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Background. With the advent of new realtime technologies such as impedance assays, extracellular field potential measurement and optical sensing for in vitro cardiac safety screening studies, researchers have now to frequently deal with analyzing voluminous amounts of complex time responses. In this context, main issues are to speed up the data analysis process and to extract accurate information for cardiotoxicity profiling. Objectives. A first objective is the development of an innovative computational method able to globally process a large set of in vitro cardiac signals (provided by 96, 384 and 1536-well microplates) instead of analyzing them once at a time. Such a statistical population approach has the advantage the account for the common characteristics between the individual responses. A second objective is to handle qualitative factors (type of cardiomyocytes, compounds and media, etc.) in the computational process. Methods. The proposed estimation method relies on the combination of a dynamic system identification method and a mixed-effect modeling technique. An output-error polynomial model structure is used for the system identification step and a stochastic approximation expectation maximization is implemented for the estimation of the hyperparameters. Input signals to be analyzed are the contractility amplitudes of cardiomyocytes submitted to compounds to be tested. Impedance signals and contractility amplitude were provided by a CardioExcyte96 system (Nanion Technologies). human iPSC-derived cardiomyocytes were provided by Cellartis Takara with 30,000 cells per well. Results. Our data-driven profiling method extracted four parameters that completely fit the contractility time variations and fully characterize the effect of compound concentration on the contractility amplitude. The proposed method not only estimates the values of the model parameters but also their uncertainty distribution. The latter allows to compute p-values associated with each effect.Conclusion. We show that the population-based estimation method developed in this study is suited to the fully characterize dynamic effects in cardiomyocyte contractility assays. Each parameter becomes a profiling characteristics of the concentration effect. It can be applied to estimate concentration and compounds effects with an optimal accuracy and could be extended directly to multielectrode array and optical sensing responses.

Human induced pluripotent stem cells (hiPSCs) are relevant for cardiac safety testing due to their validated predictivity as described in recent publications derived from the comprehensive in vitro proarrhythmia assay (CiPA) study. We combined automated patch clamp (APC), impedance and extracellular field potential (EFP) measurements to study cardiac ion channels in cell lines and hiPSC-CMs. Cell lines expressing different cardiac ion channels were recorded on two different APC instruments (8 or 384 wells simultaneously) at room and physiological temperature, at 4 different sites. Within the myocyte validation study of the CiPA initiative, hiPSC-CMs from different providers were used on a device combining impedance-based contractility and extracellular field potential (EFP) recordings. Within the ion channels working group of the CiPA initiative, ion channel data on 7 cardiac ion channel currents were measured. The effects of >20 CiPA reference compounds deemed low, intermediate and high risk by the FDA were investigated. In parallel to APC investigations, arrhythmogenicity of this and other compounds were investigated via contractility and field potential recordings in 2D monolayers of iPSC-CM. Our results show that high risk compounds such as dofetilide prolong field potential duration which resulted in the detection of arrhythmic events in both impedance and EFP recordings. Datasets from multiple sites and cell types will be shown and compared. Cross-site/cell comparisons of APC ion channel data and myocyte repolarization data was combined in order to investigate the mode of action of reference compounds. This promotes a good understanding of drug-induced arrhythmia and thus represents a valuable approach in drug development efforts.

The drug discovery process involving ion channels needs to rely on high-throughput screening (HTS) assays as well as fine-tuned characterization by electrophysiological measurements. Combining optogenetic tools with induced pluripotent stem-cells (iPSC)-derived cardiomyocytes can provide a reliable, cost-effective and highly time-resolved approach to induce electrogenic proteins activation. The aim of the OPTEL project, which combines the expertise of two leading European companies and one academic laboratory, was to develop integrated HTS-compatible platforms based on optogenetic tools (Channelrhodopsin, ChR2) for effective drug discovery in heterologous expression systems and iPS-derived cardiomyocytes. We generated HEK293 cell lines stably expressing ChR2 alone or with the cardiac sodium channel, NaV1.5 which proper functional expression was validated by fluorescence and manual patch-clamp. The best clones were used to implement light stimulation on an HTS automated patch-clamp platform (SynchroPatch 384PE, Nanion Technologies). We recorded light-induced currents and action potential-like responses in voltage and current-clamp, respectively, validating a new platform for high-throughput automated “opto-patch-clamp” assays. For optogenetic drug toxicity screening, commercially available iPSC-derived cardiomyocytes were infected with adeno-associated viruses to express ChR2 and analyzed by recording extracellular field potentials from 96 wells with a modified CardioExcyte96 system (Nanion Technologies). This was equipped with a custom designed 96 LED lid enabling pacing of cells and frequency-dependent drug screening over the physiological heart rate (1-3 Hz). Thereby we characterized adverse side effects of known ion channel blockers and pro-arrhythmogenic drugs on Na+, Ca2+ and K+ channels. These results validate new platforms that allow optogenetic control of electrical activity in HTS-compatible format with four different readouts: fluorescence, electrophysiology, impedance and extracellular field potentials. The use of these complementary readouts provides a new cost-effective and informative strategy in early stages of ion channel drug discovery, especially in the cardiac field.

Hepatic toxicity has accounted for 15 of the 47 drugs withdrawn from the market in the last two decades1. More specifically, Drug Induced Liver Injury (DILI) is the major cause of acute liver failure. DILI is classified as intrinsic (or dose-dependent) or as idiosyncratic2. A prominent example of idiosyncrasy is acetaminophen2,3, with a variable time of onset and not directly dependent on dose. We present a non-invasive DILI assay approach based on impedance measurements in monocyte-derived hepatocyte-like (MH) cells from MetaHeps®. MH cells were used on a 96-well screening system that monitors changes in impedance (or cell monolayer resistance). Cells are monitored under physiological conditions for temperature, humidity and CO2. We investigated hepatotoxic effects of paracetamol on MH cells when exposed for 24 and 48 hours. In agreement with other standard toxicity assays, such as the lactate dehydrogenase release assay, low doses of paracetamol caused transient toxicity and 'adaptation' was observed. At higher doses, hepatotoxic effects of paracetamol could be reversed upon washout after 24 hours, but continued exposure caused increased hepatotoxicity. In addition to hepatotoxicity, another validation of the principle is shown for chronic proliferation of cancer cells. In this study, murine mammary carcinoma cells (H8N8 and H8N8 T3.2) were used and changes in impedance, were used as a measure of toxicity. Intrinsic (dose-dependent) effects of the standard clinical regimen cyclophosphamide, doxorubicin and 5-fluouracil could be identified consistent with other methods of live cell analysis systems. Therefore, the utilized 96-well impedance system in combination with murine mammary carcinoma cells provides a novel tool for investigating therapy resistance of cancer cells in vitro. References: 1. Stevens, J.L, 2008. Drug Disc.Tod. 2.Roth, R.A, et al.2010, J Pharmacol Exp Ther. 3.McGill, M.R. et al 2013. Pharm Res. 30(9).

Measurement of contractility using impedance is a novel method for gaining information about a drug candidate's potential to disturb cardiac cell contraction. The impedance signal is recorded from a monolayer of cardiac cells, most commonly derived from human-induced pluripotent stem cells (hiPSCs), which are becoming an attractive model for safety testing, especially in the light of the Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative introduced in 2013. The goal of this initiative is, in part, to standardize assays, targets, and cell types but also to evaluate the potential of new technologies, in this context, such as impedance. The CardioExcyte 96 is a hybrid system that combines the impedance readout (a measure of cell contractility) with extracellular field potential (EFP) recordings. This chapter focuses on cell handling of hiPSC cardiomyocytes (CMs) and the short- and long-term investigation into pharmacological effects of a wide range of pharmacological agents, including flecainide, nifedipine, isoproterenol, and E4031 using the CardioExcyte 96.

Optical in-vitro platforms will be of particular relevance in the early stages drug discovery processes. We show recordings on impedance and extracellular field potential (EFP)-based devices with induced pluripotent stem cell (iPSC)-derived cardiomyocytes as well as automated patch clamp data. Optogenetic stimulation and the recording of electrophysiological and contractile parameters of ChR2 (channelrhodopsin 2) transfected iPS Cor.4U cardiomyocytes were performed in a new assay approach, which allows a parallel investigation of impedance and EFP signals. This allowed a mechanistic understanding of cardiomyocyte cell physiology, which has been investigated over a physiological frequency range (60-180 ppm). Frequency dependent effects on cell physiology with reference compounds such as Ranolazine and Mexiletine will be presented. Furthermore, automated patch clamp investigations in the voltage-and current-clamp mode on blue-light activated ChR2 (channelrhodopsin 2) transfected cells will be presented and discussed in association with impedance/EFP results.

We present in this paper an extensive comparison of compression methods adapted to impedance and field potential signals of cardiomyocytes. Different combinations of the traditional scheme of lossy compression have been tested and other original methods such as compressed sensing were implemented as well. All algorithms are assessed on several criteria such as compression ratio, distortion of the data, etc. We show that the selected method presents the ability and reliability to compress sensitive data with a compression ratio greater than 5: 1 while preserving the relevant information content of the recorded data.

s................................ 7 Collaboration Partner.............17 Your Nanion Team..................20 Wednesday October 26 13:00 Welcome reception 13:30 – 13:50 ▶ Welcome and introduction Niels Fertig, Nanion Technologies Label free technologies in Safety Pharmacology 13:50 – 14:20 ▶ Getting ready for CIPA – First steps with the CardioExcyte 96 Fabian Haeusermann, Hoffmann-La Roche 14:20 – 14:50 ▶ Towards exploration of 3D iPSC-CM models with the CardioExcyte 96 Matt Burnham, AstraZeneca, United Kingdom 14:50 – 15:05 ▶ News from the stem cell bench: present and future applications of cell-based assays Matthias Gossmann, Axiogenesis 15:05 – 15:20 ▶ Electrophysiology in iPSC disease Modeling: Microelectrode Array (MEA), optogenetics and impedance approaches Sonja Stölzle-Feix & Elena Dragicevic, Nanion Technologies 15:20 – 16:20 ▶ Refreshment Break & Poster Presentations Ion channel Screening goes HTS 16:20 – 16:50 ▶ Characterization of subtype selective KCNQ activators using the Synchropatch 384PE Chris Mathes, Icagen, USA 16:50 – 17:20 ▶ Automated Electrophysiology Assay Development for MEM16A, a Calcium-Activated Chloride Channel using the Whole-Cell Configuration on the SyncroPatch 384PE Kaylee Choi, Amgen Inc., USA 17:20 – 17:50 ▶ SyncroPatch 384PE: An Easy Approach to Difficult Target Andrea Brüggemann, Nanion Technologies 17:50 – 19:00 ▶ Live demonstrations 19:00 ▶ Dinner A g e n d a 3 of 20 Session 1 (chair: Niels Fertig) Session 2 (chair: Andrea Brüggemann)

The CardioExcyte 96 system provides a non-invasive, label-free, high temporal resolution approach for safety screening on iPSCMs. It is a hybrid screening instrument that combines impedance with MEA-like extracellular field potential (EFP) recordings. Furthermore, it can be either used in an incubator or, by utilizing the dedicated incubation system, directly on a lab bench. 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.

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.

The need for predictive, in vitro cardiac safety screening drives further development of automated, high-throughput-compatible drug evaluation based on cardiac cell preparations. Recently, pluripotent stem cells are evaluated as a new, more predictive model for cardiovascular risk assessment pertaining to in vitro assays. We present a new screening platform, the CardioExcyte 96, a hybrid instrument that combines impedance (cell contractility) with extracellular field potential (EFP) recordings. The electrophysiological measurements are noninvasive, label free and have a temporal resolution of 1 ms. This hybrid technology addresses the lack of easy-to-use high-throughput screening for in vitro assays and permits the reliable investigation of short- and long-term pharmacological effects. Several models of cardiomyocyte preparations were successfully validated for use with the CardioExcyte96. Furthermore, the pharmacological effects of a number of reference compounds were evaluated. Compound effects on cell monolayers of human-induced pluripotent stem cell-derived cardiomyocytes are evaluated using a quasi-simultaneous hybrid recording mode that combines impedance and EFP readouts. A specialized software package for rapid data handling and real-time analysis was developed, which allows for comprehensive investigation of the cellular beat signal. Combining impedance readouts of cell contractility and EFP (microelectrode array-like) recordings, the system opens up new possibilities in the field of in vitro cardiac safety assessment.

A new trend in the industry utilizes induced pluripotent stem cells (iPSCs) for functional assays to identify cardiac pathologies and their genetic underpinnings (such as Long QT Syndrome (LQTS)), in addition to cardiotoxicity profiling of pharmaceutical compounds. The here utilized CardioExcyte® 96 system is a hybrid instrument that combines impedance readout (a correlate of cell contractility) with electric field potential recordings of the compound signal that is generated by cellular action potentials. These electrophysiological measurements are label-free and allow investigations in High Throughput format. In addition, the instrument is capable of electrical stimulation of the cellular monolayer, while the rapid acquisition rate of 1ms allows a more accurate description of signal features and nuances stemming from pharmacological effects. A dedicated software package for rapid data handling and real-time analysis is based on novel algorithms that allow a comprehensive investigation of the cellular beat signal. A broad range of cell lines was successfully validated, such as stem cell-derived cardiomyocytes from Axiogenesis (human: Cor4U®, murine: CorAt®), GE Healthcare (Cytiva), Cellectis (human, 3D-clusters: hES-CMC™) and CDI (iCell®). Furthermore, the pharmacological effects of a number of reference compounds on these cell types were evaluated and their phenotype correctly identified. For example, the effects of compounds like Blebbistatin, which blocks contraction without disrupting the electric field potential can be easily identified since it is an inhibitor of the myosin II which is mainly responsible for the contraction of cardiomyocytes, while it has no effect to the ion channels. Other compound classes like hERG inhibitors were validated as well and example data will be shown. They induce typical arrhythmia since hERG ion channels are affected.