Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) have found utility for conducting in vitro drug screening and disease modelling to gain crucial insights into pharmacology or disease phenotype. However, diseases such as atrial fibrillation, affecting >33 M people worldwide, demonstrate the need for cardiac subtype-specific cells. Here, we sought to investigate the base characteristics and pharmacological differences between commercially available chamber-specific atrial or ventricular hiPSC-CMs seeded onto ultra-thin, flexible PDMS membranes to simultaneously measure contractility in a 96 multi-well format. We investigated the effects of GPCR agonists (acetylcholine and carbachol), a Ca²⁺ channel agonist (S-Bay K8644), an HCN channel antagonist (ivabradine) and K⁺ channel antagonists (4-AP and vernakalant). We observed differential effects between atrial and ventricular hiPSC-CMs on contractile properties including beat rate, beat duration, contractile force and evidence of arrhythmias at a range of concentrations.

As an excerpt of the compound analysis, S-Bay K8644 treatment showed an induced concentration-dependent transient increase in beat duration of atrial hiPSC-CMs, whereas ventricular cells showed a physiological increase in beat rate over time. Carbachol treatment produced marked effects on atrial cells, such as increased beat duration alongside a decrease in beat rate over time, but only minimal effects on ventricular cardiomyocytes. In the context of this chamber-specific pharmacology, we not only add to contractile characterization of hiPSC-CMs but propose a multi-well platform for medium-throughput early compound screening.

Overall, these insights illustrate the key pharmacological differences between chamber-specific cardiomyocytes and their application on a multi-well contractility platform to gain insights for in vitro cardiac liability studies and disease modelling.

In this application note, InnoVitro GmbH and Axol Biosciences Limited show that distinct pharmacological effects can be observed between axoCellsTM atrial and ventricular hiPSC-derived cardiomyocytes when using the FLEXcyte 96 platform to assess contractility, providing a powerful tool to perform in vitro drug screening and disease modelling on subtype-specific hiPSC-derived cardiomyocytes.

In order to reduce cardiovascular safety liabilities of new therapeutic agents, there is an urgent need to integrate human-relevant platforms/approaches into drug development1. Optimizing baseline function of human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs) is essential for their effective application in models of cardiac toxicity and disease2. Here, hiPSC-CMs were cultured on flexible substrates using the FLEXcyte 96 system. The promaturation environment enables observation of inotropic and chronotropic compound effects, which are typically hard to detect with 2D monolayers on overly stiff substrates3. For example, the beta-adrenergic agonist isoprenaline, or isoproterenol, is well known for its positive inotropic effects on the human heart, although common hiPSC-CM in vitro assays fail to display this physiological response by this compound.

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold promise to serve as a human-relevant model in preclinical phases of drug discovery and safety pharmacology, their maturity is still controversial in the scientific community and under constant development. We present a hybrid contractility and impedance/extracellular field potential (EFP) technology, adding significant pro-maturation features to an industry-standard 96-well platform. The impedance/EFP system monitors cellular functionality in real-time. Besides the beat rate of contractile cells, the electrical impedance spectroscopy readouts detect compound-induced morphological changes like cell density and integrity of the cellular monolayer. In the other component of the hybrid cell analysis system, the cells are cultured on bio-compliant membranes that mimic the mechanical environment of real heart tissue. This physiological environment supports the maturation of hiPSC-CMs in vitro, leading to more adult-like contractile responses including positive inotropic effects after treatment with isoproterenol, S-Bay K8644, or omecamtiv mecarbil. Parameters such as the amplitude of contraction force (mN/mm2) and beat duration also reveal downstream effects of compounds with influence on electrophysiological properties and calcium handling. The hybrid system provides the ideal tool for holistic cell analysis, allowing preclinical cardiac risk assessment beyond the current perspectives of human-relevant cell-based assays.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are attractive due to their unlimited availability and human origin, making them a promising tool for cardiac research and safety pharmacology. However, they can show an immature phenotype such as lower inward rectifier potassium current (IK1), atypical expression pattern of ion channels, divergent response to pharmacological agents and contractile behaviour compared to adult CMs. Thus, their detailed characterization and optimized recording environments are essential.

We aimed to characterize and modulate electrophysiological and contractile properties of hiPSC-CMs using automated dynamic clamp and contraction measurements on flexible substrate.

Here, we recorded iCell Cardiomyocytes2 in voltage and current clamp using a combined automated patch clamp (APC) and dynamic clamp device (Patchliner Dynamite8), and contractility recordings were made using the FLEXcyte 96. During the APC recordings simulated IK1 and seal compensation were applied to up to 8 hiPSC-CMs simultaneously, while the contractility recordings were conducted in 96-well plates. We have tested various compounds targeting cardiac ion channels and recorded their effects on action potential duration (APD), sodium, calcium and potassium currents, as well as their effect on the contraction capabilities of these cells. Additionally, different levels of static and cyclic pressure were applied to the cell monolayers with the aim to induce membrane deflection for reproducibility test of Frank-Starling mechanism and to imitate the physiological stretching experienced by CMs in the beating human heart during systolic and diastolic phases, respectively.

Seal compensation and virtual IK1 in hiPSC-CMs resulted in more stable and longer APs with low APD variability. Consequently, the dynamic clamp approach enabled reliable calcium, sodium and potassium channel pharmacology on action potentials of these cells. Culturing conditions that support contractility, i.e. flexible membrane substrates, demonstrate Ca²⁺ channel pharmacology equivalent to that expected from adult CMs while applied mechanical stimulation resulted in functional changes of hiPSC-CMs physiology.

Abstract:
Common systems for the quantification of cellular contraction rely on animal-based models, complex experimental setups or indirect approaches. Integration into standard lab procedures remains a challenge for current in vitro systems. The FLEXcyte 96 system has the potential to scale-up mechanical testing towards medium-throughput analyses. We show here that, using stem cell-derived cardiomyocytes, this system enables predictive recordings of contractile behaviour in the presence of well-known reference compounds.

A short introductory video of the feature "contractile force"

A short introductory video of FLEXcyte.

Cardiac diseases remain one of the major causes of mortality and morbidity in our society with enormous costs for the health system. Arrhythmias and cardiomyopathy diseases are difficult to prevent/cure because the molecular mechanisms behind their onset are in most cases not fully clarified. Causes and effect are often confused, even when directly studying patients’ cardiomyocytes, because of the maladaptive remodeling imposed by electro-mechanical alterations. To overcome this limitation, studying the arrhythmogenic risk associated with genetic cardiac diseases using patient-derived iPS-CMs, provides a good model.Caveolinopathies are a group of muscular diseases that arise from mutation in the caveolin-3 gene (CAV3). Several CAV3 variants have been found in patients with both skeletal and cardiac pathologies. While electrophysiological alterations behind caveolinopathies have been partly elucidated using different models, the impact of such mutations on cardiomyocyte contraction and thus on the risk of developing cardiomyopathy, although quite probable, has never been studied before. Caveolin-3 along with cholesterol, forms membrane caveolae and plays a key role in the maintenance of plasma membrane integrity and interacts with several signaling proteins and ion channels.Here, CardioExcyte 96 and FLEXcyte 96 compared relative amplitude and kinetics of contraction and relaxation in patient/control hiPS-lines in order to shed light on the relations between electrical and mechanical dysfunctions. This analysis offered various advantages, such as the possibility of electrical stimulation, recordings in an environment with an elastic surface area resembling that of the native cardiac tissue, as well as high throughput.

In order to reduce cardiovascular safety liabilities of new therapeutic agents, there is an urgent need to integrate human-relevant platforms/approaches into drug development. Optimizing baseline function of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is essential for their effective application in models of cardiac toxicity and disease. Here, hiPSC-CMs were cultured on flexible substrates using the FLEXcyte 96 system. The pro-maturation environment enables observation of inotropic and chronotropic compound effects, which are typically hard to detect with 2D monolayers on overly stiff substrates. For example, the beta-adrenergic agonist isoprenaline, or isoproterenol, is well known for its positive inotropic effects on the human heart, although common hiPSC-CM in vitro assays fail to display this physiological response by this compound.Sala et al. (2018) developed a method that allows for a comparison of cardiac contraction measurements derived from different measurement approaches. The method, called MUSCLEMOTION, builds on previously existing algorithms, is fully automated and can be used on videos, image stacks, or image sequences loaded in the open-source image-processing program ImageJ.It is an open-source, dynamic platform that can be expanded, improved, and integrated for customized applications. Dynamic changes in pixel intensity between image frames are determined and the output is expressed as a relative measure of movement during muscle contraction and relaxation. Here, we compare the previously depicted data sets from Sala et al. 2018 and compare it to FLEXcyte 96 data as recorded with Cardiosight-S® cardiomyocytes (Nexel).

High throughput Screening (HTS) scalable techniques with highly predictive cell models are needed to improve the expensive and time-consuming drug development process. Potentially dangerous consequences of side effects on the human heart make safety testing of heart related issues the main focus of pre-clinical drug development studies. However, one of the most commonly used gold standard technique for cardiac contractility measurements, the ex vivo Langendorff set-up, does not efficiently support modern drug development processes as it uses non-predictive animal models on a very low throughput level.Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combine a number of features fostering the drug development process such as high predictivity, large scale applicability with high throughput potential, low ethical concerns and cost effectiveness. Yet, when cultured on overly stiff substrates like glass or plastic, the cells are placed under unnecessary stress due to the missing auxotonic physiological environment provided by a flexible substrate.These unphysiological conditions lead to drastic transcriptional and metabolic deregulation in cardiomyocytes which affect the predictive value of this established cell model. To bridge the gap of predictive contractility measurements and HTS analysis for drug development studies, innoVitro co-developed the FLEXcyte 96 with Nanion Technologies as an add-on for the CardioExcyte 96 platform. With less than 10 μm in thickness and sophisticated surface modification, the polydimethylsiloxane (PDMS) membranes of the FLEXcyte 96 disposable plates offer physiological elasticity of native human heart tissue. As a result hiPSC-derived CM behave in an in vivo manner and finally reach their full potential as a CiPA confirmed model for drug development processes.Beta-adrenergic agonist isoproterenol and L-type calcium channel agonist S-Bay K8644 are both well known for their positive inotropic effects on the human heart, although common iPSC-CM in vitro assays fail to display this physiological response by showing negative inotropic effects instead.Here, we show that the auxotonic environment of the FLEXcyte 96 enables mature physiological responses of hiPSC-CMs on positive inotropic substances such as L-type calcium channel agonist S-Bay K8644, beta-adrenergic agonist isoproterenol and cardiac myosin activator omecamtiv mecarbil.

Background: Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) offer a promising source for heart disease modeling and drug screening. Recent developments in organoid technology have made it possible to study how hiPSC-derived CMs interact together, and this culture system mimics the tissue environment and behavior of the cardiac cells in our body. However, the similarities and differences between conventional 2-dimensional (2D) culture and 3-dimensional (3D) organoid culture systems for CM differentiation have been incompletely elucidated. Methods: To study how the individual microenvironment formed by each culture system affects the properties of CMs differentiated from hiPSCs, we conducted a comparative study between 2D monolayer and direct 3D cardiac organoid differentiation from hiPSCs throughout the sequential differentiation stages. Results: The 3D differentiation system strongly exhibited higher mesoderm commitment and cardiac induction than 2D monolayer differentiation from hiPSCs. In the late stage of differentiation, the 3D cardiac organoids showed a higher frequency of a mature myofibrillar isoform switching in sarcomere structure of differentiated CMs than was observed in monolayer culture, although over 94% of cardiac troponin T-positive cells resulted at the end point of differentiation in both systems. Furthermore, the accelerated structural maturation in 3D cardiac organoids resulted in increased expression of cardiac-specific ion channel genes and Ca²⁺ transient properties, with a high signal amplitude and rapid contractility. Conclusion: The present study provides details surrounding the 2D and 3D culture methods for CM differentiation from hiPSCs and focuses on 3D cell culture as an improved strategy for approaching and applying cardiac maturation.

In cancer research, the intense development of targeted therapeutics such as tyrosine kinase inhibitors (TKIs) has brought tremendous improvement to the survival rate of cancer patients over the last two decades. The goal to reduce diverse toxic side effects of cancer treatment with targeted therapy has been widely achieved in comparison to traditional anti-cancer treatments like anthracyclines. Nevertheless, both therapeutics, TKIs and anthracyclines, still lead to adverse cardiotoxic side effects such as left ventricular dysfunction and heart failure.The severity of these side effects depends on dosage and time span of treatment which brings chronic assessment of cardiotoxicity into focus.However, acute testing (min to hours) of cell models with low predictive value remains the primary application so far, due to the inability of common cell-based assays to analyze cellular behavior reliably over prolonged periods of time.

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.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) in monolayers interact mechanically via cell–cell and cell–substrate adhesion. Spatiotemporal features of contraction were analysed in hiPSC-CM monolayers attached to glass or plastic (Young's modulus (E) >1 GPa), detached (substrate-free) and attached to a flexible collagen hydrogel (E = 22 kPa). The effects of isoprenaline on contraction were compared between rigid and flexible substrates. To clarify the underlying mechanisms, further gene expression and computational studies were performed. HiPSC-CM monolayers exhibited multiphasic contractile profiles on rigid surfaces in contrast to hydrogels, substrate-free cultures or single cells where only simple twitch-like time-courses were observed. Isoprenaline did not change the contraction profile on either surface, but its lusitropic and chronotropic effects were greater in hydrogel compared with glass. There was no significant difference between stiff and flexible substrates in regard to expression of the stress-activated genes NPPA and NPPB. A computational model of cell clusters demonstrated similar complex contractile interactions on stiff substrates as a consequence of cell-to-cell functional heterogeneity. Rigid biomaterial surfaces give rise to unphysiological, multiphasic contractions in hiPSC-CM monolayers. Flexible substrates are necessary for normal twitch-like contractility kinetics and interpretation of inotropic interventions.

Background/ Aims:Common systems for the quantification of cellular contraction rely on animal-based models, complex experimental setups or indirect approaches. The herein presented CellDrum technology for testing mechanical tension of cellular monolayers and thin tissue constructs has the potential to scale-up mechanical testing towards medium-throughput analyses. Using hiPS-Cardiac Myocytes (hiPS-CMs) it represents a new perspective of drug testing and brings us closer to personalized drug medication.Methods:In the present study, monolayers of self-beating hiPS-CMs were grown on ultra-thin circular silicone membranes and deflect under the weight of the culture medium. Rhythmic contractions of the hiPS-CMs induced variations of the membrane deflection. The recorded contraction-relaxation-cycles were analyzed with respect to their amplitudes, durations, time integrals and frequencies. Besides unstimulated force and tensile stress, we investigated the effects of agonists and antagonists acting on Ca²⁺ channels (S-Bay K8644/verapamil) and Na⁺ channels (veratridine/ lidocaine).Results:The measured data and simulations for pharmacologically unstimulated contraction resembled findings in native human heart tissue, while the pharmacological dose-response curves were highly accurate and consistent with reference data.Conclusion:We conclude that the combination of the CellDrum with hiPS-CMs offers a fast, facile and precise system for pharmacological, toxicological studies and offers new preclinical basic research potential.

Translatability of data obtained from hiPSC-CMs to human physiology is the subject of current scientific discussion; Contractility data derived from hiPSC-CMs in an environment that reflects the mechanical properties of real human cardiac tissue in a higher throughput format (FLEXcyte 96) is physiologically relevant; Example data on commercially available cell types with the FLEXcyte 96 system show a high degree of consistency with clinical data

Drug development is a costly and time-consuming process, with high drug failure rates both in early and late stages of the development process. Pre-clinical cardiac safety, toxicity and efficacy testing, usually performed using animal models with low predictive value or primary human cells, are one of the main reasons for high drug attrition rates.

To improve the drug development process, a suitable technology is required to acquire high quality data from physiologically relevant models on high throughput level. Standard cultivation methods for stem cell-derived cardiomyocytes are still based on stiff glass or plastic surfaces, creating an unphysiological environment to what cells would experience naturally and hinder them to further mature in vitro. In contrast, the FLEXcyte 96 plates mimic flexible mechanical conditions of real biological tissue and thereby enhancing the development of a mature cardiomyocyte phenotype which cannot be elicited with other assays commonly used. In combination with the FLEXcyte 96 platform, it is possible to analyze mature cardiac contractility on a 96 well high throughput level, both after acute and chronic compound treatment, ranging from 5 minutes to 5 days.

Hence, the FLEXcyte 96 system enables high throughput at lower costs and delivers highly predictive functional information on drug candidates early in the drug development process.

In this webinar we highlight impedance-based platforms (CardioExcyte 96 and FLEXcyte 96) and a high-throughput automated patch clamp (APC) instrument, the SyncroPatch 384.

The broad range and versatility of cell-based assays easily performed with these Automated Patch Clamp and cell monitoring systems, make them an excellent choice for integration into a core cell screening center/therapy/ biomanufacturing facility and traditional academic labs around the globe.