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How to use Microelectrode Array (MEA) to assess the electrophysiological properties of iPSC-derived Atrial and Ventricular Cardiomyocytes

Introduction

Microelectrode array (MEA) is an easy-to-use, label-free and high-throughput method for identifying the electrical output from cells. MEA has many applications such as modelling network connectivity between neurons and arrhythmia in cardiomyocytes additionally, MEA is an ideal platform to investigate the effect of toxic compounds on the electrophysiology of cells.

Cardiac toxicity is one of the most prominent off-target effects that can occur during drug development and has resulted in the withdrawal of high-profile drugs from the market. The incorporation of iPSC-derived cardiomyocytes in compound screening and toxicity testing has the potential to identify cardiac toxicity and arrhythmia much earlier in drug development.

Axol’s Human iPSC-derived Atrial and Ventricular Cardiomyocytes provide consistent and reproducible models which have the potential to reduce drug attrition by providing physiologically relevant tools for use in cell based assays. These cells also offer the ability to advance the understanding of cardiovascular disease, channelopathies, cardiomyopathies and atrial fibrillation in a biologically relevant system.

Here we show how to measure the electrical parameters such as beat frequency and field potential duration (FPD)¬ of Axol’s iPSC-derived Atrial and Ventricular Cardiomyocytes.

Method:

Follow our video "How to... Culture Human iPSC-derived Atrial and Ventricular Cardiomyocytes on an Alpha MED Microelectrode Array (MEA)" to prepare your iPSC-derived cardiomyocytes for MEA.

Setting up the Alpha MED PRESTO

Step 1: Although the Alpha MED PRESTO Amplifier is well shielded from electrical noise it is susceptible to mechanical noise. Ensure the PRESTO is placed on a secure, solid base away from any sources of vibration (such as air conditioning unit or fume hood) and away from any power supply.

Step 2: Switch on and pre-warm the PRESTO to 37oC. This can take up to 30 minutes.

Step 3: Clean the contact pins with a KimWipe soaked with ethanol before every experiments

Step 4: Connect the acquisition computer with a USB cable and open the MEA Symphony software.

Step 5: Choose the Recorder -> Cardio option.

Step 6: Open your recording protocol (.moflo format) File -> Open… -> Select.

Step 7: Check recording format and make notes using the "Protocol" tab.

Step 8: Input correct output destination directory for the saved data (.modax format).

Step 9: Ensure the amplifier recalibrates before recording by clicking on "Recalibrate amplifier" changing its border to orange.

Step 10: Check the "Filtering" tab, raw data should be obtained unfiltered and with no smoothing and downsampling. The raw data can be filtered, smoothed or downsampled during the Offline Analysis phase.

Step 11: Check the "Beats" tab. Fastest recording occurs with the "Extraction off" checkbox ticked. Spike extraction can be performed later during the Offline Analysis phase.

Step 12: During initial recording Beats Extraction, Exporting and Reporting should all be disabled.

Step 13: Ensure adequate hard-drive space, the raw data will take up 1Gb/minute’s recording with the analysed data taking approximately the same amount of space again.

Step 14: Remove the MEA plate from the 37C, 5% CO2 incubator. Ensure the lid remains on throughout.

Step 15: Open the Presto’s lid and gently place the MEA plate onto the Presto. Close and lock the lid.

Step 16: If CO2 is set-up, turn on the CO2 environmental support. Without CO2 environmental support, Human iPSC-derived Atrial Cardiomyocytes and Ventricular Cardiomyocytes will remain stable enough for recording for at least 20 minutes.

Step 17: To monitor plate activity, press the "Preview" button in "Record Mode". Clicking on the "Oscilloscope" tab will display the activity of all channels. Individual electrodes and wells can be monitored in more detail by selecting the required electrode in the "Oscilloscope" tab and returning to the main screen. The oscilloscope can be set to auto-scale which will continually change the scale to maximise the recorded signal or a scale can be manually set to monitor changes in amplitude over time. It is not advised to deselect electrodes or wells during raw data acquisition as this can be done later during Offline Analysis and sometimes "noisy" electrodes will improve during the recording. Ensure all signals return to the zero baseline a constant offset could indicate the amplifier needs recalibrating.

Recording using the Alpha MED PRESTO

Step 1: Once satisfied with the activity of the plate, return to the main display and press "Stop". Ensure the "Recalibrate amplifier" button is highlighted in orange on the "Protocol" tab. Press "Record" on the main display, the amplifier will recalibrate and then automatically proceed through the recording protocol.

Step 2: Plate activity can be monitored on the "Oscilloscope" tab.

Step 3: Minimise activity around the PRESTO during recording and monitor the plate for unacceptable levels of mechanical noise.

Step 4: Once the recording is finished the timer will stop and the signals will freeze.

Analysis using the Alpha MED PRESTO

Step 5: The raw data can be analysed offline at a later date.

Step 6: Open the MEA Symphony software and select Analyzer -> Cardio.

Step 7: Select the data you want to analyse by first selecting the recording protocol (.moflo) used to acquire the data by File -> Open… -> Select.

Step 8: Ensure you are in Replay mode by toggling from Record Mode to Replay mode on the main display. Choose the data file (.modax) you wish to replay by browsing for it on the “Protocol” tab.

Step 9: The "Protocol" tab can be used to select/deselect individual wells for analysis by clicking on them or using the “All” or “None” button.

Step 10: The "Oscilloscope" tab can be used to select/deselect individual electrodes.

Step 11: Choose desired filtering settings on the "Filtering" tab. Smoothing can be used to better extract the later parts of the cardiomyocyte waveform. Downsampling can be used to reduce the data file size at the expense of losing data.

Step 12: Beats Extraction can be enabled under the "Beats" tab. The extraction thresholds can be set either positive and negative or just negative. For cardiomyocytes, it is recommended to use a positive and negative threshold. The thresholds can be calculated automatically using a percentage of the noise median or standard deviation or set manually by moving the orange levels or inputting levels on the central trace of the main display. For cardiomyocytes, the signal should far exceed the noise so automatic threshold detection of 600% is sufficient to correctly identify all spikes.

Step 13: The "Exporting" tab will, when enabled, automatically export analyse the data in a variety of ways. Alternatively, the raw data can be exported in a number of formats for analysis by other programmes. Data can be extracted in 4-well groups in a Mobius-compatible format (.modat), extracted for all electrodes in an Excel-compatible ASCII (.csv) but is limited to 52 seconds of data due Excel’s row limit or 10 minutes and more of data from up to 16 electrodes can be exported in a pClamp-compatible .bin format.

Step 14: The "Reporting" tab can be used to automatically generate reports or directly export any window within Symphony as a figure.

Results

After 8 days in culture, phase contrast and immunofluorescent imaging revealed that Axol’s Human iPSC-derived Atrial Cardiomyocytes showed expected morphology and expressed atrial-specific markers myosin light chain 2 atrial (MLC2a) and atrial natriuretic factor (ANP) and the cardiac cytoskeleton marker, Troponin T (figure 1 A-C). Similarly, Axol’s Human iPSC-derived Ventricular Cardiomyocytes showed typical morphology and expressed cardiac markers myosin light chain 2 ventricular (MLC2v) and Troponin T (figure 1 D-F).

Axol’s Human iPSC-derived Atrial and Ventricular Cardiomyocytes after 8 days of culture
Figure 1: Phase contrast (A & D) and immunocytochemistry (B, C, E & F) images of Axol’s Human iPSC-derived Atrial and Ventricular Cardiomyocytes after 8 days of culture express typical morphology and express the expected cardiac markers MLC2a (B), MLC2v (E) Troponin T and ANP (C & F).

Axol’s Human iPSC-derived Atrial and Ventricular Cardiomyocytes were cultured for a total of 29 or 32 days with electrophysiological measurements taken on alternate days. The MEA analysis of the recorded waveforms shows that Axol’s Human iPSC-derived Atrial and Ventricular Cardiomyocytes exhibit a typical field potential waveforms (figure 2A).

The inter-spike interval (ISI) shows the average time between the start of one action potential to the start of the next, recorded from one electrode. The standard deviation of the ISI recorded from both the iPSC-derived Atrial and Ventricular Cardiomyocytes is relatively small with very little variability suggesting that there is no arrhythmia present, indicating rhythmic beating (figure 2B). It is important to note that when plating the cardiomyocytes, the seeding density should be optimized to ensure that a syncytium is formed during culture. Once the Human iPSC-derived Cardiomyocytes form a syncytium they will be electrically linked and should beat synchronously.

The raster plots depict electrical events occurring from a single electrode over time, this data is representative of each experiment (figure 2C). Analysis of the raster plot shows that there is rhythmic electrical activity from both the human iPSC-derived atrial and ventricular cardiomyocytes additionally, this activity is stable over time, with recordings being measured from day 4 up to day 32 (figure 2C). The frequency of electrical events is increased in the iPSC-derived Atrial Cardiomyocytes in comparison to the iPSC-derived Ventricular Cardiomyocytes, this is indicative of the quicker beat rate expected from human atrial cardiomyocytes (figure 2C).

Axol’s iPSC-derived Atrial and Ventricular Cardiomyocytes exhibit expected electrical physiology
Figure 2: Axol’s iPSC-derived Atrial and Ventricular Cardiomyocytes exhibit expected electrical physiology. A) Representative field potential waveform exhibited by both atrial and ventricular cardiomyocytes. B) The inter-spike interval (ISI) was measured over time from DIV4 up to DIV29 (atrial) and DIV32 (ventricular), this interval measures the time interval between successive beats from the cardiomyocyte syncytium with the small standard deviation denoting rhythmic beat rate, ISI is shown as the average from 1 electrode. C) The raster plot of spontaneous electrical activity from both atrial and ventricular cardiomyocytes measurements recorded from one electrode over 1 minute.

Concurrent analysis of iPSC-derived Atrial Cardiomyocytes was conducted and shows the combination of an MEA trace with a video of Human iPSC-derived Atrial Cardiomyocytes beating in culture, this demonstrates real-time contraction with the electrical events recorded by MEA (figure 3).

Figure 3: Representative beat rate recorded from Axol’s Human iPSC-derived Atrial Cardiomyocytes using an Alpha MED Presto MEA platform. Human iPSC-derived Atrial Cardiomyocytes were analyzed at 14 DIV.

Lastly, pharmacological modulation of iPSC-derived Ventricular Cardiomyocytes was carried out at DIV32 by the addition of the hERG channel blocker, dofetilide (100 nm). After the addition of dofetilide, delayed afterdepolarisations (DADs) were observed, which is indicative of arrhythmic events occurring in the iPSC-derived Ventricular Cardiomyocytes. The prolongation of the field potential duration upon addition of doefetilide from 390ms to 510ms signalled that the ventricular repolarisation of the ventricular cardiomyocytes had been delayed compared to the untreated control (figure 4 A). The raster plot shows drug-induced instability in the beat rate of the ventricular cardiomyocytes (figure 4 B). The ISI recorded from iPSC-derived Ventricular Cardiomyocytes treated with dofetilide revealed a large standard deviation which demonstrates that there is prolongation of the interval between spikes. The presence of DADs, after a full repolarization, can trigger arrhythmia that precipitates tachycardia and is reported to be one of the main mechanisms behind LQT1/LQT5-related Torsade de pointes(TdP) demonstrating that Axol’s Human iPSC-derived Ventricular Cardiomyocytes provide a physiologically relevant cell-based research tool that has the ability to model arrhythmia in vitro.

Axol’s iPSC-derived Ventricular Cardiomyocytes respond to pharmacological modulation to model arrhythmia
Figure 4: Axol’s iPSC-derived Ventricular Cardiomyocytes respond to pharmacological modulation to model arrhythmia. A) Representative field potential waveform exhibited by ventricular cardiomyocytes after treatment with dofetilide (100nm) or under control conditions. B) The raster plot shows the frequency of spontaneous electrical activity from ventricular cardiomyocytes, prior to addition of the drug the frequency of beats was very consistent after the addition of dofetilide the frequency of electrical signals became clearly erratic. Measurements recorded from one electrode. C) The ISI recorded from ventricular cardiomyocytes treated with dofetilide shows a large standard deviation in comparison to the control (the average from 1 well).

In summary, the validation of iPSC-derived Cardiomyocytes in electrophysiological assays for high-throughput toxicology evaluation demonstrates the potential and the promise for these cells to be adopted into basic research studies to enhance the understand of cardiac disease, the drug development pathway and safety toxicity screening in pre-clinical assessment.

Axol’s Human iPSC-derived Atrial and Ventricular Cardiomyocytes have been functionally validated and characterised on the AlphaMED Presto MEA platform. This study shows that Human iPSC-derived Atrial and Ventricular Cardiomyocytes are applicable for MEA analysis and exhibit a steady and consistent beat rate that can be pharmacologically manipulated to model arrhythmia. Overall, demonstrating their suitability as a biologically relevant cell-based research tool.

Highlighted products used in this application note and where to find them:

Product Name Product Code Supplier
Human iPSC-derived Atrial Cardiomyocytes ax2515 Axol Bioscience
Human iPSC-derived Ventricular Cardiomyocytes ax2505 Axol Bioscience
Fibronectin Coating Solution ax0049 Axol Bioscience
Cardiomyocyte Maintenance Medium ax2530 Axol Bioscience
MED64 Presto Alpha MED Scientific Inc
Cloning ring 11-0162 (RING-05) Iwaki
CellSpotter24 - Comfort Alpha MED Scientific Inc
MEA 24-well Plate-comfort MED-Q2430L Alpha MED Scientific Inc