Application Note

FLEXcyte 96
Positive inotropic effects in human iPSC-derived
cardiomyocytes can be triggered through a flexible promaturation environment.
High throughput Screening (HTS) scalable
techniques with highly predictive cell models
are needed to improve the expensive and timeconsuming drug development process.
Potentially dangerous consequences of side
effects on the human heart make safety
testing of heart related issues the main focus
of pre-clinical drug development studies.
However, one of the most commonly used
gold standard technique for cardiac
contractility measurements, the ex vivo
Langendorff set-up, does not efficiently
support modern drug development processes
as it uses non-predictive animal models on avery low throughput level.
Human induced pluripotent stem cell-derived
cardiomyocytes (hiPSC-CMs) combine a
number of features fostering the drug
development process such as highpredictivity, large scale applicability with highthroughput potential, low ethical concerns andcost effectiveness. Yet, when cultured on
overly stiff substrates like glass or plastic, thecells are placed under unnecessary stress dueto the missing auxotonic physiologicalenvironment provided by a flexible substrate.
These unphysiological conditions lead todrastictranscriptionalandmetabolic
deregulation in cardiomyocytes which affectthe predictive value of this established cell
model [1].
To bridge the gap of predictive contractility
measurements and HTS analysis for drug
development studies, innoVitro co-developed
the FLEXcyte 96 (Figure 1) with Nanion
Technologies as an add-on for theCardioExcyte 96 platform. With less than
10 μm in thickness and sophisticated surface

modification,
thepolydimethylsiloxane(PDMS) membranes of the FLEXcyte 96
disposable plates offer physiological elasticity
of native human heart tissue [2]. As a result
hiPSC-derived CM behave in an in vivo mannerand finally reach their full potential as a CiPA
confirmed model for drug development
processes [3] [4].
Beta-adrenergic agonist isoproterenol and Ltype calcium channel agonist S-Bay K8644 areboth well known for their positive inotropic
effects on the human heart, although common
iPSC-CM in vitro assays fail to display thisphysiological response by showing negative
inotropic effects instead.
Here, we show that the auxotonic environmentof the FLEXcyte 96 enables maturephysiological responses of hiPSC-CMs on
positive inotropic substances such as L-type
calcium channel agonist S-Bay K8644, betaadrenergic agonist isoproterenol and cardiac
myosin activator omecamtiv mecarbil.

Figure 1. FLEXcyte 96 device for the assessment of cardiac
contractility under physiological mechanical conditions

Application Note FLEXcyte 96AIsoproterenol

B1000 nM

200%

S-Bay K8644

150%

30 nM
10 nM
control

100%

100%

0%-0,3220%
200%

00,3Time / sbeat amplitude
beat duration

50%

50%

0%

0%
-0,30,600,3 0,6Time / s1000 nM
100 nM
control100%

control

50%Omecamtiv Mecarbil

150%

150%

100 nMC-0,30,9150%

135%

140%

130%

00,3Time / s

0,6125%

180%

130%

160%

120%

115%

140%

110%

110%

120%

100%

100%

90%

80%

80%

120%

105%
100%
95%

90%

Figure 2. Recorded beat shape and corresponding beat duration / beat amplitude analysis of hiPSC-CM cultured on FLEXcyte 96
well plates after treatment with isoproterenol (A), S-Bay K8644 (B) and omecamtiv mecarbil (C). A. hiPSC-CM (Cardiosight®-S,Nexel) beat shape after treatment with 100 nM and 1000 nM isoproterenol in comparison to control. Beat amplitude and beat duration
analysis of hiPSC-CM after treatment with increasing concentrations of isoproterenol (1 nM – 1000 nM) in comparison to control.
B. hiPSC-CM (iCell® CM2, FCDI) beat shape after treatment with 10 nM and 30 nM S-Bay K8644 in comparison to control. Beat
amplitude and beat duration analysis of hiPSC-CM after treatment with increasing concentrations of S-Bay K8644 (10 nM – 300 nM)
in comparison to control. C. hiPSC-CM (Cardiosight®-S, Nexel) beat shape after treatment with 100 nM and 1000 nM omecamtiv
mecarbil. in comparison to control. HiPSC-CM beat amplitude and beat duration analysis of omecamtiv mecarbil treatment(1 nM – 1000 nM) in comparison to control.

Results
Figure 2 shows the dose-dependent effects ofisoproterenol, S-Bay K8644 and omecamtiv
mecarbil treatment on hiPSC-CM seeded onto
FLEXcyte 96 well plates.
Isoproterenol increased the contraction
amplitude in a dose-dependent manner toapprox. 210% of control at 1 µM. At the sametime, the durations of contraction andrelaxation phase were shortened, indicating anincrease in acceleration and deceleration ofthe contraction.

In contrast, S-Bay K8644 elicited a symmetry
change in the beat shape. While thecontraction phase was unaffected, theincrease in intracellular calcium concentration
resulted in a prolongation of the relaxation
phase. As a result, the total duration of thecontraction-relaxation-cycle was increased to120% of control. The amplitude reached 150%of control at 30 nM. Omecamtiv mecarbil
increased both the contraction amplitude as
well as the duration of the contraction-

Application Note FLEXcyte 96
relaxation-cycle, indicating the positive
inotropic effect based on activation of themyosin complex. The symmetry of thecontraction-relaxation-cycle was not changed.
The mature physiological responses of hiPSCCM shown here are triggered by compounds
acting at different target levels, underlining thepro-maturation effects of the FLEXcyte 96
which cannot be elicited with other assays
commonly used for drug development
purposes [5].

mediumcontaining
4x
concentrated
compound, resulting in the desired final
compound concentration.
The CardioExcyte/ FLEXcyte Control software
enables online analysis of contractility
parameters. An adaptive signal detection
algorithm extracts the positions and values ofbeating events. Besides beat intervals,
amplitudes, rising and falling time, pulse
witdhs are detected. Furthermore, integrals
and arrhythmia are identified and quantified aswell.

Methods
References
Human iPSC-derived cardiomyocytes werekindly provided by Fujifilm Cellular Dynamics
International (Madison, WA, USA) and NEXEL(Seoul, Republic of Korea).
The cells were cultured on FLEXcyte 96 wellplates according to manufacturers’ guidelines
in 200 µL maintenance medium per well. Cells
were seeded approximately 6 days before
compound treatment at 50k (Cardiosight®-S,NEXEL) or 100k (iCell® CM², FCDI) per well toallow proper monolayer and network
formation. A final media change wasconducted 4-6 hours before drug application.
For the experiments, 50 µL for the cell culture
medium was removed and replaced with 50 µL[1] Heras-Bautista et al., Acta Biomater 89,pp. 180-192, 2019.
[2] Gossmann et al., Cell Physiol Biochem 38,pp. 1182-1198, 2016.
[3] Blinova et al., Cell Reports 24, pp. 35823592, 2018.
[4] Bot et al., JPTM 93, pp. 46-58, 2018.
[5] Jiang at al., Mol Cells 41, pp. 613-621,
2018.

Notes

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