hybrid-cell-analysis-system-to-assess-structural-contractile-changes

Hybrid Cell Analysis System to Assess Structural and Contractile Changes of Human iPSC-Derived Cardiomyocytes for Preclinical Cardiac Risk Evaluation

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While ...

Nanion Technologies GmbH

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.

Microstructured apertures in planar glass substrates for ion channel research.

The patch-clamp technique is the state-of-the-art technology for the study of a large class of membrane proteins called ion channels. Ion channels mediate electrical current flow, have crucial roles in cellular physiology, and are important drug targets. However, patch clamping is a laborious process requiring a skilled experimenter and is, therefore, not compatible with the high throughput needed in drug development. The solution for automated and parallel patch-clamp measurements that is provided by microchip technology is presented here.

Microchip technology for automated and parallel patch-clamp recording.

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.

Automated ion channel screening: patch clamping made easy.

Solvent-free planar lipid bilayers were formed in an automatic manner by bursting of giant unilamellar vesicles (GUVs) after gentle suction application through micron-sized apertures in a borosilicate glass substrate. Incubation of GUVs with the purified ion channel protein of interest yielded proteoliposomes. These proteoliposomes allow for immediate recording of channel activity after GUV sealing. This approach reduces the time-consuming, laborious and sometimes difficult protein reconstitution processes normally performed after bilayer formation. Bilayer recordings are attractive for investigations of membrane proteins not accessible to patch clamp analysis, like e.g. proteins from organelles. In the presented work, we show the example of the outer membrane protein OmpF from Escherichia coli. We reconstituted OmpF in proteoliposomes and observed the characteristic trimeric conductance levels and the typical gating induced by pH and transmembrane voltage. Moreover, OmpF is the main entrance for beta-lactam antibiotics and we investigated translocation processes of antibiotics and modulation of OmpF by spermine. We suggest that the rapid formation of porin containing lipid bilayers is of potential for the efficient electrophysiological characterization of the OmpF protein, for studying membrane permeation processes and for the rapid screening of antibiotics.

Rapid screening of membrane protein activity: electrophysiological analysis of OmpF reconstituted in proteoliposomes.

Ion channels have gained increased interest as therapeutic targets over recent years, since a growing number of human and animal diseases have been attributed to defects in ion channel function. Potassium channels are the largest and most diverse family of ion channels. Pharmaceutical agents such as Glibenclamide, an inhibitor of K(ATP) channel activity which promotes insulin release, have been successfully sold on the market for many years. So far, only a small group of the known ion channels have been addressed as potential drug targets. The functional testing of drugs on these ion channels has always been the bottleneck in the development of these types of pharmaceutical compounds.New generations of automated patch clamp screening platforms allow a higher throughput for drug testing and widen this bottleneck. Due to their planar chip design not only is a higher throughput achieved, but new applications have also become possible. One of the advantages of planar patch clamp is the possibility of perfusing the intracellular side of the membrane during a patch clamp experiment in the whole-cell configuration. Furthermore, the extracellular membrane remains accessible for compound application during the experiment.Internal perfusion can be used not only for patch clamp experiments with cell membranes, but also for those with artificial lipid bilayers. In this chapter we describe how internal perfusion can be applied to potassium channels expressed in Jurkat cells, and to Gramicidin channels reconstituted in a lipid bilayer.

Planar patch clamp: advances in electrophysiology.

Ion channel dysfunction is known to underlie several acute and chronic disorders and, therefore, ion channels have gained increased interest as drug targets. During the past decade, ion channel screening platforms have surfaced that enable high throughput drug screening from a more functional perspective. These two factors taken together have further inspired the development of more refined screening platforms, such as the automated patch clamp platforms described in this article. Approximately six years ago, Nanion introduced its entry level device for automated patch clamping - the Port-a-Patch. With this device, Nanion offers the world's smallest patch-clamp workstation, whilst greatly simplifying the experimental procedures. This makes the patch clamp technique accessible to researchers and technicians regardless of previous experience in electrophysiology. The same flexibility and high data quality is achieved in a fully automated manner with the Patchliner, Nanion's higher throughput patch clamp workstation. The system utilizes a robotic liquid handling environment for fully automated application of solutions, cells and compounds. The NPC-16 chips come in a sophisticated, yet simplistic, microfluidic cartridge, which allow for fast and precise perfusion. In this way, full concentration response curves are easily obtained. The Port-a-Patch and Patchliner workstations from Nanion are valuable tools for target validation, secondary screening and safety pharmacology (for example hERG and Nav1.5 safety screening). They are widely used in drug development efforts by biotechnological and pharmaceutical companies, as well as in basic and applied biophysical research within academia.

Port-a-patch and patchliner: high fidelity electrophysiology for secondary screening and safety pharmacology.

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.

Automated patch clamp on mESC-derived cardiomyocytes for cardiotoxicity prediction.

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.

State-of-the-Art Automated Patch Clamp Devices: Heat Activation, Action Potentials, and High Throughput in Ion Channel Screening.

Neurons derived from human-induced pluripotent stem cells were characterized using manual and automated patch-clamp recordings. These cells expressed voltage-gated Na(+) (Na(v)), Ca(2+) (Ca(v)), and K(+) (K(v)) channels as expected from excitable cells. The Na(v) current was TTX sensitive, IC(50) = 12 ± 6 nM (n = 5). About 50% of the Ca(v) current was blocked by 10 µM of the L-type channel blocker nifedipine. Two populations of the K(v) channel were present in different proportions: an inactivating (A-type) and a noninactivating type. The A-type current was sensitive to 4-AP and TEA (IC(50) = 163 ± 93 µM; n = 3). Application of γ-aminobutyric acid (GABA) activated a current sensitive to the GABA(A) receptor antagonist bicuculline, IC(50) = 632 ± 149 nM (n = 5). In both devices, comparable action potentials were generated in the current clamp. With unbiased, automated patch clamp, about 40% of the cells expressed Na(v) currents, whereas visual guidance in manual patch clamp provided almost a 100% success rate of patching "excitable cells." These results show high potential for pluripotent stem cell-derived neurons as a useful model for drug discovery, in combination with automated patch-clamp recordings for high-throughput and high-quality drug assessments at human neuronal ion channels in their correct cellular background.

Characterizing human ion channels in induced pluripotent stem cell-derived neurons.

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.

Minimized cell usage for stem cell-derived and primary cells on an automated patch clamp system.

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. 

New easy-to-use hybrid system for extracellular potential and impedance recordings.

We have developed an automated patch clamp module for high-throughput ion channel screening, recording from 384 cells simultaneously. The module is incorporated into a laboratory pipetting robot and uses a 384-channel pipettor head for application of cells and compounds. The module contains 384 amplifier channels for fully parallel recordings using a digital amplifier. Success rates for completed experiments (1- to 4-point concentration-response curves for cells satisfying defined quality control parameters) of greater than 85% have been routinely achieved with, for example, HEK, CHO, and RBL cell lines expressing hNa1.7, hERG, Kir2.1, GABA, or glutamate receptors. Pharmacology experiments are recorded and analyzed using specialized software, and the pharmacology of hNa1.7 and hERG is described. Fast external solution exchange rates of <50 ms are demonstrated using Kir2.1. Short exposure times are achieved by stacking the external solutions inside the pipette of the robot to minimize exposure of the ligand on the receptor. This ensures that ligand-gated ion channels, for example, GABA and glutamate described in this report, can be reproducibly recorded. Stem cell-derived cardiomyocytes have also been used with success rates of 52% for cells that have a seal resistance of >200 MΩ, and recordings of voltage-gated Na and Ca are shown.

Automated Patch Clamp Meets High-Throughput Screening: 384 Cells Recorded in Parallel on a Planar Patch Clamp Module.

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.

Safety pharmacology studies using EFP and impedance.

Automated patch clamp (APC) devices have become commonplace in many industrial and academic labs. Their ease-of-use and flexibility have ensured that users can perform routine screening experiments and complex kinetic experiments on the same device without the need for months of training and experience. APC devices are being developed to increase throughput and flexibility. Areas covered: Experimental options such as temperature control, internal solution exchange and current clamp have been available on some APC devices for some time, and are being introduced on other devices. A comprehensive review of the literature pertaining to these features for the Patchliner, QPatch and Qube and data for these features for the SyncroPatch 384/768PE, is given. In addition, novel features such as dynamic clamp on the Patchliner and light stimulation of action potentials using channelrhodosin-2 is discussed. Expert opinion: APC devices will continue to play an important role in drug discovery. The instruments will be continually developed to meet the needs of HTS laboratories and for basic research. The use of stem cells and recordings in current clamp mode will increase, as will the development of complex add-ons such as dynamic clamp and optical stimulation on high throughput devices.

An update on the advancing high-throughput screening techniques for patch clamp-based ion channel screens: implications for drug discovery.

Current in vitro assays typically poorly predict cardiac liability as they focus on single ion channels overexpressed in cell lines. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), on the other hand, provide a unique opportunity for drug testing on human cardiomyocytes using high-throughput systems. However, these cells can differ from adult cardiomyocytes in their ion channel expression and, therefore, electrophysiologic properties. One of the main challenges of hiPSC-CMs is the physiologic expression of ion channels such as the inward rectifiers (e.g., Kir2.1-2.3), which conduct the cardiac inward rectifier potassium current (I ). I is one of the primary contributors in maintaining a stable resting membrane potential in cardiac cells, which is essential for excitability. This is only expressed in low levels, or sometimes not at all, in hiPSC-CMs as shown by patch clamp studies. Dynamic clamp is a method of electronically introducing ion currents (e.g., I ) into cells to compensate for the lack of endogenous expression, thus offering the potential to record more stable action potentials in hiPSC-CMs. In this article, we describe the method of using hiPSC-CMs on an automated patch clamp device (Patchliner) coupled with the automated dynamic clamp add-on (Dynamite ). We describe protocols for optimized cell handling and harvesting for use on the Patchliner and the steps required for automated execution of experiments and data analysis in dynamic clamp mode. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Recording action potential pharmacology from human induced pluripotent stem cell-derived cardiomyocytes in automated patch clamp combined with dynamic clamp to introduce simulated I and compensate seal resistance Support Protocol 1: Cardiomyocyte plating and culture Support Protocol 2: Cell harvesting and dissociation Alternate Protocol: Recording action potential pharmacology at physiologic temperatures.

Automated Dynamic Clamp for Simulation of I in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Real Time Using Patchliner Dynamite.

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 FLEXcyte96 technology with respect to physiological responses of hiPSC-CMs to pharmacological compounds with known inotropic and/or cardiotoxic effects.

Michael George

Nanion Technologies GmbH

Hybrid Cell Analysis System to Assess Structural and Contractile Changes of Human iPSC-Derived Cardiomyocytes for Preclinical Cardiac Risk Evaluation