Application Note

Channels:
Cells:
Tools:

hERG, ß-adrenergic
receptorhuman iPSC
Cardiomyocytes
CardioExcyte 96

Impedance and EFP recordings of iCell®
Cardiomyocytes2 on the CardioExcyte 96
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cardiomyocytes kindly provided by Cellular Dynamics International.

SummaryResults

Cardiomyocytes
pluripotent

derivedfrom

human

inducediCell Cardiomyocytes2 were plated on the NSP-96 plates

stem cells (hiPSCs) are gaining interest inon the CardioExcyte 96 and measurements were taken

cardiac safety screening. Given their recapitulationon days 1, 4 and 6 after plating. The cardiomyocyteof native behavior, availability, ease of use andelectrical and contractile activity is stable after 4 DIV, as

standardized production, they are likely to provide asuggested by the manufacturer and indicated by theviable alternative to acutely isolated cardiomyocytes

impedance signal which changes little between day 4toand day 6 (Figures 1 & 2).

assessthe

pro-arrhythmic

potentials

ofdrugcandidates. Although automated patch clamp can
provideexcellent information about theeffects ofcompounds on cardiac ion channels and possible
effectsonthecardiacactionpotential1,

other

outputs such as extracellular field potential (EFP) andimpedance, also provide crucial and complementary
information about complex physiological parameterssuch as beat rate, amplitude and duration. The
CardioExcyte

96

isa

new

hybridscreening

tool combining impedance (cell contractility) and
EFP recordings2. These measurements are non-invasive,
label-free and have a temporal resolution of 1 ms. The
recordings are made from cells within a network thus
providing a physiologically relevant environment formeasuring

drug-inducedchanges

incontractile

parameters. This hybrid technology is a high-throughput
screening tool which permits the reliable investigation ofshort- and long-term pharmacological effects.
Herewepresent

data

recordedon

theCardioExcyte 96 using iCell® Cardiomyocytes2 from
Cellular Dynamics International (CDI). When handledaccordingto

themanufacture’sinstructions,impedance and EFP signals were stable 4 days after
plating and compound effects could be robustlymeasured and analyzed.
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Figure 1: Impedance measurements on the CardioExcyte 96 at different
timepoints. A Screenshot (4 out of 12 columns) of the CardioExcyte 96
software during measurements in the impedance mode on day 1 (left), 4(middle) or 6 (right) after plating. B Impedance mean beat on day 1 (left),4 (middle) and 6 (right) after plating. Impedance mean beat changes
little between day 4 and day 6 indicating that the cells can be used forexperiments 4 days after plating as suggested by the manufacturer.

Application Notenge

Figure 2 shows the development of the impedanceexchaparameter beat rate over time. After 4 days beatmrate had stabilized and the cells could be used forBeatRate
[BPM]

M

ed
iu
mex
changeEFP Amplitude
[µV]

6040
30
20
100-10-20M

ediuexperiments.

040

123Time [Days]

45620
00123Time [Days]

456Figure 2: Impedance beat rate shown over time course of several days.
Medium exchanges were made as indicated by the vertical red lines. Beatrate was stable after 4 days at which point experiments can be started.

Figure 4: EFP amplitude shown over time course of several days. Medium
exchanges were made as indicated by the vertical red lines. EFP amplitude
was stable after 4 days at which point experiments can be started.

Figure 5 shows the effect of the specific hERG
inhibitor,

This phenomenon was also mirrored in the EFP signal.
Figure 3 shows a screenshot of the CardioExcyte 96
software during a recording in the EFP mode on day 1, 4
and 6 after plating. The EFP mean beat displayed in Panel
B shows that the EFP signal is stabilized after day 4 - 6.

E4031,ontheimpedanceandEFP

signals. E4031 has a profound effect on the beatrateinimpedance

afterdepolarizationsmode(EADs)

andwhichinduces
canearly

leadtopotentially fatal ventricular arrhythmias. This can beclearly recognized using the CardioExcyte 96. The
EFP signal is also greatly affected by E4031 in aconcentration-dependent manner. In this experiment,impedance measurements were taken at the start of theexperiment (control) and then every 5 minutes for 30 s
after E4031 application. The time point shown in Figure 5
is 13 mins after the start of E4031 application. EFPmeasurements were taken at the start of the experiment(control) and then at 30 mins after the start of E4031
application.

Figure 3: EFP measurements on the CardioExcyte 96 at different time points.
A Screenshot (4 out of 12 columns) of the CardioExcyte 96 software during
measurements in the EFP mode on day 1 (left), 4 (middle) or 6 (right) after
plating. B EFP mean beat on day 1 (left), 4 (middle) and 6 (right) after plating.
EFP mean beat changes little between day 4 and day 6 indicating that thecells can be used for experiments 4 days after plating as suggested by themanufacturer.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9

fax80636 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960

Application Note

Figure 5: Effect of the specific hERG blocker, E4031, on the impedance andEFP signals. A Impedance signal of 12 wells in control conditions (left) andthe same 12 wells after 13 mins incubation in E4031 at the concentrations
indicated (right). E4031(300 nM, 3 mins incubation) induces EAD, shown in
the inset, which can lead to potentially fatal ventricular arrhythmias. B EFP
signal of 12 wells in control conditions (left) and the same 12 wells after 13
mins incubation in E4031 at the concentrations indicated (right). E4031(300nM, 30 mins incubation) also causes arrhythmic effects in the EFP modeshown in the inset.

Figure 6 shows the effect of the ß-adrenergic agonist,
isoproterenol, on the impedance signal. Isoproterenol
is used predominantly as a bronchodilator and heartstimulant,

particulary

after

myocardialinfarct,

cardiac surgical procedures or to treat congestive heart
failure3. Isoproterenol acts on the ß-1 adrenergic
receptors in the myocardium increasing heart rate
and,thereby,cardiacoutput3.

Thereisaclear

increase in beat rate in the impedance mode caused byisoproterenol recorded on the CardioExcyte 96 using theiCell Cardiomyocytes2. In the same way, in EFP mode,
field potential duration (FPD) is shortened resulting in an
increase in beat rate.

Figure 6: Effect of the ß-adrenergic receptor agonist, Isoproterenol, on
the impedance and EFP signals. A Impedance signal of 12 wells in controlconditions (left) and the same 12 wells after 13 mins incubation in isoproterenolat the concentrations indicated. Isoproterenol acts as a heart stimulant, it
increases heart rate, an effect which is highlighted in the inset where thetrace in the presence of isoproterenol (300 nM; black) and the control trace(red) are shown overlaid. In the presence of even low concentrations ofisoproterenol (1 nM), an increase in the beat rate is observed. B Bar graphshowing beat rate before (dark blue) and after (light blue) incubation inisoproterenol at the concentrations indicated. At all concentrations, a clear
increase in beat rate is observed. C EFP signal of 12 wells in control conditions(left) and the same 12 wells after 30 mins incubation in isoproterenol at
the concentrations indicated (right). Isoproterenol shortens the FPD, aneffect which is highlighted in the inset where the trace in the presence ofisoproterenol (300 nM; black) and the control trace (red) are shown overlaid.

Nanion Technologies GmbHphone +
 49 89 218997972

Gabrielenstr. 9fax80636 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960

Application Note
In this experiment, impedance measurements wereimpedance modes, causing an increase in the beat ratetaken at the start of the experiment (control) and thenat low concentrations. Therefore, the CardioExcyte 96

every 5 minutes for 30 s after isoproterenol application.

provides a complementary device to automated

The time point shown in Figure 6 is 13 mins after the start ofpatch clamp for high throughput cardiac safety andisoproterenol application. EFP measurements were taken

cardiotoxicity studies.

at the start of the experiment (control) and then at 30
mins after the start of isoproterenol application.
Weare

confident

that

theCardioExcyte

96

inIn conclusion, the CardioExcyte 96 can be used tocombination with hIPSCs will have a great impact on thereliably perform EFP and impedance measurements

field of pharmacology, toxicology, and cardiac safety

on hiPSCs from CDI (iCell Cardiomyocytes2). The

screening in the light of the CIPA (Comprehensive In

manufacturer claims that the cells can be used for

Vitro Pro-Arrhythmia Assay) proposal. This project aims

experiments 4 days after plating, and this claim isto define a new, integrated preclinical in vitro/in silico

validatedparadigm in which the potential proarrhythmic risk of a

here

using

theCardioExcyte

96.

The

impedance and EFP signals are stable after about

new drug will be assessed using hIPSCs.

4 days of plating. The hERG blocker, E4031, hadprofound effects on the EFP signal and the beat rate in
impedance mode, causing EAD which could be clearly
identified on the CardioExcyte 96. The ß-adrenergic agonist, isoproterenol, also affected beat rate in both EFP andReferences

Medium was exchanged from iCell Cardiomyocytes
Plating to Maintenance Medium 4 hours post thaw, and

1. Stoelzle, S., et al., 2011. Front. Pharmacol. doi: 10.3389/fphar.2011.00076.
2.

Doerr,

L.,

etal.,

2014.

J.

Lab.

Autom.

pii:2211068214562832.
3.http://pubchem.ncbi.nlm.nih.gov/compound/isoproterenol#section=Drug-Indication

then every other day until the conclusion of the assay.
The cells could be used for pharmacology experiments 4days after plating but were typically used 7 days afterplating. This enabled the formation of a dense monolayer
and a suitable network of cell-cell interconnections toensure a steady propagation of the excitation andsynchronized beating. At least 4 hours before drugMethods

application the medium was completely removed from

Cardiomyocytes

experiments, a 2x concentration of compound was

the wells and 100 μl fresh medium was added. For theiCell® Cardiomyocytes2 were kindly provided by CDI.

prepared, 50 μl of solution was removed from the welland 50 μl of the 2x compound was added to give thefinal concentration indicated in the wells, although

Impedance and EFP measurements
Impedance and EFP measurements were conductedaccording to Nanion’s standard procedures for theCardioExcyte 96. Cardiomyocytes were seeded at 50,000
viable cells per well on fibronectin-coated Sensor Plates.

different methods of addition (e.g. 90 μl addition of fresh
medium and then 10x concentration of compoundadded) could be used. All signals were normalized to agroup of control measurements (n=5-11) on the sameplate using DMSO (0.01%) as vehicle.

Nanion Technologies GmbHphone + 49 89 218997972Gabrielenstr. 9fax

80636 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960