Category: Assay Development and Screening
[Introduction] For drug selection at early stages of development, most of the common electrophysiological assays used for cardiac safety studies lack a reasonable throughput (manual patch-clamp recordings from primary cardiomyocytes or electrode recordings from multicellular preparations) or a high informational content (automated patch-clamp of recombinant cell lines). This can be overcome by combining the high level of information of HumaniPS-derived cardiomyocytes (hiPSCM), which are increasingly used for the assessment of drug-induced proarrhythmic adverse effects, into HTS assay systems. Here we describe a method for the recording of membrane potential changes and calcium transients using fluorescent dyes in 96 well format with the Hamamatsu FDSS, an advanced kinetic plate reader system.
[Methods] The assay was established with commercially available hiPSCM including Cor.4U and Pluricytes from Ncardia. The cells were seeded and cultured in 96 well plates according to provider’s instructions or received as a ready-to-use pre-seeded 96 well plate (Cardioplate 96). The cells were loaded with Cal520 or FluoVolt dyes, respectively, in serum-free assay buffer before the dyes were washed-out. Recordings were performed with the FDSS 7000 or µCell plate readers at 37°C. All 96 wells were recorded simultaneously with a sample rate of 62.5 Hz (16 ms interval) and signals were analysed using the Wavechecker software (Hamamatsu).
[Results] Voltage and calcium signals were easily obtained with a very good signal to noise ratio. The signal of a whole well was close enough to single cell or a small cluster of cell activity to show the same properties of genuine action potentials and calcium transients. It allowed deep analysis for precise pharmacological effects characterization.
[Conclusion] We show here that voltage- and calcium-sensitive fluorescent dye recordings from hiPSCM with the Hamamatsu FDSS kinetic plate reader are predictive and robust assays for the detection of cardiotropic effects. The semi-automatic analysis allows a rapid and intuitive overview of the effects of the drugs in the plates with a very good throughput. Both hiPSCMs, Cor.4U and Pluricytes, are suitable for this assay and the possibility of pre-seeded plate provides additional easiness and reproducibility.
Greg Luerman– Technical Director, Ncardia, Plymouth Meeting, PA