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Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.


Introduction
Human Pluripotent Stem Cells (hPSCs) have the capacity to self-renew and generate virtually any cell type of the human body through differentiation 1,2 . Detailed protocols on how to direct differentiation of hPSCs into several cardiac lineages (ventricular, atrial, pacemaker-like cardiomyocytes) have been described 3,4,5,6,7 . Cardiomyocytes are electrically active cells and detailed knowledge of their electrophysiological activity can be extremely informative for understanding heart development and disease 8 . Patient-specific hiPSC-derived cardiomyocytes (hiPSC-CMs) have been successfully used to model and study the cellular, molecular, and electrical features of several cardiac arrhythmias, including Long QT Syndrome (LQTS) 9,10,11,12,13 , Brugada syndrome 14 , and cathecolaminergic polymorphic ventricular tachycardia 15,16 . Furthermore, multiple drugs have been added to diseased hiPSC-CMs to recapitulate therapeutic intervention and to rescue the cellular pathological phenotypes 10,15,20,21,22 . More recently, screening platforms based on WT hiPSC-CMs have been developed, in response to the need for human systems to the early phases of drug discovery 23,24,25 as rodent cardiomyocytes differ profoundly from humans in ion channel expression and biophysics 26 .
For this purpose, technologies suitable for medium-to high-throughput application are being developed and implemented. These include optical recordings of membrane potential, Ca 2+ transients and strain, impedance measurements (as an indirect measure of cell contractility), and extracellular field potential (FP) measurements (for review see reference 24 ). Multi-electrode Arrays (MEA) devices allow recording of the electrical waveform signals (or FPs) generated and shaped by monolayers or small clusters of cardiomyocytes. FP contour correlates with the cardiac action potential and, to some extent, with the electrocardiogram (ECG) recordings 27 ; they typically show an initial rapid upstroke corresponding to the Na + influx and membrane depolarization (R/Q peak), a slow wave/plateau phase likely corresponding to the Ca 2+ influx, and a repolarization phase corresponding to a predominant K + efflux (T peak). Perturbation of the FP waveform can be correlated with changes in specific action potential phases 28 .
Although patch clamp recordings of action potentials could be more informative, especially for parameters as upstroke velocity and resting membrane potential, manual measurements are not feasible for experiments at medium-and high-throughput scale, while automated patch clamp has only recently been applied to hPSC-CMs 5. Rinse the well with 1 mL LI-BPEL to collect all the remaining cells and cell clumps. 6. Add another 2-3 mL of LI-BPEL to reach a final volume of 5-6 mL and gently pipette up and down 3-5x with a 5 mL pipette to dissociate cell clumps. NOTE: Dissociation into single cells at this point is not necessary, since it will affect cell survival. Presence of small clusters will ensure higher cell viability. 7. Centrifuge the cells at RT for 3 min at 300 x g. 8. Remove the supernatant, trying to remove most of the surplus fluid but without dislodging the cell pellet. 9. Resuspend the cell pellet in 250 µL LI-BPEL (using a P1000 and pipetting extremely gently).

Check Signal Quality (Figure 4)
1. Switch on the computer and launch the software suite linked to the MEA set up: TCX-Control, MC_MEA Select and MC_Rack. Set the temperature to 37 °C in TCX-Control to record measurements at physiological temperature. 2. Remove the dish containing the MEA chip from the incubator. Open the lid, take out the MEA chip, and place it on a tissue to absorb residual water. 3. Carefully wipe the external contacts of the plate with a tissue and clean them using a cotton swab moistened with 100% (v/v) ethanol to remove any residual water or debris, which might cause signal noise. 4. Transfer the MEA plate to the heated (37 °C) recording head-stage to detect the spontaneous activity (e.g., hardware: see Table of Materials; software: MC_Rack).

Start Experiment and Recording
1. Click 'record' and then 'play', and acquire data for 10 min under baseline conditions to determine the steady state. Annotate the electrodes that have the best signal so that they can be easily identified and exported later for the analysis. 2. For drug-response assessment, add increasing concentrations of drug at every 10 min. As an example, add the hERG blocker E4031 at a final concentration of 1 μM. For this, remove 100 μL of medium and add the same volume of 10 μM E4031 dissolved in the medium. NOTE: As previously demonstrated by Cavero and colleagues 31 , a wise choice of the volume in which the drugs are dissolved is important, since it can profoundly alter the drug response curve. 3. Repeat step 7.2 for all the other drug concentrations of interest. 4. Click 'stop' to conclude the recordings at the end of the protocol.

Representative Results
One day after dissociation and plating, the layer of hPSC-CMs will be visible as a dense and white film covering the center of the MEA chamber ( Figure 3A). After removal of the ring (Figure 3B), the layer should remain in place and inspection on a light microscope will show the MEA electrodes covered by the (contracting) hPSC-CM layer ( Figure 3C). Because of physical and electrical coupling of the cells, only one electrode (golden electrode) will be used for analysis.
Alternatively, when working with 3D structures, such as embryoid bodies or microtissues, these can be plated so that they are physically and electrically uncoupled. Visual inspection at the microscope could confirm no physical connection between the clusters and nonsynchronized R waves at MEAs confirm no electrical coupling. In this case, multiple independent electrodes can be analyzed. Figure 4. In particular, a good quality trace can be defined by the presence of a clear peak corresponding to the Na + influx and membrane depolarization (R/Q peak), a clear repolarization phase corresponding to K + efflux (T peak), and a high signal to noise ratio ( Figure 4A, left: note y-axis scale and Figure 4B). Bad quality traces (Figure 4A, middle) may be the result of failure of hPSC-CMs to attach to the MEA plate or of weak hPSC-CM electrical activity. Waiting 1-3 days for better attachment may improve the signal; however, if no signal improvement is visible, excluding this MEA from experiments is recommended. Noisy traces (Figure 4A, right) may be analyzed after filtering.

Typical recordings of FP traces are shown in
Successful analysis of RR interval can be identified by visual inspection of the screen showing peak detection (Figure 5A, 5B). Within the time interval defined by the vertical cursors, blue marks corresponding to each peak should be present. In case the program does not identify one or more peaks, try to move the horizontal cursor and run the analysis again or adjust the detection settings. Similarly, successful analysis of QT interval can be identified by visual inspection of the screen showing FP detection (Figure 7). Within the time interval defined by the vertical cursors, blue marks corresponding to each FP detection should be present. In case the program does not identify one or more FPs, try to redefine the FP template (Figure 6) or adjust the detection settings and run the analysis again.

Discussion
This protocol shows how to dissociate and prepare hPSC-CMs for measuring their FP using MEAs. hPSC-CMs usually display spontaneous electrical activity, which can be measured as FP and can provide meaningful data with respect to beating frequency, QT interval duration, and arrhythmic events.
Dissociation of 2D cardiac differentiated cultures is needed for recreating a beating layer on the MEA and it represents a critical step. Mechanical stress by repeated pipetting and/or aggressive dissociation enzyme treatments may result in high cell mortality, failure to attach to the MEA plate, and lack of spontaneous electrical activity. This protocol has been optimized for monolayer cultures. However, a similar approach can be used for three-dimensional (3D) cultures (e.g., embryoid bodies or EBs) with minor modifications, such as collection of the EBs followed by PBS wash and longer incubation time with the dissociating enzyme. Importantly, in both 2D and 3D differentiated cultures, the older the differentiated cells, the longer incubation time required might be to detach the cells because of increased extracellular matrix deposition.
The protocol described here for quantifying FP parameters can be used to generate dose-response curves for cardioactive drugs. As recently described by Cavero et al. 31 , the starting concentration of a drug might profoundly affect the outcome of a MEA measurement. Therefore, to improve accuracy and reliability of the results, we suggest the following: 1) in case of irreversible activators/blockers, use relatively large volumes of medium containing the drug to be tested. More in detail, remove 10-50% of medium volume from the MEA chip and add an equal volume of medium in which the drug was previously dissolved at the appropriate concentration. In this case, for calculating the final drug concentration, it is critical to consider the change in concentration after medium removal. 2) In case of reversible activators/blockers, add 10 µL of each drug dose from a 100X stock solution.
The majority of the cardiac differentiation protocols results in a variable mixed population of nodal-like, atrial-like and ventricular-like cardiomyocytes, with the ventricular type being the most represented. This might constitute a limitation when modeling cardiac diseases affecting a specific cardiomyocyte subtype or drugs acting on cardiac subtype-specific ion channels. Although several studies have optimized conditions to direct more controlled specification during cardiac differentiation 3,5,37,38 , their broader applicability is still under investigation.
Cardiomyocyte-enriching strategies based on surface protein expression 35,45 (by florescence-assisted cell sorting or by magnetic-bead selection 46,47 ), and metabolic selection 44,48 may represent valid strategies that can be applied to any (genetically modified or unmodified) hPSCline prior plating of the hPSC-CMs, to improve the electrical signal.
Although hPSC-CMs are notoriously immature as compared to human adult cardiomyocytes 4,49 , they have proven to be valuable in recapitulating and identifying specific disease-related changes (e.g., in channelopathies) 19,20,50 and drug-induced responses (e.g., cardiac ion channel blockers) 4,51 . Furthermore, immature cells are easier to dissociate, and recover better than adult cardiomyocytes after dissociation and plating 44 therefore, hPSC-CM immaturity may be rewarded as an advantage in this respect. However, to be able to recapitulate e.g. late onset cardiac diseases and faithfully reproduce drug responses of adult cardiomyocytes, a more mature mechanical, metabolic, and electrical hPSC-CM state should be obtained. Methods to mature these cells include prolonged time in culture 52 , mechanical strain 53 , electrical pacing 54 , addition of small molecules 55 , 3D-culture 56 , co-culture with other cell types 57 , and even a combination of these approaches 58 ; to date, none of these approaches has led to an adult-like phenotype.