The new xCELLigence® RTCA CardioECR instrument combines high frequency measurement of cell-induced electrical impedance with multielectrode array technology to simultaneously assess cardiomyocyte contractility, viability, and electrophysiology. This model is similar to our other xCELLigence® instruments in its use of noninvasive electrical impedance monitoring to assess cellular morphology change and attachment quality in a label-free and real-time manner. Because cell-induced electrical impedance is dependent on cell size/shape and how strongly the cell interacts with the plate bottom, the contraction-relaxation cycle of cardiomyocytes gives rise to a distinct, rhythmic fluctuation in impedance that is readily captured on the millisecond time scale. Changes in the intensity and periodicity of this “beating” pattern can be monitored in the short (seconds) to long (days) time regimes to assess cardiomyocyte contractility and viability in the presence of different drugs. The CardioECR (ExtraCellular Recording) model differs from our other xCELLigence® instruments, including the first generation Cardio model, in the following ways: (1) It has an enhanced impedance data acquisition rate (every 1 millisecond), (2) it uses a separate pair of electrodes to measure field potential at 10 kHz, (3) it provides a pacing stimulus, and (4) it uses an electronic 48-well microtiter plate (E-Plate® CardioECR 48). The simultaneous recording of impedance and field potential by the xCELLigence® RTCA CardioECR instrument provides a view of cardiomyocyte health at an unprecedented level of detail, enabling a deeper understanding of the mechanisms underlying drug-induced cardiac liability.
The xCELLigence® RTCA CardioECR instrument is placed in a standard CO2 cell culture incubator and interfaces via a cable with analysis and control units that are housed outside the incubator. User friendly software allows for real-time control and monitoring of the instrument, and includes real-time data display and analysis functions.
The xCELLigence® RTCA CardioECR instrument, regularly used in combination with human iPSC-derived cardiomyocytes, enables in vitro assays that are highly predictive of drug induced cardiac liability. It offers:
• A high-throughput method for detecting functional cardiotoxicity (effects on short-term and long-term cardio beating activity) and general toxicity in vitro
• A test of integrated ion channel activity
• Data that display excellent correlation with clinical arrhythmogenic risk
• Pacing function for more tightly controlled assays
• More thorough understanding of drug mechanism of action
Building upon the impedance-only xCELLigence® RTCA Cardio system, the new CardioECR system combines impedance recording (for evaluating contractility and viability) with both multi electrode array (MEA) technology and a pacing function (for evaluating integrated ion channel activity). Cardiomyocytes are seeded in a 48-well electronic microtiter plate (E-Plate® CardioECR 48) that contains gold microelectrode arrays fused to the bottom of each well (Figures 1A-C). Application of a low voltage (less than 20 mV) establishes an electric current between the electrodes, which is differentially modulated by the number of cells covering the electrodes, the morphology of those cells, and the strength of cell attachment. Because the cardiomyocyte contraction/relaxation cycle involves substantial changes in cell morphology and adhesion, it can be dynamically monitored using impedance (Figures 2A-B). The enhanced impedance measurement rate (every 1 ms) of the xCELLigence® RTCA CardioECR system provides extremely high temporal resolution for viewing subtleties of the cardiomyocyte contraction/relaxation continuum. In addition to this ability to monitor cell viability and contractile activity, additional point electrodes in the well bottoms (Figure 1C) allow for extracellular field potential (FP) measurements at 10 kHz, which can be performed in tandem with impedance recording (Figure 2B).
Figure 1. E-Plate CardioECR 48 compatible with the xCELLigence RTCA CardioECR system. (A) E-Plate CardioECR 48. The footprint of this plate complies with ANSI/SBS 1-2004 requirements, and the spacing of the wells in each column is 9 mm center-to-center as per the ANSI/SBS 4-2004 standard. (B) Zoomed in view of E-Plate CardioECR 48 wells. (C) Graphic depiction of a confluent layer of cardiomyocytes interacting with both types of recording electrodes (impedance and field potential electrodes).
Figure 2. Simultaneously using impedance and field potential to monitor cardiomyocyte health and function. (A) Comparison of the contracted vs. relaxed states of two cardiomyocytes adhered to a single electrode. The differences in cell size/shape, and the manner in which cells contact the electrode cause these two states to impede the flow of electric current differently. (B) Simultaneously monitoring cardiomyocyte contraction (red, green, blue, and pink traces) and field potential (integrated ion channel activity; black traces) in real-time. Compared to the negative control (upper set of traces), the three different drugs being evaluated here (bottom three sets of traces) have distinct effects on both contraction and field potential.
Building upon the impedance-only xCELLigence® RTCA Cardio system, the new CardioECR system combines impedance recording with multi electrode array (MEA) technology and a pacing function. With the ability to evaluate cardiomyocyte electrical, contractile, and structural toxicity simultaneously, the xCELLigence RTCA CardioECR system is specifically designed for comprehensive in vitro cardiotoxicity screening. The pacing function enables studies to be performed under normal vs. stressed conditions.
Workflow of the xCELLigence RTCA CardioECR System: Cardio-Safety Testing
No cell labeling required, fully automated, physiological conditions
Shown in the workflow above, cryopreserved iPSC cardiomyocytes are directly plated in a 48-well microtiter E-Plate® CardioECR. Post seeding, the cardiomyocytes are given sufficient time to form gap junctions and organize into a synchronized beating monolayer. The electrode layout on the E-Plate CardioECR 48 facilitates visual inspection of the general health of the cardiomyocytes under a microscope. The ability to simultaneously monitor overall cell health (using cellular impedance), cell contractile activity (using beating rate and amplitude), and electrical activity (using FPD and spike amplitude) provides an extremely effective means of evaluating a drug’s cardiac liability.
The Cardio/CardioECR system is capable of performing all xCELLigence RTCA applications, except chemotactic cell migration and invasion. The most cited applications of the Cardio/CardioECR system are in the following in vitrocardio-safety research areas.
Figure 1. Zoomed in screen shot of table for recording the contents/conditions of each well in an E-Plate.
Pre-defined protocols guide you through experimental set-up and analysis in seconds.
The CardioECR Software enables facile experiment setup and execution along with powerful data analysis, while still remaining efficient and intuitive. A general synopsis of how the software is used to run and analyze an experiment is shown below.
Step 1: Record Plate Layout
Using an intuitive graphical interface the contents/conditions of each well in the electronic microtiter plate (E-Plate®) are recorded (Figure 1). Information fields for the wells include parameters such as cell type, cell number, drug identity, drug concentration, etc. Table autofilling functions, similar to what are available in Excel or other spreadsheet programs, enable rapid data entry and automatic establishment of drug concentration gradients, cell number titrations, etc. Even when multiple cell types and assay conditions are being examined, it takes just minutes to record the information for every well in a plate.
Step 2: Define Data Acquisition Parameters
Using a second table the details of electrical stimulation and data acquisition are defined. These include:
Step 3: Running the Experiment
Press “Run” and watch as impedance and field potential data are acquired simultaneously in real-time for every well in the plate. Even as data is being acquired it can be viewed and graphically manipulated.
Step 4: Data Plotting and Analysis
Using an intuitive graphical interface the real-time impedance and field potential data for all the wells, or a subset of wells, from the E-Plate can be plotted (Figure 2A). Data from multiple wells can be averaged and the coefficient of variation automatically calculated and plotted. The viewing window for the x- and y-axes can be readily adjusted, and data traces can be normalized to a specific time point (immediately before drug addition, for example). By zooming in on a short time range it is possible to view the rhythmic fluctuation in impedance and field potential associated with cardiomyocyte beating (Figure 2B). Impedance and field potential traces can be viewed individually, or can be overlaid on one another.
Figure 2. Data plotting and analysis using the CardioECR Software. (A) Screen shot of data plotting/analysis window. Here all of the curves have been normalized to the time point immediately preceding drug treatment (denoted by the bold back vertical line). Error bars represent coefficients of variation. (B) Viewing cardiomyocyte contraction (red, green, blue, and pink traces) and field potential (black traces). By zooming in on a shorter time range (the time points inside the red box in part “A”), it is possible to view the rhythmic fluctuation of impedance and integrated ion channel activity associated with cardiomyocyte beating. Though shown overlapped here, the impedance and field potential traces can be viewed individually.
Built-in data analysis tools enable characterization of drug-induced early afterdepolarization (EAD) and arrhythmia, as well as calculating field potential duration (Figure 3A). Additionally, the real-time impedance trace can be interrogated to quantify cardiomyocyte beating rate and amplitude (Figure 3B) in the presence of different drugs.
Figure 3. Data analysis using the CardioECR Software. (A) Quantifying field potential duration in the presence and absence of drug. (B) Using impedance traces to quantify cardiomyocyte beating rate and amplitude in the presence and absence of drug.