Application Note

Cells:
Tools:

Cardiosight-S®
FLEXcyte 96

FLEXcyte 96 recordings in comparison to invivo and in vitro cardiac muscle contraction
via MUSCLEMOTION dataNanion Technologies GmbH, Munich.
Human iPSC-derived cardiomyocytes kindly provided by Nexel.
SummaryResults

In order to reduce cardiovascular safety liabilities of newMean

therapeutic agents, there is an urgent need to integrate

for hiPSC-CMs grown on the flexible substrate of thehuman-relevant

andvelocity

parameters

drug

FLEXcyte 96 were calculated and compared with beatparameters from the MUSCLEMOTION software pro-induced pluripotent stem cell-derived cardiomyocytes

gram as previously published by Sala et al, 20184.

(hiPSC-CMs) is essential for their effective application inFigure 1A shows the contraction and velocity profiles

models of cardiac toxicity and disease2. Here, hiPSC-CMsof isolated hPSC-CMs, hPSC-CM monolayers, cardiac

were cultured on flexible substrates using the FLEXcyteorganoids, engineered heart tissue (EHTs) and adult

96 system. The pro-maturation environment enables

rabbit cardiomyocytes. Figure 1B shows the contractionobservation of inotropic and chronotropic compoundand velocity profiles of hiPSC-CM monolayers on theeffects, which are typically hard to detect with 2D

FLEXcyte 96. Interestingly, the contraction and velocity

monolayers on overly stiff substrates3. For example, theprofiles are highly comparable to adult cardiomyocytes

beta-adrenergic agonist isoprenaline, or isoproterenol, isindicating a more mature phenotype of these cells.
Amethod, called MUSCLEMOTION, builds on previously
existing algorithms, is fully automated and can be usedon videos, image stacks, or image sequences loaded in
the open-source image-processing program ImageJ5.
It is an open-source, dynamic platform that can beexpanded, improved, and integrated for customized
applications.

Dynamicchanges

inpixelintensity

between image frames are determined and the output
is expressed as a relative measure of movement during
muscle contraction and relaxation. Here, we compare
the previously depicted data sets from Sala et al. 20184
and compare it to FLEXcyte 96 data as recorded with
Cardiosight-S® cardiomyocytes (Nexel).

Download more Application Notes from www.nanion.deB

25 50 75 100
Frames (n)

060
20 40
Frames (n)

60
20 40
Frames (n)

1.00
0.75
0.50
0.25
0.00

0060
2040
Frames (n)60
2040
Frames (n)

1.00
0.75
0.50
0.25
0.00

00

10 20 30 40 50
Frames (n)10 20 30 40 50
Frames (n)Contraction (a.u.)0

Contraction (a.u.)Contraction (a.u.)

1.00
0.75
0.50
0.25
0.00

1.00
0.75
0.50
0.25
0.00

Adult CMs

Velocity (a.u.)

025 50 75 100
Frames (n)

1.00
0.75
0.50
0.25
0.00

EHTs

Velocity (a.u.)

derived from different measurement approaches. The0Contraction (a.u.)

a comparison of cardiac contraction measurements

1.00
0.75
0.50
0.25
0.00

1.00
0.75
0.50
0.25
0.00

Cardiacorganoids

Velocity (a.u.)

Sala et al. (2018)4 developed a method that allows for

1.00
0.75
0.50
0.25
0.00

Velocity (a.u.)

display this physiological response by this compound.

hPSC-CMmonolayers

Isolated
hPSC-CMs

Contraction (a.u.)

heart, although common hiPSC-CM in vitro assays fail to

1.00
0.75
0.50
0.25
0.00

1.00
0.75
0.50
0.25
0.00

01020 30 40
Frames (n)01020 30 40
Frames (n)hiPSC-CMs on FLEXcyte 96
1.00
0.75
0.50
0.25
0.00

0.0

1.00
Velocity (a.u)

well known for its positive inotropic effects on the human

Velocity (a.u.)

intocontraction

development1. Optimizing baseline function of humanContraction (a.u.)platforms/approaches

beat

0.1

0.2

0.3 0.4
Time (s)

0.5

0.6

0.75
0.50
0.25
0.00

0.0 0.1 0.2 0.3 0.4 0.5 0.6
Time (s)Figure 1: A Contraction (top) and velocity (lower) individual beat profile
generated using MUSCLEMOTION for hPSC-CMs, hPSC-CM monolayers,
cardiac organoids, engineered heart tissue (EHTs) and adult rabbit CMs(adapted from Ref. 4). B Contraction and velocity of mean beats ofhiPSC-CMs recorded on the FLEXcyte 96.

Application Note

MUSCLEMOTION analysis of optical images are shown inFigure 2A. Average concentration response curves for[Isoprenaline]

nM
3s

nM

ebkr

1

Normalized
Relaxation Time (%)

Adult CMsµM
3600nMnM

30060

icle30

response is shown for contraction amplitude only in adult50rabbit CMs and EHTs, whereas this increase in contraction

0amplitude can also be detected in hiPSC-CMs recorded
3nM

60060

30030clehi

µM

100nM

are shown in Figure 2B. Importantly, a positive inotropicnM

150nMline obtained for hiPSC-CMs recorded on the FLEXcyte 96

VeNormalized
Ba Contraction Amplitude (%)
se1 lin en
3 M
10 nM30nM
10 nM
030 nM0nM[Isoprenaline]

relaxation time and contraction amplitude for isoprena-[Isoprenaline]
Contraction Amplitude
200

CMs and on hiPSC-CMs recorded on the FLEXcyte 96indicating a more mature or adult-like profile of thesecells when grown on the flexible substrate of the FLEXcyte 96.

[Isoprenaline]

In summary, the FLEXcyte 96 in combination with hiPSCCMs can be used to investigate the effect of positive (andnegative) inotropic compounds on parameters such

[Isoprenaline]

Figure 2: A Average concentration response curves (black) andsingle measurements (red) for isoprenaline for relaxation time (left)
and contraction amplitude (right) generated using MUSCLEMOTION for hPSC-CMs, hPSC-CM monolayers, cardiac organoids,
engineered heart tissue (EHTs) and adult rabbit CMs (adapted fromRef. 4). B Average concentration response curves (black) and single
measurements (red) for isoprenaline for relaxation time (top) andcontraction amplitude (lower) recorded on the FLEXcyte 96.

References
2. Birket, M.J., et al. 2015. Cell Rep. 13(4): 733–745.
3. Stoelzle-Feix, S., et al. 2019. White Paper: https://www.
nanion.de/en/products/flexcyte-white-paper-download.html

Moreover, hiPSC-CMs grown on the flexible substrate of
the FLEXcyte 96 plates (FLX-96) display more adult-like
contraction and velocity profiles compared with hiPSCCMs grown on rigid surfaces. In this way, hiPSC-CMs canbe used for more predictive assays for pro-arrhythmiceffects.

Contraction measurements were conducted according
to Nanion’s standard procedures for the FLEXcyte 96.
Cardiosight-S® were cultured on FLX-96 plates for at least
6 days before compound addition. Compounds wereapplied in the external media. About 4-6 hours before

4. Sala, L., et al. 2018. Circ. Res. 122(3): e5–e16
5. Ribeiro, A.J., et al. 2015. PNAS. 112: 12705–12710.

Methods

drug application a final media exchange was performed,
leaving 200 µl media in each well. For compoundaddition, 50 μl media was removed and replaced with50Cells

thankas contraction amplitude, velocity and relaxation time.

Contraction measurements

1. Pang, L., et al. 2019. Circ. Res. 125: 855–867

We

on the FLEXcyte 96 (Figure 2B), albeit at high concentrations. A reduction in relaxation time is also seen in adult[Isoprenaline]

Normalized
Normalized
Contraction Amplitude (%) B Contraction Amplitude (%)
aselineb
1 ens
3 M
110 nMnMnM
30
310 nM
nM0nM

Ba

seli
1 nen
3 M
10 nM
n30 M
10 nM
0nM

Normalized
Relaxation Time (%)

EHTsnMand adult rabbit cardiomyocytes obtained using the60nM

80[Isoprenaline]

kr[Isoprenaline]

CMs, hPSC-CM monolayers, cardiac organoids, EHTs

100

Normalized
Contraction Amplitude (%)[Isoprenaline]

known positive inotropic compound, for isolated hPSC-120

Normalized

Normalized
Relaxation Time (%)

se1 lin en
3 M
10 nM30nM
10 nM
030 nM
0nMBaBa

se1 lin en
3 M
10 nM
30nM
10 nM
030 nM
0nM

Normalized
Relaxation Time (%)

Cardiac organoids

time and contraction amplitude for isoprenaline, a well-140

hPSC-CM monolayers[Isoprenaline]

Average concentration response curves for relaxation

hiPSC-CMs on FLEXcyte 96
Relaxation Time160

Veh[Isoprenaline]

B

Normalized
Relaxation Time (%)[Isoprenaline]

Contraction Amplitude

Normalized
Contraction Amplitude (%)
DM
0. SO
31 nMn3 M
10 nM
30nM
10 nM
0nM

Normalized
Relaxation Time (%)
DM0. SO
31 nM
n3 M
10 nM30nM
10 nM
0nM

Isolated hPSC-CMs
Relaxation TimeBa Contraction Amplitude (%)
se1 lin en3 M
10 nM30nM
10 nM
030 nM0nM

A

NEXELforproviding

thehiPSC-CMs(Cardiosight-S®).

μl

media

containing

thecompoundat

4x

concentration, resulting in the desired final concentration.
All signals were normalized to a group of control
measurements on the same plate.

Nanion Technologies GmbHphone +
 49 89 219 095-0

Ganghoferstr. 70A

fax80339 Munich, Germany

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