Cell Monitoring using Multi-Frequency Impedance Recordingsfor Label-free and Time-resolved Cell Response Analysis
Sonja Stölzle-Feix1, Krisztina Juhasz1,2, Michael Skiba3, Joachim Wegener3, Ronald Knox4, Marta Lemme1, Ulrich Thomas1, Max Engelstädter1, Elena
Dragicevic1, Michael George1, Niels Fertig1
1Nanion Technologies, Munich, Germany; 2Institute for Nanoelectronics, Technische Universität München, Germany, 3Institute of Analytical Chemistry, Chemo- and Biosensors, Faculty of Chemistry andPharmacy, University of Regensburg, Regensburg, Germany, 4Nanion Technologies Inc., Livingston, NJ, The United States of America

Cell proliferation and cell toxicity
experiments

Impedance-based assay
technology

Abstract
Reading the impedance of planar gold-film electrodes that are used as growth
substrate for adherent cells reveals changes in electrode coverage or cellmorphology. Thus, real time impedance data provide insights in various cellcharacteristics, such as cell morphology, proliferation, lateral migration orcytotoxicity even over prolonged periods of time. A crucial advantage overstandard assays is the continuous cell monitoring. Advanced information content isobtained by using multi-frequency impedance readouts as they allow zooming inon changes in membrane topography, cell-cell or cell- matrix junctions
deconvoluting the complex whole cell response, for instance, to G-proteincoupled receptor (GPCRs) activation.

AA

B

Cell-covered electrode

IZI (Ω)Low AC frequency
High AC frequency

Multi-frequency impedance measurements were used to monitor cell adhesion,
cell specific structural changes, proliferation and cytotoxicity. The multi-frequency
character of the data allows selecting the most sensitive frequency for a particularapplication, cell type or detail of the cell response after data acquisition iscomplete.

Culture medium

Gold electrode
Culture substrate

B
Frequency (kHz)

Impedance spectra were calculated for HeLa, HEK293 and CHO cells todemonstrate cell-type specific differences under stationary conditions. Dataacquisition was performed using the CardioExcyte96 impedance recording system.
Cells covering the electrode impede the applied current due to insulating
properties of their membranes and the individual tightness of their cell junctions.
Cell responses to experimental challenges will induce corresponding impedancechanges. Multi- frequency impedance analysis provides a stronger basis for datainterpretation as it highlights the changes in specific cell structures.

Fig. 2 The frequency dependent impedance of a cell-free and cell-covered
electrode in log-log presentation (A) reflects the impedance increase due to thecell layer between ~ 1 – 100 kHz. For the experimental setup presented here, thesignal is dominated by the paracellular resistance of the cell layer between
about 1 – 10 kHz (range indicated by light blue frame) and is increasingly
influenced by capacitive currents across the cell membranes between 10 – 100
kHz (range indicated by darker blue frame). B Schematic side view of a cellcovered electrode.

HeLa, HEK293 and CHO cells show different proliferation curves. Frequencydependent impedance data reveal individual drifts of the most sensitivefrequency, i.e. of the maximal responses indicating cell type specific changes incell junctions and proliferation dynamics. The expected concentration dependent
toxicity was observed for DMSO, Escin and Triton-X by multi-frequency impedance
recordings showing decreasing impedance values with exposure time and driftingof the maximal responses to lower frequencies. GPCR-mediated signaltransduction was investigated using cells recombinantly expressing the dopamine
D2 receptor. Agonist mode measurements using the endogeneous agonist
dopamine and antagonist mode measurements using the specific antagonist
haloperidole returned potencies that are in line with the literature. Multi-frequency
impedance data allow for non-invasive continuous monitoring of cells with theoption to zoom in on certain cell aspects or to tailor the readout to individual cell
types for improved data interpretation.

Impedance denotes the ratio between the AC voltage applied to a given
system and the current through the system that is induced by this applied
voltage. In contrast to DC, capacitors have a finite impedance as they become
more conductive with increasing AC frequency. Thus, analysis of the frequency
dependent impedance reveals the resistive and capacitive properties of thesystem under test. Therefore, impedance spectra of cells and tissues covering abroad frequency range disclose the passive electric properties of the cell layer.

Multi-parametric cell monitoring with
CardioExcyte 96

Fig. 3 A Time-dependent impedance measurements at a single fixed
frequency (e.g 10 kHz) reveal the time course of cell adhesion,
proliferation or cell death. Such cell type-specific profiles of various
cell phenotypes are e.g. relevant in cancer research. B Typical cell
death experiment. Effect of Escin on the impedance signal of CHO
cells in concentrations of 6.8 μM, 13.6 μM, 27.2 μM and 54.4 μM (n = 4).
Effect of Triton X-100 (2 %) is also shown. Data are normalized to thelast data point prior to addition of chemicals or control treatments
and the time point of compound addition.

Frequency spectra reveal the sweet spots:“Where do I find the maximum resolution of my effect?”
ABCDEF

CardioExcyte 96

Nanion´s cell monitoring technology is widening its application area. Next toelectrophysiology, contractility and viability recordings now the deconvolutionof complex whole cell responses is possible with the new software toolSpectraControl running on CardioExcyte 96. In addition to high quality
consumables and controlled temperature and environment, the systemcomes with an automated liquid handling system for cell seeding, compoundapplications and medium exchange.
Reference electrode

Impedance /EFP electrode40 xFig. 4 “Which frequency reports on the highest impedance change?” Averaged relative changes of the impedance signal with associated standard deviation due to cellproliferation of CHO (A), HEK (B) and HeLa (C) cells with initially seeded cell densities of 8k, 16k and 32k / well over a time period of 72 h (n=12 each). Data is normalized tomedium reference without cells and to the time point of cell addition to wells. Frequency spectra at time points 12 h, 24 h, 36 h, 48 h, 60 h and 72 h for CHO cells with initialcell densities of 2k (D), 4k (E) and 16k / well (F). Data is normalized to medium reference without cells and cell addition time point. Sweet spots, maximum of amplitudechange, is marked with “x”.

Fig. 1 Multiple functional assays can be combined in a single plate.
Transparent NSP-96 sensor plates are compatible with standard plate readers
and automated instrumentation, thereby giving you freedom to combine yourassays in one plate.

Conclusions

Stimulation of GPCRs is mirrored in time-dependent impedance profiles• Electric cell-substrate impedance sensing withfrequencies ranging from 0.1 kHz – 100 kHz are
successfully integrated in Nanion´s cell monitoring
technology (CardioExcyte 96/FLEXcyte 96).

The CardioExcyte 96 software
provides a SpectraControl tool torecord and analyze impedance
spectra.
Changes in cell shape and cellnumber can be investigated. The
method is suitable for monitoring celladhesion, cell specific structuralchanges, proliferation and toxic
effects of compounds.

Cell free(typical hockey stickshape)• Nanion´s experimental setup reveals the kinetics
of cell behavior and allows an in-depth
mechanistic understanding in real-time.
B

600
10-3 M C
10-4 M500
10-5 M
400-610 M
300
10-7 M200
10-8 M
10-9 M
100-10
10 M
0-1110 M-100
Control (L15 buffer)

600
500
400

1E-5 M
1E-6 M
1E-7 M
1E-8 M
1E-9 M
1E-10 M
1E-11 M
Control (L15 buffer,
DMSO - 0.05 %)|Z|12 kHz / 

|Z|12 kHz / AWith cells• Continuous and multi-parametric cell monitoring
has an advantage over time- and cost- intensive
individual endpoint assays.

3002001000-10001020t / min

304001020t / min

3040

Institute of Analytical
Chemistry, Chemo- andBiosensors
Prof. Joachim Wegener

Fig. 5 Frequency-dependent impedance recordings are the basis for many applications of impedance technology in cell analysis. A shows simulated impedance
spectra illustrating cell-type specific dielectric properties of the listed cell lines for a 0.6mm recording electrode. Additionally, we were able to monitor specific GPCR
(here dopamine D2 receptor) activation by applying the endogeneous agonist dopamine in agonist (B) and antagonist mode (C) using haloperidole as specificantagonist. Concentrations in B refer to dopamine concentratoins in absence of antagonist. Concentrations in C denote concentrations of haloperidol pre-incbation
prior to addition of 10 µM dopamine at time zero. Agonist mode measurements returned a pEC50 of (7.3  0.1) for dopamine whereas the antagonist modemeasurements returned a pIC50 of (6.8  0.2) for haloperidole.

• Multiple research areas benefit from impedance
recordings at multiple frequencies.