Is there an alternative workflow for MEA and xCELLigence applications with iCell Cardiomycytes2 other than a 4-day assay from Monday to Friday?

We have an Application Protocol that describes how to handle iCell® Cardiomyocytes2 for use on the FDSS and provides basic instructions for compound treatment, data acquisition, and analysis. We demonstrated culture of iCell Cardiomyocytes2 on 96- or 384-well plates, where they formed a stable, electrically and mechanically active syncytium amenable to measuring drug-induced perturbations for in vitro screening of compound efficacy and toxicity. Experiments should be conducted after 7 days in culture, where screening can be performed on the same day the calcium dye is added to reduce potential for toxicity.

Others have demonstrated the utility of the FDSS/μCELL system (Hamamatsu Photonics) with our iCell Cardiomyocytes in posters (Saito et al. and Roman et al.) and in the peer-reviewed publication by Puppala et al.

For both of these screening systems, assay timing is critical, and we recommend that you adhere to the workflow in the application protocol. Ideal timing for FDSS analysis of iCell Cardiomyocytes2 is after day 7 post-thaw, which shows consistent and uniform results across wells when measuring beat rate and dose response. To reduce the potential for toxicity, we recommend that you screen on the same day that you add calcium dye.

It is also important to pay attention to temperature with cardiomyocytes, as deviations from a controlled environment can reduce the beat rate of the cells. To overcome temperature variations, keep run times short and always return plates to 37°C incubator as soon as possible during incubation steps.

General Protocol Recommendations:

  • Assay timing: day 7
  • Assay density (direct plating): 50,000 cells plated in 100 μl per well in a 96-well plate; 16,500 cells plated in 33 μl per well in a 384-well plate
  • Assay media: iCell Cardiomyocytes Maintenance Medium
  • Assay ECM: 0.1% Gelatin

For more information about optimizing culture conditions for iCell Cardiomyocytes, including edge effects and environmental issues, see Solution 00000043E.

References:

  1. Application Protocol: Measuring Cardiac Activity: Intracellular Calcium Flux Detection on the FDSS System Application Protocol
  2. Application Note (Molecular Devices):EarlyTox Cardiotoxicity Kit Provides Biologically Relevant Cardiotoxicity Data Earlier in the Drug Discovery Process Application Note (Molecular Devices)
  3. Poster Presentation: Sirenko et al, 2013 Multiparameter In Vitro Assessment of Compound Effects on Cardiomyocyte Physiology Using iPSC Cells
  4. Poster Presentation: Saito et al. (SLAS 2014) Electric Field Stimulation of iPSC-Derived Cardiomyocytes Using Hamamatsu FDSS/μCELL with Fast Data Acquisition
  5. Poster Presentation: Roman et al. (SPS 2014) Determination of Pro-arrhythmic Effects of Compounds in Human iPSC-Derived Cardiomyocytes Using FDSS/μCell Imaging Platform
  6. Puppala et al. 2013 Comparative Gene Expression Profiling in Human-Induced Pluripotent Stem Cell–Derived Cardiocytes and Human and Cynomolgus Heart Tissue
  7. Bedut et al. 2016 High-throughput Drug Profiling with Voltage- and Calcium-Sensitive Fluorescent Probes in Human iPSC-Derived Cardiomyocytes