Development of an Impedance Based Screening Assay for Cardiac Safety and Cardiotoxicity Detection in Stem Cell-derived Cardiomyocytes

Robert W. Kirby, Said El Haou, Edward Humphries, John Ridley and Marc Rogers

1. Metrion Biosciences Ltd, Riverside 3; Suite 1, Granta Park, Cambridge, CB21 6AD, United Kingdom

Validation of chronic cardiotoxicity assays

Cardiac toxicity remains the leading cause of new drug safety side-effects. Current preclinical cardiac safety assays rely on in vitro cell-based ion channel assays and ex vivo and in vivo animal models⁽¹⁾. These assays provide an indication of acute risk but they do not always predict the effect of chronic compound exposure, as recently seen with oncology drugs⁽²⁾. Therefore, new assays are required to characterise chronic structural and functional effects in human cells earlier in drug discovery⁽²̛ ³⁾. Impedance-based technology can provide more accurate chronic cardiotoxicity measurements in an efficient manner using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs)⁽¹̛ ³⁾.

Here we outline validation of chronic cardiotoxicity assays using an impedance platform in combination with commercial hiPSC-CMs:

1. Impedance readouts can be reliably measured and are stable to allow acute (< 24 hrs) as well as longer duration (72 hrs) testing.
2. Identify the acute and chronic effects of clinical cardiac ion channel modulators against cardiomyocyte contractility and viability.
3. Confirm that structural and functional cardiotoxicity of clinical compounds arising from multiple cellular and signalling mechanisms could be detected in an acute and/or chronic assay timeframe.

Cardiomyocyte culture and impedance monitoring

hiPSC-CMs from commercial vendors (Ncardia and CDI) were plated onto CardioExcyte96 well plates (Nanion) according to manufacturers’ instructions. Plates were incubated at 37 °C (5 % CO₂ ) for 7-10 days and 100 % medium exchanges were performed daily. Impedance signals were recorded using the CardioExcyte96 (CE96) platform and the formation of a spontaneous beating syncytial network was monitored with 30 s impedance recording every hour (1 hr) during the first week in culture.

A single concentration of compound was then applied to each well (N ≥ 4) every 24 hrs as a 1:1000 fold dilution into media (final = 0.1 %) for a maximum of 72 hours before a washout period. The outputs (Figure 1) were recorded at 60 minute intervals to assess acute vs. chronic effects (< 24 hrs vs 24 – 72 hrs).

Predictive power and translational relevance of the cardiotoxicity assay

• The combination of CE96 impedance platform and commercial human stem cell-derived cardiomyocytes delivers a stable and consistent assay with a high success rate across every 96 well plate, enabling the reliable screening of acute and chronic cardiotoxicity effects of test compounds.
• Surprisingly, we detected chronic functional and structural effects of known cardiac ion channel modulators, in addition to their expected acute effects on cell excitability and contraction.
• This assay can reliably detect the chronic cardiotoxicity effects of clinical compounds with diverse mechanisms-of-action, indicating its utility as part of a translational cardiac safety testing regime.

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