Application NoteiCell® Cardiomyocytes2:

Lot-to-lot Robustness Study of Commercial Human
iPSC-derived Cardiomyocytes
New approach methodologies (NAMs) employing
human-based cell types hold great promise for
improving drug attrition rates early in the drug development process¹. Advantages of these approaches overcurrent animal-based testing methods encompass
functional as well as financial reasons, such as elimination of the animal-to-human translational gap andincreased throughput for cost-efficient data generation.
Cardiac-related side effects display one of the major
causes of high drug attrition rates, demonstrating theurgent need of the cardiovascular field for
integrating human-relevant platforms to reliably
analyse preclinical safety risks². Commercial humaninduced pluripotent stem cell-derived cardiomyocytes(iCell Cardiomyocytes²) represent an ideal cell modelfor this matter with constant availability of well characterized cardiomyocytes³. Nevertheless, consistentfunctional behaviour amongst different lots needs tobe assessed adequately to prove this cell models’ reliability for the use in preclinical drug development.
Here we demonstrate the robustness of iCell
Cardiomyocytes² on the FLEXcyte 96 system, a NAM
that mimics physiological human heart conditions withflexible membranes as substrates for the cells on a96-well plate4,5. Cardiac contraction behaviour of ten
iCell Cardiomyocytes² lots was assessed before andafter compound treatment with nifedipine and sotalol,
not only demonstrating the reliability of this cell model,
but also the robustness and combined power of iCell
Cardiomyocytes² and the FLEXcyte 96 system.

Mean Beat Rate of iCell Cardiomyocytes2 Before CompoundFigure 1. Mean beat rate of iCell Cardiomyocytes² on FLEXcyte 96 plates before compound addition. Ten different lots
collected over a time span of 1 year were assessed for beat rate after 6 days in culture. Bar graph shows the beat rate/bpm(beats per minute) of the lots with only minor fluctuations ranging from 47 bpm – 53 bpm.

Figure 2. Amplitude of iCell Cardiomyocytes2 analyzed with the FLEXcyte 96 system after compound treatment withnifedipine. Control condition (blue) is normalized to 100%. Nifedipine concentrations of 10 nM (green) and 30 nM (pink) are
shown. Dotted lines in respective colors represent standard deviations.

Results
Over a time course of one year, iCell Cardiomycytes2
vials from ten different lots were cultured on FLEXcyte-96 plates according to manufacturer’s guidelines2.
The cells were maintained in culture for 6 days to allow
proper syncytium formation. To analyze the stability of
the general beating behaviour amongst the lots, contractility was assessed with the FLEXcyte 96 system.
The data demonstrates a stable performance of each
lot with similar beating behaviour ranging from 47 – 53bpm (Fig.1).
After contraction analysis of iCell Cardiomyocytes²,
acute drug-induced effects were analyzed using two
concentrations of gold standard compounds nifedipine and sotalol, respectively. Amplitude of contraction
force and beat duration served as contractility-relatedparameters to determine the functional behaviour ofiCell Cardiomyocytes² upon compound treatment.
Treatment with calcium channel blocker nifedipine
showed a concentration-dependent decrease in meancontraction amplitude from 1.0 (control) to 0.8 ± 0.05
upon 10 nM, and a decrease to approximately 0.5 ±0.09 upon 30 nM nifedipine treatment.
The low variability in between lots is shown by thestandard deviation not exceeding 10% ( Fig. 2).

Additionally, assessing the effect of nifedipine on thebeat duration further demonstrates the functional
consistency of iCell Cardiomyocytes², as a concentration-dependent shortening of the beat duration⁶ wasobserved for 10 nM and 30 nM nifedipine, respectively(Fig. 3).

Control
Nifedipine 10 nM
Nifedipine 30 nM

Figure 3. Nifedipine effect on iCell Cardiomyocytes2
downstroke duration. 10 nM (green) and 30 nM (pink)
nifedipine concentrations are shown compared to the control(blue). Higher concentrations of nifedipine lead to a shortening in beat duration. (Extract of downstroke duration isdepicted).

Figure 4. Amplitude of iCell Cardiomyocytes2 treated with sotalol. Mean amplitudes are shown after 10 μM (green) and100 μM (pink) sotalol treatment compared to the control condition (blue) set to 100%. Dotted lines in respective colors represent standard deviation.

The effect of hERG channel blocker sotalol on ten iCell
Cardiomyocytes2 lots was also carried out with focus
on amplitude, beat duration and respective standarddeviations. As drug-treatment occurs in a serum-freebuffer, insignificant fluctuations of the mean contraction amplitude at 10 μM and 100 μM sotalol wereobserved, presumably as a result of a reduced/missing
ß-adrenergic pre-stimulation. The low standard deviations not exceeding 10% demonstrate once more therobust reaction window of the cells (Fig.4).

The known torsadogenic risk potential of sotalol7 could
be shown with the analyzed beat duration after 10 μMand 100 μM treatment. Here, a concentration-dependent duration prolongation was detected representing
the contractile response to QT-prolongation (Fig.5).
Together, these data demonstrate the robustnessof this cell-based assay system for reliable preclinical cardiotoxicity assessment, uniting cost effective
high-throughput analysis with human-based
cardiomyocytes.
MethodsiCell Cardiomyocytes2 were cultured on FLEXcyte 96
well plates in 200 μl maintenance medium per well.
Cells were seeded 6 days before compound treatment
at 100,000 (iCell Cardiomyocytes2, FCDI) per well toallow proper monolayer and network formation. A final
media change was conducted 4-6 hours before drug
application.

Figure 5. Sotalol effect on iCell Cardiomyocytes2 downstroke duration. 10 μM (green) and 100 μM (pink) sotalol
concentrations are shown compared to control (blue). Higher
concentrations of sotalol lead to a prolongation in beat duration. (Extract of downstroke duration is depicted).

Measurements were performed acutely over a periodof 20 minute after compound addition. The CardioExcyte / FLEXcyte Control software enables direct
analysis of contractility parameters. An adaptive signal
detection algorithm extracts the positions and valuesof beating events. Beat intervals, amplitudes, risingand falling time, pulse widths are detected as well as
integrals and arrhythmia.

References

For More Information

1. Fermini et al., SLAS Discovery 23 (8), 765–776(2018).
2. Pang et al., Current Opinion in Toxicology. 23, 5055 (2020).
3. Burnett et al., Expert Opin Drug Metab Toxicol.
17(8), 887–902 (2021).
4. Gossmann et al., J Pharm Tox M. 105, 106892,(2020).
5. Lickiss et al., J. Vis. Exp. 188, (2022).
6. Guo et al., Cell Physiol Biochem. 27 (5), 453-62(2011).
7. Ando et al., J Pharm Tox M. 84, 111–127, (2017).

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