Combined impedance and extracellular field potential recordings on iPS cardiomyocytes

• ¹ Nanion Technologies, Germany
• ² Nanion Technologies Inc., United States
• ³ Nanion Technologies, Germany
• ⁴ Nanion Technologies, Germany
• ⁵ Nanion Technologies, Germany

Motivation Drug induced arrhythmia is one of the most common causes of drug development failure. Human induced pluripotent stem cell-derived cardiomyocytes (iPSCMs) have a great potential for cardiovascular research and predictive in-vitro cardiac safety screening when it comes to early detection of arrhythmic compounds. Material and Methods The CardioExcyte 96 system (Nanion Technologies GmbH) provides a non-invasive, label-free, high temporal resolution approach for safety screening on iPSCMs (Figure 1)(1). It is a hybrid screening instrument that combines impedance with MEA-like extracellular field potential (EFP) recordings (2,3). It can be either used in an incubator or, by utilizing the dedicated incubation system, directly on a lab bench. The system is able to electrically pace the cells, allowing screening of cells which beat with individual frequencies and, in addition, investigations of frequency dependent compound inhibition. All hiPSC-CMs (iCell Cardiomyocytes2 from Cellular Dynamics International, Cor.4U from Axiogenesis AG, Pluricytes from Pluriomics and Cellartis® hiPS-CM from Takara Bio Europe AB) attached onto the NSP-96 sensor plates whether thawed from frozen or harvested from live cultures, were beating and subsequently recorded and analyzed. Data was analyzed using DataControl 96 (Nanion Technologies GmbH), using online analysis parameters as previously published (1,4). Results We describe the development and optimization of a cell-based assay that is sensitive and provides reproducible results for safety pharmacology. Changes in the impedance signal indicate effects on cell contractility and shape whereas the field potential parameters provide information about the electrophysiological activity of the beating network of cells. We performed recordings of hiPSC-CMs obtained from four different cell providers, demonstrating stable and reproducible signals with cells forming a monolayer and establishing synchronous beating (4). Additionally, in accordance with the Comprehensive In Vitro Proarrhythmia Assay (CiPA) guidelines, standard reference compounds were tested on these same cells. We have tested the effects of classical hERG blockers (E-4031, Figure 2 and dofetilide, data not shown (4)), CaV blocker (nifedipine, Figure 3) and beta-adrenergic receptor agonist (isoproterenol, data not shown (4)), on the impedance and EFP signals of hiPSC-CMs. In addition to these compound, we have tested six more compounds with similar signal stability. A color-coded summary of the effects of 10 compounds on impedance and EFP parameters (up- or downregulation) is given in Table 1 (4). Discussion We have shown combined impedance and EFP measurements using a commercially available platform (CardioExcyte 96) can be made reliably from a number of different hiPSC-CMs including Cor.4U (Axiogenesis AG), iCell Cardiomyocytes2 (Cellular Dynamics International), Pluricytes (Pluriomics) and Cellartis® hiPS-CM (Takara Bio Europe AB) (4). These cells are becoming increasingly important for cardiac safety testing due to their abundance, purity, recapitulation of native behavior and suitability for HTS techniques. Our data show a highly relevant drug safety assay, which assesses the potential cardiac liabilities of a drug candidate with two functional readouts, the contractility and electric field potential, using stem cell-derived cardiomyocytes. The data presented documents the suitability of this safety assay format in terms of predictability and ease-of-use. Together with the availability of a variety of stem cell-derived cardiomyocytes from different providers, we demonstrate the potential for the assay to become a routine/valuable tool in drug safety assessment under the future CiPA paradigm. Conclusion The CardioExcyte 96 is, so far, the only platform on the market capable of recording in combination impedance and EFP measurements from same cell. This provides a unique opportunity to detect changes in both contractility and ion channel function at a high throughput which may prove crucial for cardiac safety screening particularly in the light of the new CiPA guidelines. References 1. Doerr, L., Thomas, U., Guinot, D. R., Bot, C. T., Stoelzle-Feix, S., Beckler, M., George, M., Fertig, N., J. Lab. Autom., 20(2), 175–188. (2015). 2. Giaever, I., & Keese, C. R., Proc. Natl. Acad. Sci. U. S. A., 88(17), 7896–900. (1991). 3. Clements, M., & Thomas, N., Toxicol. Sci., 140(2), 445–61. . (2014). 4. Obergrussberger, A., Juhasz, K., Thomas, U., et al. J. Pharmacol. Toxicol. Methods, In Press, (2016). Figure Legend Figure 1: The CardioExcyte 96 with incubation chamber, NSP-96 in recording position and hand lever for removal of the NSP-96. By using the incubation chamber, a constant temperature and humidity can be maintained without the need for a separate cell culture incubator. The use of the incubation chamber minimizes temperature changes when exchanging solutions, e.g. by media changes or addition of compounds. Figure 2: Effect of the specific hERG blocker, E4031, on the impedance and EFP signals recorded from iCell Cardiomyocytes2. Impedance signal of 12 wells in control conditions (left) and the same 12 wells after 30 min incubation in 300nM E4031 (right). E4031 (300 nM, 30 min incubation) caused arrhythmic effects in EFP mode shown in the inset. Figure 3: Effect of nifedipine on the impedance and EFP signals recorded from Cor.4U cardiomyocytes. Figure represents 10 s excerpt from example Cor.4U cell in impedance mode in control conditions (left, black trace) and in the presence of 300 nM nifedipine (left, red trace), as well as exemplary 10 s excerpt from a Cor.4U cell in EFP mode in control conditions (right, black trace) and in the presence of 300 nM nifedipine (right, red trace). Table 1: Changes of hiPSC-CMs (Cor.4U) beating pattern measured by impedance and extracellular field potential methods. Red (darkest is highest effect) indicates upregulation of parameter, blue (darkest blue is highest effect) indicates downregulation, white indicates no effect. Amp = amplitude; Rate = beat rate; PW50 = pulsewidth 50%; BRRI = beat rate regularity index; FPD = field potential duration.