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Introduction For reliable identification of cardiac safety risk, compounds should be screened for activity on cardiac ion channels in addition to hERG, including NaV1.5 and CaV1.2. We identified different parameters that might affect IC50s of compounds on NaV1.5 peak and late currents recorded using automated patch clamp (APC) and suggest outlines for best practices. Methods APC instruments SyncroPatch 384 and Patchliner were used to record NaV1.5 peak and late current. Up to 24 CiPA compounds were used to investigate effects of voltage protocol, holding potential (−80 mV or − 95 mV) and temperature (23 ± 1 °C or 36 ± 1 °C) on IC50 values on hNaV1.5 overexpressed in HEK or CHO cells either as frozen cells or running cultures. Results The IC50s of 18 compounds on the NaV1.5 peak current recorded on the SyncroPatch 384 using the CiPA step-ramp protocol correlated well with the literature. The use of frozen or cultured cells did not affect IC50s but voltage protocol and holding potential did cause differences in IC50 values. Temperature can affect Vhalf of inactivation and also compound potency. A compound incubation time of 5–6 min was sufficient for most compounds, however slow acting compounds such as terfenadine required longer to reach maximum effect. Discussion We conclude that holding potential, voltage protocol and temperature can affect IC50 values and recommend the use of the CiPA step-ramp protocol at physiological temperature to record NaV1.5 peak and late currents for cardiac safety. Further recommendations include: a minimum compound incubation time of 5 min, a replicate number of 4 and the use of positive and negative controls for reliable IC50s.

Induction Despite increasing acceptance of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in safety pharmacology, controversy remains about the physiological relevance of existing in vitro models for their mechanical testing. We hypothesize that existing signs of immaturity of the cell models result from an improper mechanical environment. With the presented study, we aimed at validating the newly developed FLEXcyte 96 technology with respect to physiological responses of hiPSC-CMs to pharmacological compounds with known inotropic and/or cardiotoxic effects. Methods hiPSC-CMs were cultured in a 96-well format on hyperelastic silicone membranes imitating their native mechanical environment. Cardiomyocyte contractility was measured contact-free by application of capacitive displacement sensing of the cell-membrane biohybrids. Acute effects of positive inotropic compounds with distinct mechanisms of action were examined. Additionally, cardiotoxic effects of tyrosine kinase inhibitors and anthracyclines were repetitively examined during repeated exposure to drug concentrations for up to 5 days. Results hiPSC-CMs grown on biomimetic membranes displayed increased contractility responses to isoproterenol, S-Bay K8644 and omecamtiv mecarbil without the need for additional stimulation. Tyrosine kinase inhibitor erlotinib, vandetanib, nilotinib, gefitinib, A-674563 as well as anthracycline idarubicin showed the expected cardiotoxic effects, including negative inotropy and induction of proarrhythmic events. Discussion We conclude that the FLEXcyte 96 system is a reliable high throughput tool for in vitro cardiac contractility research, providing the user with data obtained under physiological conditions which resemble the native environment of human heart tissue. We showed that the results obtained for both acute and sub-chronic compound administration are consistent with the respective physiological responses in humans.

Drug-drug interactions pose a difficult drug safety problem, given the increasing number of individuals taking multiple medications and the relative complexity of assessing the potential for interactions. For example, sofosbuvir-based drug treatments have significantly advanced care for hepatitis C virus-infected patients, yet recent reports suggest interactions with amiodarone may cause severe symptomatic bradycardia and thus limit an otherwise extremely effective treatment. Here, we evaluated the ability of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) to recapitulate the interaction between sofosbuvir and amiodarone in vitro, and more generally assessed the feasibility of hiPSC-CMs as a model system for drug-drug interactions. Sofosbuvir alone had negligible effects on cardiomyocyte electrophysiology, whereas the sofosbuvir-amiodarone combination produced dose-dependent effects beyond that of amiodarone alone. By comparison, GS-331007, the primary circulating metabolite of sofosbuvir, had no effect alone or in combination with amiodarone. Further mechanistic studies revealed that the sofosbuvir-amiodarone combination disrupted intracellular calcium (Ca2+) handling and cellular electrophysiology at pharmacologically relevant concentrations, and mechanical activity at supra-pharmacological (30x Cmax) concentrations. These effects were independent of the common mechanisms of direct ion channel block and P-glycoprotein activity. These results support hiPSC-CMs as a comprehensive, yet scalable model system for the identification and evaluation of cardioactive pharmacodynamic drug-drug interactions.

Introduction Screening compounds for activity on the hERG channel using patch clamp is a crucial part of safety testing. Automated patch clamp (APC) is becoming widely accepted as an alternative to manual patch clamp in order to increase throughput whilst maintaining data quality. In order to standardize APC experiments, we have investigated the effects on IC50 values under different conditions using several devices across multiple sites. Methods APC instruments SyncroPatch 384i, SyncroPatch 384PE and Patchliner, were used to record hERG expressed in HEK or CHO cells. Up to 27 CiPA compounds were used to investigate effects of voltage protocol, incubation time, labware and time between compound preparation and experiment on IC50 values. Results All IC50 values of 21 compounds recorded on the SyncroPatch 384PE correlated well with IC50 values from the literature ( Kramer et al., 2013 ) regardless of voltage protocol or labware, when compounds were used immediately after preparation, but potency of astemizole decreased if prepared in Teflon or polypropylene (PP) compound plates 2–3 h prior to experiments. Slow acting compounds such as dofetilide, astemizole, and terfenadine required extended incubation times of at least 6 min to reach steady state and therefore, stable IC50 values. Discussion Assessing the influence of different experimental conditions on hERG assay reliability, we conclude that either the step-ramp protocol recommended by CiPA or a standard 2-s step-pulse protocol can be used to record hERG; a minimum incubation time of 5 min should be used and although glass, Teflon, PP or polystyrene (PS) compound plates can be used for experiments, caution should be taken if using Teflon, PS or PP vessels as some adsorption can occur if experiments are not performed immediately after preparation. Our recommendations are not limited to the APC devices described in this report, but could also be extended to other APC devices.

Drug discovery is challenged by poor predictability of proarrhythmic effects of present safety screening approaches. The Comprehensive in Vitro Proarrhythmia Assay (CiPA), an FDA-directed initiative, is specifically focused on improved preclinical assessment of torsade de pointes (TdP) risk, striving to optimize the drug discovery process.In recent years, human stem cell-derived cardiomyocytes have proven to recapitulate key features of human cardiac electrophysiology in vitro. Furthermore, it has become apparent that the intact ensemble of cardiac ion channels is necessary to determine proarrhythmic effects reliably. Hence, due to their relatively easy availability, stem cell-derived cardiomyocytes have become the preferred choice of cardiac cells.We present chip-based approaches allowing parallel patch clamp recordings without compromising data quality or technical sophistication. Culture and harvesting protocols have been optimized on cardiomyocytes for success rate but also the number of cells used.We show patch clamp data from two automated patch clamp platforms (medium throughput - Patchliner and high throughput - SyncroPatch 384/768PE). The Patchliner and the SyncroPatch 384/768PE both boast temperature control to enable recordings at physiological temperature - an important feature since drug efficacies may vary with temperature. Here, we present pharmacological data on voltage-dependent channels of the CiPA panel at different temperatures.The effect of drugs on action potentials is important for assessing the interaction of the cardiac ion channel ensemble. Both patch clamp platforms have amplifiers capable of current clamp. Compound effects on action potentials will also be presented.Patchliner and SyncroPatch 384/768PE are both planar patch-clamp platforms combining all the necessary features for successful drug discovery (well-suited for stem cell-derived cardiomyocytes, temperature control, and current clamp mode).

Introduction While extracellular field potential (EFP) recordings using multi-electrode arrays (MEAs) are a well-established technique for monitoring changes in cardiac and neuronal function, impedance is a relatively unexploited technology. The combination of EFP, impedance and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has important implications for safety pharmacology as functional information about contraction and field potentials can be gleaned from human cardiomyocytes in a beating monolayer. The main objectives of this study were to demonstrate, using a range of different compounds, that drug effects on contraction and electrophysiology can be detected using a beating monolayer of hiPSC-CMs on the CardioExcyte 96. Methods hiPSC-CMs were grown as a monolayer on NSP-96 plates for the CardioExcyte 96 (Nanion Technologies) and recordings were made in combined EFP and impedance mode at physiological temperature. The effect of the hERG blockers, E4031 and dofetilide, hERG trafficking inhibitor, pentamidine, β-adrenergic receptor agonist, isoproterenol, and calcium channel blocker, nifedipine, was tested on the EFP and impedance signals. Results Combined impedance and EFP measurements were made from hiPSC-CMs using the CardioExcyte 96 (Nanion Technologies). E4031 and dofetilide, known to cause arrhythmia and Torsades de Pointes (TdP) in humans, decreased beat rate in impedance and EFP modes. Early afterdepolarization (EAD)-like events, an in vitro marker of TdP, could also be detected using this system. Isoproterenol and nifedipine caused an increase in beat rate. A long-term study (over 30 h) of pentamidine, a hERG trafficking inhibitor, showed a concentration and time-dependent effect of pentamidine. Discussion In the light of the new Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative to improve guidelines and standardize assays and protocols, the use of EFP and impedance measurements from hiPSCs may become critical in determining the proarrhythmic risk of potential drug candidates. The combination of EFP offering information about cardiac electrophysiology, and impedance, providing information about contractility from the same area of a synchronously beating monolayer of human cardiomyocytes in a 96-well plate format has important implications for future cardiac safety testing.

Cardiovascular side effects are critical in drug development and have frequently led to late-stage project terminations or even drug withdrawal from the market. Physiologically relevant and predictive assays for cardiotoxicity are hence strongly demanded by the pharmaceutical industry. To identify a potential impact of test compounds on ventricular repolarization, typically a variety of ion channels in diverse heterologously expressing cells have to be investigated. Similar to primary cells, in vitro-generated stem cell-derived cardiomyocytes simultaneously express cardiac ion channels. Thus, they more accurately represent the native situation compared with cell lines overexpressing only a single type of ion channel. The aim of this study was to determine if stem cell-derived cardiomyocytes are suited for use in an automated patch clamp system. The authors show recordings of cardiac ion currents as well as action potential recordings in readily available stem cell-derived cardiomyocytes. Besides monitoring inhibitory effects of reference compounds on typical cardiac ion currents, the authors revealed for the first time drug-induced modulation of cardiac action potentials in an automated patch clamp system. The combination of an in vitro cardiac cell model with higher throughput patch clamp screening technology allows for a cost-effective cardiotoxicity prediction in a physiologically relevant cell system.

Drug induced arrhythmia is one of the most common causes of drug development failure. Human induced pluripotent stem cell-derived cardiomyocytes (iPSCMs) show great promise for cardiovascular research and predictive in-vitro cardiac safety screening.We present data recorded on the CardioExcyte 96, a hybrid screening instrument that combines impedance (cell contractility) with MEA-like extracellular field potential (EFP) recordings. A case study showing impaired excitation-contraction coupling will be presented. This indicates the importance of testing compounds in assays complementary to patch clamp electrophysiology to provide a more complete safety profile. Furthermore, in accordance with the Comprehensive In Vitro Proarrhythmia Assay (CiPA) guidelines, standard reference compounds were tested on iPS-derived cardiomyocytes. 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. Pharmacological effects of a number of reference compounds including those designated high risk (e.g. Dofetilide), intermediate risk (e.g. Cisapride) or low risk (e.g. Verapamil) were evaluated and summarized in this study.The CardioExcyte 96 system provides a non-invasive, label-free, high temporal resolution approach for safety screening. It can be either used in an incubator or, by utilizing the dedicated incubation system, directly on a lab bench. Furthermore, the system is capable of electrically pacing the cells, which allows for screening of cells which beat with individual frequencies and, in addition, investigations of frequency dependent compound inhibition.

Ion channels are essential in a wide range of cellular functions and their malfunction underlies many disease states making them important targets in drug discovery. The availability of standardized cell lines expressing ion channels of interest lead to the development of diverse automated patch clamp (APC) systems with high-throughput capabilities. These systems are now available for drug screening, but there are limitations in the application range. However, further development of existing devices and introduction of new systems widen the range of possible experiments and increase throughput. The addition of well controlled and fast solution exchange, temperature control and the availability of the current clamp mode are required to analyze standard cell lines and excitable cells such as stem cell-derived cardiomyocytes in a more physiologically relevant environment. Here we describe two systems with different areas of applications that meet the needs of drug discovery researchers and basic researchers alike. The here utilized medium throughput APC device is a planar patch clamp system capable of recording up to eight cells simultaneously. Features such as temperature control and recordings in the current clamp mode are described here. Standard cell lines and excitable cells such as stem cell-derived cardiomyocytes have been used in the voltage clamp and current clamp modes with the view to finding new drug candidates and safety testing methods in a more physiologically relevant environment. The high-throughput system used here is a planar patch clamp screening platform capable of recording from 96 cells in parallel and offers a throughput of 5000 data points per day. Full dose response curves can be acquired from individual cells reducing the cost per data point. The data provided reveals the suitability and relevance of both APC platforms for drug discovery, ion channel research, and safety testing.

Efficient high resolution techniques are required for screening efforts and research targeting ion channels. The conventional patch clamp technique, a high resolution but low efficiency technique, has been established for 25 years. Recent advances have opened up new possibilities for automated patch clamping. This new technology meets the need of drug developers for higher throughput and facilitates new experimental approaches in ion channel research. Specifically, Nanion's electrophysiology workstations, the Port-a-Patch and the Patchliner, have been successfully introduced as high-quality automated patch clamp platforms for industry as well as academic users. Both platforms give high quality patch clamp recordings, capable of true giga-seals and stable recordings, accessible to the user without the need for years of practical training. They also offer sophisticated experimental possibilities, such as accurate and fast ligand application, temperature control and internal solution exchange. This article describes the chip-based patch clamp technology and its usefulness in ion channel drug screening and academic research.

We have developed planar glass chip devices for patch clamp recording. Glass has several key advantages as a substrate for planar patch clamp devices. It is a good dielectric, is well-known to interact strongly with cell membranes and is also a relatively in-expensive material. In addition, it is optically neutral. However, microstructuring processes for glass are less well established than those for silicon-based substrates. We have used ion-track etching techniques to produce micron-sized apertures into borosilicate and quartz-glass coverslips. These apertures, which can be easily produced in arrays, have been used for high resolution recording of single ion channels as well as for whole-cell current recordings from mammalian cell lines. An additional attractive application that is greatly facilitated by the combination of planar geometry with the optical neutrality of the substrate is single-molecule fluorescence recording with simultaneous single-channel measurements.

Nicotinic acetylcholine receptors (nAChR) are ligand gated cation channels that are opened by agonists like Acetylcholine and Nicotine. They are widely distributed in neurons all over central nervous system, where they are mainly found at postsynaptic locations of cholinergic nerve endings.The most abundant homomeric nicotinic acetylcholine receptors in the mammalian brain are the pentameric alpha7 nAChRs which consist of five alpha7 subunits. Each subunit provides an orthosteric low affinity binding site for its endogenous ligand, acetylcholine. Several lines of evidence suggest a link between the alpha7 neuronal nicotinic acetylcholine receptor and brain disorders including schizophrenia, Alzheimer's disease, and traumatic brain injury. Alpha 7 receptors are also a target for non-CNS indications like diabetes and asthma.View Large Image | View Hi-Res Image | Download PowerPoint SlideHere we show the results of the screen of allosteric modulators on alpha 7 receptors. High parallelization without a compromise in data quality combined with fast and accurate solution exchange allows characterization of many compounds. A second analysis at physiological temperatures showed a pronounced effect on the allosteric modulation.

Nicotinic acetylcholine receptors (nAChRs) belong to the superfamily of ligand-gated ion channels. The most abundant homomeric nAChR in the mammalian brain are the pentameric α7 nAChRs, which consist of five α7 subunits. Positive allosteric modulators (PAMs) of nAChRs have been implicated to be putative therapeutics in the pharmacological treatment of Alzheimer's disease, schizophrenia and Parkinson's disease.Recently, Sitzia et al. (2011) showed a striking temperature dependence for positive allosteric modulation of PNU-120596 on nAChRs. The aim of our study was to investigate the temperature dependence of other PAMs on nACHRs with automated patch clamp techniques. For this study, we have used GH4C1cells expressing human nAChR α7 subunits. Electrophysiological recordings were performed on the Patchliner, a robotic patch clamp device allowing temperature controlled recordings from up to 8 cells simultaneously.View Large Image | View Hi-Res Image | Download PowerPoint SlideFirst experiments on GABA A receptors allude to a similar but weaker effect on the temperature dependence of PAMs.These data suggest that experiments on the efficacy of PAMs conducted at room temperature have to be interpreted with caution.Sitzia et al (2011) FrontPharmacol;2:81.

Introduction Chip-based automated patch clamp systems are widely used in drug development and safety pharmacology, allowing for high quality, high throughput screening at standardized experimental conditions. The merits of automation generally come at the cost of large amounts of cells needed, since cells are not targeted individually, but randomly positioned onto the chip aperture from cells in suspension. While cell usage is of little concern when using standard cell lines such as CHO or HEK cells, it becomes a crucial constraint with cells of limited availability, such as primary or otherwise rare and expensive cells, like induced pluripotent stem (IPS) cell-derived cardiomyocytes or neurons. Methods We established application protocols for CHO cells, IPS cell-derived neurons (iCell® Neurons, Cellular Dynamics International), cardiomyocytes (Cor.4U®, Axiogenesis) and pancreatic islet cells, minimizing cell usage for automated patch clamp recordings on Nanion's Patchliner. Use of 5 μl cell suspension per well for densities between 55,000 cells/ml and 400,000 cells/ml depending on cell type resulted in good cell capture. Results We present a new cell application procedure optimized for the Patchliner achieving > 80% success rates for using as little as 300 to 2000 cells per well depending on cell type. We demonstrate that this protocol works for standard cell lines, as well as for stem cell-derived neurons and cardiomyocytes, and for primary pancreatic islet cells. We present recordings for these cell types, demonstrating that high data quality is not compromised by altered cell application. Discussion Our new cell application procedure achieves high success rates with unprecedentedly low cell numbers. Compared to other standard automated patch clamp systems we reduced the average amount of cells needed by more than 150 times. Reduced cell usage crucially improves cost efficiency for expensive cells and opens up automated patch clamp for primary cells of limited availability.