Tackling Chronic Compound Responses of hiPSC-CMs for Preclinical Cardiac
Risk Evaluation: Defined Serum-Free Medium and Long-Term Culture on theFLEXcyte 96
Matthias Gossmann1, Bettina Lickiss1, Marta Lemme2, Elena Dragicevic2, Ravi Vaidyanathan3, Peter Linder1, Ulrich Thomas2, Sonja Stoelzle-Feix2, Michael George2 George Okeyo4,Ronald Knox4, Rodolfo Haedo4 and Niels Fertig2
1innoVitro GmbH, Juelich, Germany, 2Nanion Technologies GmbH, Munich, Germany and 3FUJIFILM Cellular Dynamics, Inc., Madison, WI, USA, Nanion Technologies Inc., NJ, USAResults
Cardiomyocytes serum-free medium on the FLEXcyte 96 system.

SF

1,40,60,480%
60%
40%

0,220%

Technology
The FLEXcyte technology is based on a special 96 well plate that contains high-precision, ultra-thin andhyper-elastic silicone membranes instead of stiff plastic surfaces as basis for human iPSC-CMs. ThisFLEXcyte 96 plate is analysed in the FLEXcyte 96 device (Fig.1A), an add-on system for theCardioExcyte 96 (Nanion Technologies).
In the FLEXcyte 96-well plate (Fig.1B), the cells
adhere as monolayers on flexible substrates.
While being deflected by the weight of theculture medium, rhythmic contraction of the
cardiomyocytes lifts the membranes in the 96well upwards. These changes in deflection arequantified by means of capacitive distancesensing (Fig.1C). The unique Mean Beat
Function of the software automaticallyvisualizes the average beat of traces from one
well per sweep, enveloped by the standard
deviation. Parameters measured are (Fig.1D):beat rate

beat duration

5.C

Amplitude over time

upstrokeslope

day 44

125%
100%
75%
50%
25%
0%
-0,30,2Time / s0,7Beat rate over time

60

195%

50

175%
155%135%
115%

40302095%

1075%

001020304050010Days of culture20304050

Days of culture

downstroke
slope

Figure 5.. (A) Graph shows mean beat comparison of iCell® Cardiomyocytes² cultured for 7, 24 and 44 dayson FLEXcyte 96 plates. Data is normalized to amplitude on day 7. (B) Graph shows rising amplitude of iCell®
Cardiomyocytes² over a time span from 7 – 44 days in culture. Data is normalized to amplitude on day 7. (C)Graph shows beat rate of iCell® Cardiomyocytes² cultured with Geltrex on FLEXcyte 96 plates over a timespan of 7 – 44 days.

SummaryCB

Chronic compound analysis of iCell® Cardiomyocytes² cultured in iCell®
Cardiomyocytes serum-free medium on the FLEXcyte 96 system.

• iCell® Cardiomyocytes serum-free medium for chronic cardiotoxic studies and prolonged cell culture timesof iCell® Cardiomyocytes² on FLEXcyte 96 plates to promote further maturation in vitro were successfullyassessed.

4.A• A defined iCell® Cardiomyocytes serum-free medium was successfully assessed using iCell®
Cardiomyocytes², with a goal to find a solution for scientific issues arising from working with serum orserum free culture media, as the presence of serum has the potential to alter the uptake and relative
potencies of several compounds, and lack of serum eliminates a protein source, necessary for
cardiomyocytes to keep stable conditions over prolonged cell culture times.

Chronic compound analysis of iCell® Cardiomyocytes² cultured
in iCell® Cardiomyocytes serum-free medium

1,6

Amplitude

Beat Rate

Downstroke Slope

1,4
1,2D1

Amplitude of Contraction Force (N/mm2)•• Beat Duration• Upstroke and Downstroke Velocity• Upstroke and Downstroke Area under Curve(AUC)• Beat Rate
• Beat rate Variations• Arrhythmic Events

0,80,60,40,20

3µM erlotinib

300nM doxorubicin

Methods
Workflow of iCell® Cardiomyocytes² on FLEXcyte 96 System.

4.B

4.C
Erlotinib SF mean beat

Doxorubicin SF mean beat

1,21,2.

1
10,80,60,40,2-0,25-0,15-0,050,050,15

Time / sDMSO SF

Erlotinib SF 300nMErlotinib SF 3uM

Erlotinib SF 10uM

0,2500,350,2-0,25-0,15-0,05-0,2Erlotinib SF 1uM

0,050,150,25-0,2Time / s
DMSO SF

Doxorubicin SF 100nM

Doxorubicin SF 300nM

Doxorubicin SF 1uM

Doxorubicin SF 3uM

00,35

Amplitude

0,40,8

Amplitude

0,6Figure 2. Human iPSC-CMs (iCell® Cardiomyocytes², 01434 FUJIFILM Cellular Dynamics Inc.) were cultured
on FLEXcyte 96 well plates at 100k cells per well according to manufacturers’ guidelines in 200 µL serum
containing maintenance medium. Cells were seeded 6 days to allow proper monolayer and network
formation. A final media change was conducted 24 hours before drug application with half the plate . For
compound treatment, 50 µL of the cell culture medium was removed and replaced with 50 µL medium
containing 4x concentrated doxorubicin or erlotinib, resulting in the desired final compound concentration.
Measurements were performed for a maximum of 5 days.

AUC

Figure 3. (A) Comparison of mean beats are shown of iCell® Cardiomyocytes² cultured in iCell® Cardiomyocytes
serum-free and serum containing iCell® Cardiomyocytes Maintenance medium 24 hours after medium switch. (B)Bar graph of contractile parameters (amplitude, beat rate, beat duration, Area under Curve, upstroke anddownstroke slopes) of iCell® Cardiomyocytes² cultured in iCell® Cardiomyocytes serum-free medium after 24hours. Data is normalized to iCell® Cardiomyocytes² cultured in iCell® Cardiomyocytes Maintenance Medium.

Figure 1. FLEXcyte 96 TechnologyA

amplitude-0,2

day 24

150%

0%

0,35

Time / sday 7

175%

-25%

00,15

Mean beats

200%

100%

0,85.B215%

120%

1-0,055.A

140%

1,2-0,25

Contractile parameters of iCell® Cardiomyocytes² in iCell®
Cardiomyocytes serum-free medium

Beat rate / bpm

MM3.B

Mean beats

Amplitude normalized to day 7

3.A

Here we assessed the effects of relatively defined medium (serum-free) and prolonged cell
culture times on iCell® Cardiomyocytes² maturation with focus on functional contractile
properties using the FLEXcyte 96 technology. Analysis of iCell® Cardiomyocytes² cultured iniCell® Cardiomyocytes serum-free medium compared to regular serum-containing iCell®
Cardiomyocytes Maintenance Medium showed similar results for most contractile parametersand expected effects of gold standard compounds assessed over 5 days.
In addition, iCell® Cardiomyocytes², cultured on FLEXcyte 96 plates and analyzed for 40 days,
showed an increase in amplitude and a decrease in beat rate over time, indicating the promaturation effect of a physiological environment over time.

Analysis of maturity indicators amplitude and beat rate of iCell®
Cardiomyocytes² cultured on FLEXcyte 96 plates over 40 days.

Analysis of iCell® Cardiomyocytes² contractile parameters cultured in iCell®

Amplitude normalized to day 7In pre-clinical drug development, cardiac contraction analysis of potential drug candidates is oneof the crucial steps to ensure a successful and reliable transition to clinical stages. The use ofhuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) continues to increasein the assessment of safety and toxicological side effects of newly developed compounds, due totheir reproducibility and low ethical concern. However, the obstacles of an immature phenotype
as well as the need of serum-containing media for chronic assays raise concerns over nonphysiological responses in preclinical drug development. To solve this issue, a variety of methods
are currently tested to foster hiPSC-CM maturation in vitro and to conduct chronic studies without
the need of serum containing medium.

amplitude

Abstract

Figure 4. (A) Bar graph ofiCell
Cardiomyocytes²
culturedin
iCell
Cardiomyocytes
Serumfree Medium (SF) on
FLEXcyte 96 plates andtreated with gold standard
compoundsErlotinib(3μM) and Doxorubicin(300nM) for 5 days.
Depicted bar graph results
show amplitude, beat rateand downstroke slope
reactions after 48 hours.
Data is normalized to iCell
Cardiomyocytes² cultured
in iCell Cardiomyocytes
MaintenanceMedium(MM). (B-C) Comparisonof mean beats are shownof iCell Cardiomyocytes²
treatedwitherlotinib(300nM–10μM)
anddoxorubicin (100nM–3μM)
in iCell Cardiomyocytes
Serum-free Medium. Data
represents mean beat ofiCell
Cardiomyocytes²
reactions 24 hours after
compound treatment.

• iCell® Cardiomyocytes Serum-free medium did not alter most contractile properties of the cells assessedwith the FLEXcyte 96 technology.
• An increase in the downstroke slope, referring to the relaxation phase of the contraction was detected,indicating a higher sensitivity towards intracellular calcium levels.
• Cardiosafe (erlotinib) and cardiotoxic (doxorubicin) compound treatment using iCell® Cardiomyocytes²
showed expected human relevant reactions over a time span of 5 days, indicating an ideal serum-freemedium composition to keep the cells stable for chronic cardiac risk assessment.

✓ Additionally, by applying known maturation promoting cues, such as time and a physiologicalenvironment (flexible membranes of the FLEXcyte 96 plates), one can reliably increase theamplitude and decrease the beat rate of iCell® Cardiomyocytes² over time. Both, well known
indicators for cardiomyocyte maturation (Yuxuan et al., 2020).

Conclusion
The displayed adult-like hiPSC-CM contractile parameters upon prolonged cell culture time further
underline the known Gossmann et al., 2020) pro-maturation effect of the physiological environment
created by the flexible membranes and the technologies potential for long-term cultures of human iPSCCMs.
Serum-free chronic cardiotoxic compound analysis of iCell® Cardiomyocytes² assessed on the FLEXcyte
96, demonstrates the combined power of iCell® Cardiomyocytes² and the FLEXcyte technology for(sub)chronic safety and toxicity evaluation of new drug candidates.

References
Gossmann M. Journal of Pharmacological and Toxicological Methods. 2020; 106892
Yuxuan G., William T. P. Circulation Research. 2020;126:1086–1106