Agilent xCELLigence RTCA CardioECR User Manual

Application Note

Cardiovascular Research

Assessment of Cardiomyocyte
Disease Models Using the Agilent
xCELLigence CardioECR System

Authors

Xiaoyu Zhang, Jeff Li, and
Yama Abassi
Agilent Technologies, Inc.

Abstract

The Agilent xCELLigence RTCA CardioECR system combines simultaneous
measurement of field potential signal using extracellular recording (ECR) electrodes
as well as contractility and viability using impedance electrodes. The real-time
multiplexed evaluation of the functional activity of beating cardiomyocytes using
the CardioECR system allows for depicting and recapitulating disease phenotype of
cardiomyocytes differentiated from patient-specific induced pluripotent stem cell
(PS-iPSC) and investigating their pharmacological responses, which could potentially
provide information about underlying disease mechanisms.

Introduction

B

ECR/FP

In 2006, Yamanaka and his colleagues
established the first induced pluripotent
stem cells (iPSC) from murine
fibroblasts via transduction of four
specific transcriptional factors. This
novel discovery was also translated to
human somatic cells and has created
a new frontier in the medical field in
many respects. The characteristics of
iPSCs, including infinite self-renewal
and multipotency, allow them to be
potentially used in a wide variety of
applications, including disease modeling,
drug screening, and regenerative
therapy.
iPSCs obtained from patients maintain
patient-specific genetic lesions, providing
a rationale for disease modeling using
patient-specific iPSCs (PS-iPSCs).
Even though primary patient cells have
previously been used for studying
disease mechanisms, certain cell types
such as neurons and cardiomyocytes,
are rather difficult to obtain. The
scalability of primary diseased cells
and the duration of maintaining these
cells in culture has also been a hurdle
for investigating disease mechanisms.
PS-iPSC differentiated target cells
provide researchers with a stable,
scalable, and physiologically relevant
cell source for disease modeling.5
To date, numerous studies have
reported that these target cells can
recapitulate disease phenotypes similar
to those observed from patients. The
pathophysiological cellular phenotypes
of genetically heritable heart diseases,
such as cardiomyopathies and
channelopathies, have been successfully
modeled invitro using patient-specific
iPSC-derived cardiomyocytes
(PS-iPSC-CMs).6 These model systems
are promising tools for understanding
disease mechanisms and can potentially
be used in drug discovery, personalized

1,2

It is well established that

3,4

medicine, and cardiac liability
assessment in specific populations that

may carry geneticanomalies.

This application note used the

AgilentxCELLigence RTCA CardioECR

system (CardioECR system) to compare
functional profiles of contractility and
electrophysiology between a PS-iPSC­CM disease model and its isogenic
control. After identifying baseline
phenotypes at the cellular level, this
study investigated pharmacological
responses of diseased and isogenic
cardiomyocytes to compounds with
established mechanisms. As a further
approach to assessing the responses
of the wild type and diseased CM, both
cell lines were subjected to electrical
stimulation followed by treatment with
isoproterenol.

components, CardioECR control unit
(laptop), CardioECR analyzer, CardioECR
station, and E-plate CardioECR 48
(CardioECR plate) (Figure 1A). Two
sets of electrodes, interdigitated
impedance (IMP) micro-electrode
arrays as well as two individual field
potential electrodes are integrated
into the bottom of each well of the
CardioECR plate (Figure 1B). Similar to
other Agilent xCELLigence platforms, the
CardioECR System uses IMP electrodes
to measure cellular impedance, which
is affected by the number of cells
covering the electrode, the morphology
of the cells, and the extent of cell
attachment. The fast sampling rate of

IMP measurement (2ms) allows for

capturing of the temporal rhythmic
changes in cell morphology and
degree of cell attachment to the plate,

Assay principle

The xCELLigence RTCA CardioECR
system is a dual-mode instrument that
includes both simultaneous monitoring
of hiPSC-CM viability, contraction,
and field potential (FP) in real time
as well as directed electrical pacing
of hiPSC-CMs. It consists of four

A

Figure 1. (A) The Agilent xCELLigence RTCA CardioECR system consists of four components: Control
unit/laptop, CardioECR analyzer (left), CardioECR station (right), and E-PlateCardioECR 48. (B) A close-up
image of E-Plate CardioECR48. Inset: a close-up of the wells reveals the layout of the electrodes,
impedance (IMP) electrode assays, and two field potential (FP) electrodes.

which is a hallmark of contraction
of cardiomyocytes. Therefore, the
physical contraction of cardiomyocytes
is monitored and recorded in real
time with high temporal resolution.
Additionally, the millisecond time
resolution can be performed at regular
intervals over a prolonged duration of

IMP

2

time providing beating and viability

Da

information of cardiomyocytes in real
time. Furthermore, two field potential
electrodes are used to measure
integrated ion channel activities at a data
acquisition rate of 10 kHz simultaneously
while IMP recording is taking place via
IMP electrodes. A typical workflow of
cardiomyocyte assay using CardioECR
system is shown in Figure 2.

One of the other critical features of
the CardioECR system is its ability
to electrically pace cardiomyocytes.
During electrical pacing, electrical
pulses are directly applied to the cells
through the IMP electrodes. For most
cardiomyocytes, the length of each
electrical pulse employed by the IMP
electrode is less than 2 ms, which allows
the contractile activities of cells to be
immediately captured and recorded

while the cells are being paced by IMP
electrodes. The optimal conditions for
electrical pacing are dependent on the
cell type, the inherent beating frequency,
and the experimental context. The pacing
functionality of the CardioECR system is
suitable for both acute pacing regimens
to evaluate compound effects on
contractility under controlled beating rate
and long-term stimulation used for the
improvement of functional maturation of
hiPSC-CMs.

Raw data collected from IMP electrodes
and ECR electrodes were analyzed
offline using Agilent xCELLigence RTCA
CardioECR data analysis software. The
software provides users with more
than 25 analysis parameters based on
IMP and FP signals to assess cardiac
cell beating and electric activities. The
fundamental parameter for contractility

using cellular impedance measurement
are beating amplitude (BAmp) and
beating rate (BR). The beating rate
is defined as the number of beats
per unit of time and is expressed as
beats/minute. BAmp is defined as the
absolute (delta) Cell Index (CI) value
between the lowest and highest points
within a beating waveform (Figure 3A).
For analysis of FP signal, the FP spike
amplitude (FP-Amp) is derived, which
is the absolute (delta) value in mV from
the lowest point of the initial spike to
the highest point of the spike. The FP
Duration (FPD) is defined as the period
between the negative peak of the FP
spike to the maximum or minimum point
of the reference wave. The reference
wave can be negative or positive
depending on how the cells are situated
to the FP electrodes (Figure 3B).

Cell viability

Thaw cellsand plateinto

E-plateCardioECR 48

ys in culture

Figure 2. (A) The workflow of hiPSC cardiomyocyte assay using the Agilent xCELLigence RTCA CardioECR system: cell seeding on day 0, start to measure cell
performance on day 3, start cell status QC before compound addition, add compounds to the cells if they pass QC. Alternatively, after cells start to generate stable
and robust functional activity, approximately 5 days after seeding, progressive electrical pacing is applied to the cells for consecutive 15 days to achieve functional
maturation before compound addition.15 After treatment, cell viability, contraction, and electrophysiology are evaluated via IMP and FP readouts measured by
CardioECR system.

0

Signal acquisition

3

Medium change

Cell status QC

5 to 14

Progressive

electrical pacing

Compound

addition

Contractility/IMP

Cell Index

(impedance)

ECR(mV)

Fieldpotential

3

Materials and methods

Beating amplitude

A

Cell culture

Patient-specific iPSC-derived
cardiomyocytes (PS-iPSC CMs) were
purchased from FUJIFILM Cellular
Dynamics International (FCDI, Madison,
WI, USA). The cells were stored in liquid
nitrogen until thawed and cultured
according to manufacturer instructions.
Briefly, each well of the E-Plate
CardioECR 48 (Agilent Technologies,
Santa Clara, CA, USA) was coated with
50 µL of a 1:100 diluted fibronectin
(FN) solution at 10 µg/mL (F1114,
Sigma-Aldrich, St. Louis, MO, USA) and

incubated at 37 °C for at least 1hour.

Next, the fibronectin solution was
replaced with 50 µL of prewarmed iCell
cardiomyocyte plating medium. Cells
were thawed and diluted in prewarmed
plating medium at the manufacture’s
recommended concentration. 50 µL
of the cell suspension was transferred
using a multichannel pipette and
seeded directly onto a precoated E-Plate
CardioECR 48 (20,000 viable and
plateable cells/well) in a laminar hood.
The plates containing PS-iPSC CMs were
kept in the hood at room temperature for
30 minutes, then placed and cultivated
in a humidified incubator with 5% CO2 at
37 °C. The plating medium was replaced
with iCell cardiomyocyte maintenance
medium 48 hours postseeding. A
medium change was performed every
other day afterward.

Chemical reagents

All the chemical reagents were
purchased from Tocris (Minneapolis, MN,
USA), Sigma-Aldrich (St. Louis, MO, USA),
or provided by the Chemotherapeutic
Agents Repository of the National Cancer
Institute. The 1,000-fold chemical stock
solutions were prepared in DMSO and
stored at –20 °C. The serially diluted
chemicals (1,000-fold) were further
prepared in DMSO immediately before
compound addition. The 10-fold final
dilution of the chemicals was prepared
with the culture medium for a single-time
use only. The final concentration of
DMSO in the treated well was 0.1%.

Results and discussion

Evaluation Brugada channelopathy
model using CardioECR system

Brugada syndrome (BrS) is one of
the more common forms of familial
arrhythmic syndromes characterized
by a dynamic or persistent ST
segment elevation, an enhanced risk of
syncope, and sudden cardiac death in
young adults without structural heart
disease.
genes identified so far, L-type Ca2+
channelopathy of loss-of-function
mutations in the CACNA1C (Cav1.2a1)
has been reported to give rise toBrS
Type 3(BrS3).

7,8

Among several responsible

8,9

This study used used BrS3
cardiomyocytes (BrS3 CMs) purchased
from Fujifilm CDI (Fujifilm CDI, part
number R1136). This BrS3 CM line
possesses a change of amino acid 490
from glycine-to-arginine (G490R), which
was genetically engineered into the
genome of iCell cardiomyocytes (iCell
CMs) derived from a healthy donor with
no known disease-related genotypes.
iCell CMs (Fujifilm CDI, part number
R11320) were used as the isogenic
control for BrS3 CMs. To investigate
the functional consequences of the
mutation, the BrS3 CMs and WT iCell
CMs were seeded first on the same
E-Plate CardioECR 48 at the same
seeding density (20,000 cells/well). The
cell performance, including attachment,
growth, and functional activities were
monitored and recorded immediately
after cell seeding. Figure 4A shows
that BrS3 had slower kinetics of cell
attachment and spreading than the
control line reflected by the lower
slope at the exponential phase of the
Cell Index curve. Overall, Cell Index
values of BrS3 were generally smaller
than the control line. In addition to the
attachment and growth profiles, the
excitation-contraction profiles of the
BrS3 CM and WT iCell CM displayed
significant differences. As shown in
Figures 4B and 4D, the BAmp of BrS3
CMs was smaller than control cells by
27 ±13% while the BR of BrS3 was faster

Figure 3. Definition of main parameters used to evaluate cell contractile and field potential activities. (A) The typical IMP waveform/contraction pattern and (B) the
typical field potential (FP) waveforms were obtained from diseased and WT CM. The reference point can be negative or positive.

+

–––

4

+

B