Mechanical Stimulation in a 2D High-Throughput Contractility System Induces
Functional Changes in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes
Marta Lemme1, Peter Linder2, Ulrich Thomas1, Elena Dragicevic1, Michael George1, Niels Fertig1, Matthias Gossmann2, Sonja Stoelzle-Feix1
1 Nanion Technologies, Munich, Germany
2 innoVitro GmbH, Juelich, Germany

Maturation of hiPSC-CMs
can be enhanced by mechanical stress

FLEXcyte 96 can reproduce theFrank-Starling mechanism

The use of human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs) to assess efficacy and cardiac safety of new drug candidates continues toincrease. In this context, a new 2D high-throughput system, FLEXcyte 96, allows forcontractility measurements of cells cultured in a more physiological environment,
reflecting the mechanical conditions of the native human heart1.
This study aims to show the influence of mechanical stimulation on functional
behavior of hiPSC-CMs. This aspect is of particular importance, as mechanicalload acts as crucial growth regulator in the developing heart.

Increasing levels of applied strain resulted inhigher contraction amplitudes of the cellmonolayer. The amplitude increase under higher
strain could be observed in both serum-free andmaintenance medium culture conditions, but it
was more prominent in serum-free conditions. Thisconfirms the reproducibility of the Frank-Starlingmechanism with the FLEXcyte 96.

Mechanical stimulation modulates the contractility of hiPSC-CMs

FLEXcyte 96 allows applicationof constant and cyclic stretching to cell monolayer
iCell Cardiomyocytes2 (FUJIFILM Cellular Dynamics) were cultured on fibronectincoated silicone membrane of the FLEXcyte plate (105 cells/well). The cellmonolayer was further mechanically stimulated through application ofpneumatic pressure, resulting in increased deflection and mechanical strain of
the cell monolayer. Through this feature, the FLEXcyte 96 allows biaxial loadconditions and simultaneous contractility evaluation of cell monolayers in a 96
well format. Different levels of strain (%) inducing membrane deflection wereapplied to the cell monolayer in order to test the reproducibility of Frank-Starlingmechanism. In addition, 1Hz cyclic pressure stimulation, inducing 5% membrane
deflection, was applied to investigate its effect on hiPSC-CMs monolayer during 3days. This cyclic stretching of the silicone membranes aims to imitate thephysiological stretching experienced by CMs in the beating human heart duringsystolic and diastolic phases.

Frank-Starling mechanism shows contraction amplitude ofiCell Cardiomyocytes2 cultured in maintenance medium
and serum-free buffer at increasing level of membranestrain. The amplitude values were normalized to theamplitude at 0% strain.

Serum-free buffer

Amplitude (a.u)

4

FLEXcyte 96 plate.

3210

Picture of FLEXcyte 96 modified to have an active
pressure regulation (a prototype). This system allows toapply a mechanical stress to the cell monolayer.

0%
0.5%
1.3%
1.8%
2.2%
2.6%
2.8%

FLEXcyte 96 mechanical stimulation promotes maturation of hiPSC-CMs
0.2

0.4

0.6

0.8

1.0

Time (s)-1Contraction amplitude over time in serum-free buffer atincreasing level of membrane stretching (%).
The bottom of a FLEXcyte 96 plate
has silicone membranes
characterized by a thickness ~6 μm
and an elastic modulus 30KPa.

Mainteinance medium
0%0.5%1.3%
1.8%
2.2%
2.6%
2.8%

Amplitude (a.u)

432

The Frank-Starling mechanism could be successfully reproduced in the FLEXcyte 96. Applying higher steps of pressure
induces higher membrane strain, which in turn resulted in higher contraction amplitude. The higher amplitude increase inserum-free buffer could be associated to the basal weaker contractile activity of the hiPSC-CMs when cultured without
serum. In other words, hiPSC-CMs cultured in serum-free buffer offer a higher window of potential amplitude increase2.
We conclude, that FLEXcyte 96 is a reliable high throughput tool for in vitro cardiac contractility research, which allows
culturing hiPSC-CMs under more physiological conditions and introduces dynamic change to their mechanicalenvironment. In this study, the applied mechanical stimulation resulted in higher contraction amplitude and faster
upstroke/relaxation velocity. In hiPSC-CMs physiology, these changes indicate higher level of maturation towards adult
hiPSC-CMs3. FLEXcyte 96 thereby offers the potential to measure real values of contraction force in an in vitro 2D highthroughput format and model cardiac pathological conditions, such as acute hypertension.

510-1

Pneumatic pressure mechanism in a single well of FLEXcyte 96.

Comparison of iCell Cardiomyocytes2 contractility parameters under control condition and after 3 days of 5% cyclicstretching: 3 days of cyclic stretching resulted in a significant increase of the contraction amplitude (3.1±0.14 a.u, n=47vs. 1.7±0.07 a.u, n=41; p<0.0001), upstroke velocity (23±0.9 a.u/s, n=47 vs. 13.5±0.6 a.u/s, n=41; p<0.0001) and relaxation
velocity (-20.4±0.8 a.u/s, n=47 vs. -17.3±0.8 a.u/s, n=41; p=0.0172) compared to non-stimulated cells. Accordingly, therising and falling times decreased significantly. Unexpectedly, also the beat rate increased after 3 days of cyclicstretching (87.8±0.7 bpm, n=47 vs. 60±0.6 bpm, n=41; p<0.0001), which resulted in a shortening of the pulse width.

0.2

0.4

0.6

0.8

1.0

Time (s)Contraction amplitude over time in maintenance medium
at increasing level of membrane stretching (%).

References
1. Gossmann M. Journal of Pharmacological and Toxicological Methods. 2020; 106892
2. H. Naito. Circulation. 2006;114:I-72–I-78
3. Yuxuan G., William T. P. Circulation Research. 2020;126:1086–1106

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