Calcium Transient Assay for Cardiac Arrhythmic Potential Evaluation: using human iPS-Derived Cardio Myocytes

Commonly used acronym: CTCM on hiPS-CMs

Scope of the method

The Method relates to
  • Human health
  • Other
The Method is situated in
  • Regulatory use - Routine production
  • Translational - Applied Research
  • Other
Safety Pharmacology
Type of method
  • In vitro - Ex vivo
This method makes use of
  • Human derived cells / tissues / organs
Specify the type of cells/tissues/organs
hiPSC-derived cardiomyocites


Method keywords
  • Calcium transient
  • Proarrhythmia
  • Stem cell
  • Fluorescent Dye Calcium-sensitive Imaging
  • Cardiomyocyte
  • hiPS-CMs
  • Human induced Pluripotent Stem Cell
Scientific area keywords
  • Safety Pharmacology
  • Side effects
  • medium-high throughput assay
  • cardiovascular derisking
  • regulatory
Method description

Cell Culture and Reagents hiPSC-CMs can be obtained commercially either as living pre-plated cells seeded onto fibronectin-coated 96-well mClear plates (Greiner Bio-One, No. 655090) or can be plated in house at a density (~25,000 cells/well) suited to forming a confluent synchronously beating mono-layer. Most commercially offered cardiomyocyte lines represent a mix of ventricular, atrial and nodal cells and derived from one human donor. Cells were cultured with commercial culture medium (optimized for the specific cell line) in a humidified incubator at 37oC and 5% CO2, with medium being changed once a day. On the day of the experiment, the culture medium was replaced with Tyrode’s solution (Sigma, No. T2397) supplemented with 10 mM HEPES together with KCl to represent isokalemic (4.2 mM K+) conditions. As a calcium-sensitive fluorescence dye Cal-520 AM (Cat. No. 36,338; AAT Bioquest) was used to capture the intracellular calcium transients. Accordingly, Cal-520 was incubated for 45 min followed by a washout and a 30-min recovery before starting the experiments. Calcium Transient Measurements Spontaneous beating activity of hiPSC-CMs was assessed through measurement of the Ca2+ fluorescence signal integrated over the whole well. Fluorescence signals were measured using the Hamamatsu FDSS/mCell platform and the records subsequently analyzed offline using NOTOCORD-hem software (version 4.3), containing EXT modules and an algorithm developed by XiTechniX to detect beat-by-beat Amp, BR, and CTD90 parameters. All wells within a plate were measured simultaneously using the following FDSS/mCell settings: sampling frequency 66.7 Hz, exposure time 14.6 ms, excitation wavelength 480 nm, emission wavelength 540 nm, temperature controlled at 37oC. First, the experimental plates were put into the FDSS/mCell to stabilize for 10 min. Next, a baseline recording was run for 3 min followed by compound addition. The effect of a compound was recorded (5-min recording time) around 15 and 30 min after compound addition. CTD90, BR, and Amp were quantified for baseline and 30-min compound effects as the median value of all beats (calcium transients) measured within a 1-min interval of the recording. The recording around 15 min was used only for observation of Early-After-Depolarisation (EADs) or fibrillation-like events. EADs were manually monitored and evaluated. Cessation of beating was defined after 30 min in case BR was <5 beats/min. Wells that temporarily stopped beating during compound addition but recovered at the 30-min time point were not defined as beat stop.

Lab equipment
  • Laminar flow hood ;
  • Cell Incubator ;
  • amamatsu FDSS/mCell platform ;
  • Analysis software.
Method status
  • Internally validated
  • Published in peer reviewed journal
  • Validated by an external party (e.g. OECD, EURL ECVAM,…)

Pros, cons & Future potential


Early (fast) evaluation of arrhythmic potential using human cells

  • Maturation of cells remains debate ;
  • Cell-layer don’t reflect 3D-complexity of a (human) heart ;
  • Commercial cell line represents n = 1.
  • Use of more mature cells (due to improved culture conditions ?) ;
  • Improved phenotypic appearance (obtaining closer cellular resemblance of the human heart).
Future & Other applications

Calcium transient method can potentially be applied for measuring activity of other cell types as well

References, associated documents and other information


Lu HR et al., Assessing Drug-Induced Long QT and Proarrhythmic Risk Using Human Stem-Cell-Derived Cardiomyocytes in a Ca2+ Imaging Assay: Evaluation of 28 CiPA Compounds at Three Test Sites. Toxicol Sci. 2019 Aug 1;170

Kopljar I. et al., Development of a Human iPSC Cardiomyocyte-Based Scoring System for Cardiac Hazard Identification in Early Drug Safety De-risking. Stem Cell Reports. 2018 Dec 11;11(6):1365-1377

Kopljar I. et al., Impact of calcium-sensitive dyes on the beating properties and pharmacological responses of human iPS-derived cardiomyocytes using the calcium transient assay. J Pharmacol Toxicol Methods. 2018 May - Jun; 91: 80-86.

Contact person

Ard Teisman


Janssen Pharma of JNJ
Global Safety Pharmacology
Flemish Region