaries (b) for an improved cell or tissue positioning and electric impedance spectroscopy via two or multiple electrode arrangements. In the case of capillary multiarrays, positioning of the cells or spheroids is facilitated by a medium permeable membrane (green). Cavities can be produced with different diameters ranging from 10 to 1000 |jm, whereas capillaries may have diameters of 100 |m, 200 |m, 300 |m, and 400 |m. (Copyright © BBZ/Andrea Robitzki, Maik Schmidt).

(transient gene silencing) manipulated to decrease proliferation and to induce apoptosis, the changed cell and tissue properties would correlate with a fivefold decrease (Fig. 3.7b) of the relative extracellular resistance as measured by impedimetric spectroscopy [14, 22, 23].

A further important application area of impedance spectroscopy is the testing of blood-brain barrier (BBB) function and permeability with respect to the application of novel drugs or toxicological chemicals. Here, the barrier function of endothelial or epithelial cell layers can be quantified by determining the transcellular electric resistance (TER). To measure the TER, cells are either placed directly on the surface of planar gold electrodes (similar to ECIS) [24-26] or, by an alternative approach of preculturing cells on multi-well filter inserts which then can be

Tumor Spheroid Shape
Fig. 3.7 Cellular properties contributing to the impedance of biological cells or tissues are detectable in a frequency range of 1 KHz to 1 MHz (a), as revealed by the impedance measurement of covered and noncovered

recording sites. When proliferation is decreased in tumor spheroids by genetic manipulation, a fivefold decrease can be measured in the relative extracellular resistance (b).

transferred into the measuring chamber for frequency-dependent impedance recording [27]. At the bottom of this chamber a thin, gold film is evaporated as a measuring electrode, whilst a ring-shaped platinum counter electrode is dipped into the culture medium above. In seeking an automated approach, Wegener et al. [28] developed a multi-well measuring chamber that could be loaded with cell-covered filter inserts where the TER is measured on up to 24 different sites simultaneously [28]. Improved and modulated software may provide a system that enables the screening in a 96-array format

Another approach to investigating membrane properties in terms of drug transport is to use membrane transistors with lipid vesicles or lipid rafts on silicon chips. Lipid rafts or bilayers on silicon substrates represent the future of bioelectronic devices for measuring and monitoring the drug transport if the junction is sufficiently insulated (Fig. 3.8).

Planar Bilayers
Fig. 3.8 Schematic illustration of an open gate field transistor coated with a giant lipid vesicle for testing membrane properties before and after application of drugs. (Copyright © BBZ/Andrea Robitzki, Maik Schmidt).

An open gate of a field transistor is touched with a preformed giant lipid vesicle; the membrane is bound by a polyelectrolyte interaction [29]. At a contact area of 0.1 mm ' the sheet resistance along the junction is 100 G^ and the membrane resistance was above 100 G^. This contact area of the membrane and solid phase reflects a planar electrical core-coat conductor. The insulation of the junction can be determined by the resistance of the membrane and by the resistance of the cleft between membrane and substrate. Such a membrane-based semiconductor can be generated by: (1) depositing monomolecular lipid films or (2) by spreading lipid vesicles [30-34]. Such compound lipid-silicon microstructures are suitable for coupling semiconductor and electroactive proteins. Using such hybrid devices, biomolecules can be addressed by micro- and nanostructures in a semiconductor. In case the close contact of a bilayer and substrate interferes with the function of a biomolecule - for example, its lateral motion and conformational changes - a local blister on the lipid film can be created by a local coating of the chips via immobilization of proteins that enhances the distance between the chip and the lipid bilayer.

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