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JoVE Journal Medicine

Medicine

Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Published: August 4, 2022 doi: 10.3791/64265
Automatic Translation
1,2, 1,2, 1,2, 1,2, 1,2,3
1Stanford Cardiovascular Institute, Stanford University School of Medicine, 2Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, 3Department of Radiology, Stanford University School of Medicine

Summary

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising in vitro model for drug-induced cardiotoxicity screening and disease modeling. Here, we detail a protocol for measuring the contractility and electrophysiology of hiPSC-CMs.

Abstract

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety assessment models is a critical step during drug development. Currently, cardiac safety assessment is still highly dependent on animal studies. However, animal models are plagued by poor translational specificity to humans due to species-specific differences, particularly in terms of cardiac electrophysiological characteristics. Thus, there is an urgent need to develop a reliable, efficient, and human-based model for preclinical cardiac safety assessment. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as an invaluable in vitro model for drug-induced cardiotoxicity screening and disease modeling. hiPSC-CMs can be obtained from individuals with diverse genetic backgrounds and various diseased conditions, making them an ideal surrogate to assess drug-induced cardiotoxicity individually. Therefore, methodologies to comprehensively investigate the functional characteristics of hiPSC-CMs need to be established. In this protocol, we detail various functional assays that can be assessed on hiPSC-CMs, including the measurement of contractility, field potential, action potential, and calcium handling. Overall, the incorporation of hiPSC-CMs into preclinical cardiac safety assessment has the potential to revolutionize drug development.

Introduction

Drug development is a long and expensive process. A study of new therapeutic drugs approved by the US Food and Drug Administration (FDA) between 2009 and 2018 reported that the estimated median cost of capitalized research and clinical trials was $985 million per product1. Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market2. Notably, cardiotoxicity is reported among multiple classes of therapeutic drugs3. Therefore, cardiac safety assessment is a crucial component during the drug development process. The current paradigm for cardiac safety assessment is still highly dependent on animal models. However, species differences from the use of animal models are increasingly recognized as a primary cause of inaccurate predictions for drug-induced cardiotoxicity in human patients4. For example, the morphology of cardiac action potential differs substantially between humans and mice due to the contributions from different repolarizing currents5. In addition, differential isoforms of cardiac myosin and circular RNAs that can impact cardiac physiology have been well documented among species6,7. To bridge these gaps, it is imperative to establish a reliable, efficient, and human-based model for preclinical cardiac safety assessment.

The groundbreaking invention of induced pluripotent stem cell (iPSC) technology has generated unprecedented drug screening and disease modeling platforms. Over the past decade, methods to generate human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become well established8,9. hiPSC-CMs have attracted great interest in their potential applications in disease modeling, drug-induced cardiotoxicity screening, and precision medicine. For instance, hiPSC-CMs have been utilized to model the pathologic phenotypes of cardiac diseases caused by genetic inheritance, such as long QT syndrome10, hypertrophic cardiomyopathy11,12, and dilated cardiomyopathy13,14,15. Consequently, key signaling pathways implicated in the pathogenesis of cardiac diseases have been identified, which can shed light on potential therapeutic strategies for effective treatment. Moreover, hiPSC-CMs have been used to screen drug-induced cardiotoxicity associated with anticancer agents, including doxorubicin, trastuzumab, and tyrosine kinase inhibitors16,17,18; strategies to mitigate the resultant cardiotoxicity are under investigation. Finally, the genetic information retained in hiPSC-CMs allows for the screening and prediction of drug-induced cardiotoxicity at both individual and population levels19,20. Collectively, hiPSC-CMs have proven to be an invaluable tool for personalized cardiac safety prediction.

The overall goal of this protocol is to establish methodologies to comprehensively and efficiently investigate the functional characteristics of hiPSC-CMs, which are of great importance in applying hiPSC-CMs toward disease modeling, drug-induced cardiotoxicity screening, and precision medicine. Here, we detail an array of functional assays to assess the functional properties of hiPSC-CMs, including the measurement of contractility, field potential, action potential, and calcium (Ca2+) handling (Figure 1).

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Protocol

1. Preparation of media and solutions

  1. Prepare hiPSC-CM maintenance medium by mixing a 10 mL bottle of 50x B27 supplement and 500 mL of RPMI 1640 medium. Store the medium at 4 °C and use it within a month. Equilibrate the medium to room temperature (RT) before use.
  2. Prepare hiPSC-CM seeding medium by mixing 20 mL of serum replacement and 180 mL of hiPSC-CM maintenance medium (10% dilution, v/v). While freshly prepared seeding medium is preferred, it can be stored at 4 °C for no more than 2 weeks. Equilibrate the medium to RT before use.
  3. Prepare extracellular matrix coating solution by thawing one bottle (10 mL) of basement membrane matrix at 4 °C overnight and aliquoting 500 µL of basement membrane matrix in sterile 1.5 mL tubes. Store at -20 °C for further use. Mix 500 µL of basement membrane matrix and 100 mL of ice-cold DMEM/F12 medium (1:200 dilution, v/v) to make basement membrane matrix coating solution.
  4. Prepare 50 mL of Tyrode's solution containing 135 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM glucose, and 10 mM HEPES. Adjust the pH to 7.4 with NaOH. Prepare fresh Tyrode's solution on the day of the experiment.
  5. Prepare 50 mL of intracellular pipette solution containing 120 mM KCl, 1 mM MgCl2, 10 mM EGTA, and 10 mM HEPES. Adjust the pH to 7.2 with KOH. Aliquot the intracellular pipette solution in sterile 5 mL tubes and store it at -20 °C. On the day of the experiment, freshly add MgATP to the solution to achieve a final concentration of 3 mM.
  6. Prepare 1 mL of Fura-2 AM loading solution containing 2 µM Fura-2 AM and 0.1% Pluronic F-127. Prepare fresh loading solution on the day of the experiment and protect it from the light.

2. Measurement of hiPSC-CM contraction motion

  1. Prepare basement membrane matrix-coated plates by adding 100 µL of extracellular matrix coating solution to each well of 96-well plates. Incubate the 96-well plates in a humidified cell culture incubator at 37 °C, 5% CO2 overnight.
  2. Enzymatic dissociation of hiPSC-CMs
    1. Request hiPSCs from Stanford Cardiovascular Institute iPSC Biobank (http://med.stanford.edu/scvibiobank.html). Generate hiPSC-CMs according to the previous publications8,9. Observe the hiPSC-CMs cultured in six-well plates under a microscope at 10x magnification.
      NOTE: Do not dissociate the cells when they are still proliferative. Otherwise, the cells will overgrow on the wells after replating and lead to inaccurate results of the functional assays. Usually, hiPSC-CMs stop proliferation at days 18-23.
    2. Make sure the hiPSC-CMs are beating strongly and have a purity of over 95%. Assess the purity by immunostaining against cardiac troponin T, a specific marker of cardiomyocytes8,9.
    3. Aspirate the maintenance medium and wash the cells with 1x DPBS twice. Add 1 mL of cell detachment solution to each well of the six-well plates and incubate for 10 min at 37 °C.
      NOTE: The incubation duration should be regularly monitored. Make sure not to over-digest the cells as it will reduce cell viability.
    4. Gently pipette hiPSC-CMs up and down several times using a 5 mL pipette to generate a single-cell suspension. Add an equal volume of hiPSC-CMs seeding medium to neutralize the cell detachment solution.
    5. Centrifuge hiPSC-CMs at 300 x g for 3 min at RT. Aspirate the supernatant and resuspend the cell pellet in 1-2 mL of hiPSC-CM seeding medium.
    6. Quantify the cell density and viability using a trypan blue exclusion method. Briefly, mix 10 µL of cell suspension with an equal volume of trypan blue, transfer to a cell counting slide, and then count using a cell counter, as previously described21.
  3. Seeding hiPSC-CMs
    1. Remove the extracellular matrix coating solution from the 96-well plates.
    2. Seed 50,000 cells per well in 100 µL of hiPSC-CM seeding medium and place the 96-well plates in a humidified cell culture incubator at 37 °C, 5% CO2 overnight.
    3. Replace the hiPSC-CM seeding medium with 100 µL of hiPSC-CM maintenance medium 1 day after plating. Change the hiPSC-CM maintenance medium every 2 days until recording.
  4. Data acquisition
    1. Measure the hiPSC-CM contraction motion at least 10 days post-plating (recommended).
    2. Turn on the temperature controller and set 37 °C as the preferred temperature. Turn on the microscope, camera, and CO2 supply.
    3. Fill the water jacket of the plate holder with an appropriate amount of sterile deionized water (Figure 2A). Place the 96-well plate on the plate holder and allow cells to acclimate for at least 5 min.
    4. Open the view software, set the camera frame rate to 75 frames per second (fps) and the video/image resolution to 1024 × 1024 pixels. Apply the auto-focus and auto-brightness functions. Record videos and images of hiPSC-CMs.
      NOTE: Avoid vibration of the microscope table during recording.
    5. Open the analyzer software for automatic analysis.

3. Measurement of hiPSC-CM field potential

  1. Preparation of basement membrane matrix-coated plates
    1. Carefully place an 8 µL droplet of extracellular matrix coating solution over the recording electrode area of each well of the 48-well microelectrode array (MEA) plates. See Figure 3A for the appropriate electrode area for droplet placement. This step is critical to keeping the hiPSC-CM monolayer concentrated on the electrodes. Avoid touching the electrodes in any circumstances.
    2. Add 3 mL of sterile deionized water to the area surrounding the wells (Figure 3A) of the 48-well MEA plates to prevent coating solution evaporation. Incubate 48-well MEA plates in a humidified cell culture incubator at 37 °C, 5% CO2 for 45-60 min.
      NOTE: Do not incubate for too long to prevent the basement membrane matrix from drying.
  2. Perform enzymatic dissociation of hiPSC-CMs as described in step 2.2.
  3. Seeding hiPSC-CMs
    1. Remove most of the extracellular matrix medium from the well surface without touching the electrodes using a 200 µL pipette tip within the same single row or column.
    2. Seed 50,000 cells per well in an 8 µL droplet of hiPSC-CM seeding medium over the recording electrode area of each well of the same row or column.
    3. Repeat the last two steps until all wells have been plated with cells. Check under the microscope that all wells have the hiPSC-CM suspension. For the desired density, see Figure 3B.
      NOTE: Attachment of hiPSC-CMs will be compromised if the basement membrane matrix is completely dried out, making cell attachment suboptimal.
    4. Incubate the 48-well MEA plates in a humidified cell culture incubator at 37 °C, 5% CO2 for 60 min.
    5. Slowly and gently add 150 µL of hiPSC-CM seeding medium to each well of the 48-well MEA plates.To add medium without dispersing previously seeded hiPSC-CMs, hold the plate at a 45° angle, lean the pipette tip against the wall of each well, and release the medium slowly.
      NOTE: Adding medium too quickly or with high pressure will dislodge the adhered cardiomyocytes. Avoid direct contact with the hiPSC-CM drop suspension.
    6. Incubate the 48-well MEA plates in a humidified cell culture incubator at 37 °C, 5% CO2 overnight.
    7. Refresh the hiPSC-CM seeding medium with 300 µL of hiPSC-CM maintenance medium 1 day after plating. Change the hiPSC-CM maintenance medium every 2 days before recording.
  4. Data acquisition
    1. Perform MEA measurement at least 10 days post-plating (recommended). On day 10 post-plating, check the density of spontaneous beating hiPSC-CM monolayer under a microscope at 10x magnification, which should be as shown in Figure 3C.
    2. Place the 48-well MEA plate in the recording instrument. The touch screen allows to observe the temperature (37 °C) and CO2 (5%) status.
    3. Open the navigator software. In the experimental setup window, select Cardiac Real-time Configuration and Field Potential Recordings. Apply spontaneous or paced beating configurations.
    4. In beat detection parameters, set detection threshold to 300 µV, minimum beating period to 250 ms, and maximum beating period to 5 s. Select Polynomial Regression for field potential duration (FPD) calculation.
    5. Check whether the baseline electrical activity signal is mature and stable.
      NOTE: The mature waveforms recorded by each electrode from the multi-well MEA plate must reflect the cardiac field potential with easily identifiable characteristics coinciding with the cardiac depolarization spike and repolarization phase, as shown in Figure 3D-F.
    6. Acquire the baseline cardiac activities for 1-3 min. If drug treatment is needed, add compounds to the wells after baseline recording. Place the plate in an incubator for at least 30 min before the next recording.

4. Measurement of hiPSC-CM action potential

  1. Prepare basement membrane matrix-coated dishes by placing 500 µL of extracellular matrix coating solution on the glass-bottom part of 35 mm dishes (Figure 4A). Incubate the 35 mm dishes in a humidified cell culture incubator at 37 °C, 5% CO2 overnight.
  2. Perform enzymatic dissociation of hiPSC-CMs as described in step 2.2.
  3. Seeding hiPSC-CMs
    1. Remove the extracellular matrix coating solution from the 35 mm dishes. Seed 50,000 cells per well in 500 µL of hiPSC-CM seeding medium on the glass bottom part of 35 mm dishes.
    2. Incubate the 35 mm dishes in a humidified cell culture incubator at 37 °C, 5% CO2 overnight. Remove the hiPSC-CM seeding medium and add 2 mL of hiPSC-CM maintenance medium to the 35 mm dishes.
      NOTE: It is recommended to put a 200 µL non-filtered tip over the 2 mL aspiration pipette to remove the spent medium to avoid touching cells.
    3. Change the hiPSC-CM culture medium every 2 days before recording.
  4. Data acquisition
    1. Perform action potential measurement at least 10 days post-plating (recommended). Prepare fresh Tyrode's solution as described in step 1.4. Thaw one aliquot of intracellular pipette solution and freshly add MgATP to a final concentration of 3 mM.
    2. Pull the micropipettes from borosilicate glass capillaries using a micropipette puller.
      NOTE: A resistance of 2-5 MΩ of the micropipettes is preferred.
    3. Replace the hiPSC-CM maintenance medium with Tyrode's solution and allow the cells to acclimate to Tyrode's solution for 15 min (Figure 4C). Insert the temperature sensors in the chamber, put the 35 mm dish in the chamber as shown in Figure 4C, and adjust the temperature to 37 °C (Figure 4D).
    4. Fill the micropipettes with intracellular solution and insert them into the holder connected to the patch-clamp amplifier headstage (Figure 4E). Open the stimulation/acquisition software, load the action potential recording protocol, and select the voltage-clamp configuration.
    5. Insert the micropipette into the bath solution, select appropriate single hiPSC-CMs, and adjust and lower the position of the micropipette next to the selected cell using a micromanipulator. When the pipette tip is inserted into the bath solution, check for pipette resistance that can be observed on the oscilloscope monitor.
    6. Position the pipette close to the cell, and the current pulses will decrease slightly to reflect the increasing seal resistance. Apply gentle suction with negative pressure to increase the resistance, which will form a gigaseal. Apply the Seal function.
    7. Apply additional suction to break the membrane to get a whole-cell recording configuration. Once the membrane portion within the pipette area is ruptured by suction, the internal solution is in equilibrium with the cell cytoplasm. At this point, switch from voltage-clamp mode (V-clamp) to current-clamp mode or no current injection (C-Clamp) using the amplifier dialog. Press the Record button to generate and save the action potential recording files.
      NOTE: The mode change provides a continuous recording of the spontaneous action potentials of hiPSC-CMs via no trigger and no stimulation recording protocol, which can be monitored at the V-mon output of the patch-clamp amplifier.

5. Measurement of hiPSC-CM Ca2+ transient

  1. Refer to the previous protocol for preparing customized cell chambers for Ca2+ imaging21. For the preparation of the basement membrane matrix-coated cell chamber, place 200 µL of extracellular matrix coating solution in the cell chambers. Incubate the cell chambers in a humidified cell culture incubator at 37 °C, 5% CO2 overnight.
  2. Perform enzymatic dissociation of hiPSC-CMs as described in step 2.2.
  3. Seeding hiPSC-CMs
    1. Remove the extracellular matrix coating solution from the cell chambers. Seed 20,000 cells per well in 200 µL of hiPSC-CM seeding medium.
    2. Replace the hiPSC-CM seeding medium with 200 µL of hiPSC-CM maintenance medium 1 day after plating. Change the hiPSC-CM maintenance medium every 2 days before recording.
  4. Data acquisition
    1. Perform Ca2+ transient measurement at least 10 days post-plating (recommended). Prepare fresh Tyrode's solution as described in step 1.4. Prepare fresh Fura-2 AM loading solution as described in step 1.6.
    2. Aspirate the hiPSC-CM maintenance medium and wash with Tyrode's solution twice. Apply 100 µL of Fura-2 AM loading solution and incubate for 10 min at RT.
    3. Replace the Fura-2 AM loading solution with Tyrode's solution and allow at least 5 min for complete de-esterification of Fura-2 AM.
    4. Add a drop of immersion oil on the 40x objective of an inverted epifluorescence microscope equipped with a 40x oil immersion objective (0.95 NA). Place the cell chamber in the stage adapter of the microscope (Figure 5A).
    5. Install the temperature controller wires to the bath chamber and set it to 37 °C. Install the electrical field stimulation electrodes and set the pulses at 0.5 Hz with a 20 ms duration.
    6. Turn on the ultra-high-speed wavelength-switching light source and set it to 340 nm and 380 nm fast-switching mode according to the instrument's instructions.
    7. Apply an exposure time of 20 ms for both wavelengths and a recording time of 20 s in the software. Record the video reflecting real-time changes of Ca2+ transient in hiPSC-CMs. Export the data to a spreadsheet and analyze the data as previously described23.

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Representative Results

This protocol describes how to measure the contraction motion, field potential, action potential, and Ca2+ transient of hiPSC-CMs. A schematic diagram including the enzymatic digestion, cell seeding, maintenance, and functional assay conduction is shown in Figure 1. The formation of the hiPSC-CM monolayer is necessary for the contraction motion measurement (Figure 2B). A representative trace of the contraction-relaxation motion of hiPSC-CMs is shown in Figure 2C. The analyzer software enables the analysis of contraction-relaxation motion traces in an automated fashion by detecting the contraction start, contraction peak, contraction end, relaxation peak, and relaxation end using the default setting (Figure 2D). The velocities of contraction and relaxation peaks are used to assess the contraction and relaxation capacities of hiPSC-CMs. In addition, contraction and relaxation deformation distances can also be obtained.

MEA assay is the extracellular recording of action potentials, known as field potentials. Successful measurement of field potentials requires full coverage of the hiPSC-CM monolayer on all the electrodes within each well of the MEA plates (Figure 3C). The mature waveforms recorded by each electrode must reflect the cardiac field potential with easily identifiable characteristics, as shown in Figure 3D-F. The specified quality standards are 1) the baseline activity should show a spontaneous beating within 20-90 beats per min, 2) the beating rate should be within 6 SDs calculated for the baseline beating rate on all of the wells, 3) the coefficient of variation of the beat period should be less than 5%, and 4) the depolarization amplitude should be over 0.3 mV, as previously published22. Figure 3E shows representative field potential traces of hiPSC-CMs. The navigator software detects and identifies beats and FPD automatically. After analysis, beat period, FPD, and spike amplitude can be obtained (Figure 3F). Specifically, the beat period is determined by the depolarizing peak-to-peak interval, FPD is determined by the interval between depolarizing peak to repolarization peak, and spike amplitude is measured by the distance between the depolarizing positive peak to the negative peak, as shown in Figure 3F.

Single-cell patch clamp is the gold standard for assessing the electrophysiological properties of hiPSC-CMs (Figure 4B). Whole-cell patch-clamp recordings in current-clamping mode can record the spontaneous action potentials of single hiPSC-CMs (Figure 4F). After analysis, several parameters, including the amplitude, max diastolic potential (MDP), and action potential duration (APD) at 30%, 50%, and 90% repolarization can be obtained (Figure 4G).

For Ca2+ imaging, fluorescence intensity changes of individual hiPSC-CMs over time can be recorded by the software. A representative area of Fura-2-loaded hiPSC-CMs is shown in Figure 5B. The usage of an ultra-high-speed wavelength-switching light source allows for the recording of Ca2+ transients from hundreds of hiPSC-CMs via frame mode scanning. After the analysis by a spreadsheet-based program23, the F340/F380 ratio over time for each cell can be obtained. Then, raw traces of stimulated Ca2+ transients can be plotted (Figure 5C). In addition, parameters such as amplitude, diastolic Ca2+, and Ca2+ decay tau can be obtained (Figure 5D).

Figure 1
Figure 1. Overall schematic diagram of the protocol. On day 0, digest and seed hiPSC-CMs on basement membrane matrix-coated 96-well plates/48-well MEA plates/35 mm dishes/cell chambers. Allow a recovery period of at least 10 days before performing the functional assays using a cell motion imaging system, MEA, patch-clamp, and Ca2+ imaging system. Abbreviations: MEA = microelectrode array. Please click here to view a larger version of this figure.

Figure 2
Figure 2. Measurement of contraction motion. (A) The cell motion imaging system. (B) A representative region of the hiPSC-CM monolayer. Scale bar: 20 µm. (C) Representative contraction-relaxation traces of hiPSC-CMs. (D) Schematic diagram of a contraction-relaxation trace showing the contraction start, contraction peak, contraction end, relaxation peak, and relaxation end. Please click here to view a larger version of this figure.

Figure 3
Figure 3. Measurement of field potential. (A) Top views of (left) a 48-well MEA plate, (middle) one well with 16 electrodes, and (right) the area that a basement membrane matrix-droplet needs to cover. Add sufficient distilled water to the water reservoir to make up for evaporation. (B) hiPSC-CM suspension and the desired density after replating. (C) A representative region of the hiPSC-CM monolayer. (D) Continuous waveform plots from the 16 electrodes in one well of the 48-well MEA plate. (E) Representative field potential traces of hiPSC-CMs. (F) Schematic diagram of a field potential trace showing how the parameters were analyzed. Abbreviation: FPD = field potential duration. Please click here to view a larger version of this figure.

Figure 4
Figure 4. Measurement of action potential. (A) Basement membrane matrix-coating and hiPSC-CMs suspension placement on 35 mm dishes. (B) A representative region of single hiPSC-CMs. Scale bar: 20 µm. (C-E) Patch-clamp system setup. (F) Representative action potential traces of hiPSC-CMs. (G) Schematic diagram of an action potential trace showing how the parameters were analyzed. Abbreviations: APD = action potential duration, MDP = maximum diastolic potential. Please click here to view a larger version of this figure.

Figure 5
Figure 5. Measurement of Ca2+ transient. (A) The Ca2+ imaging system. (B) A representative region of Fura-2-loaded single hiPSC-CMs. Scale bar: 100 µm. (C) Representative Ca2+ transient traces of hiPSC-CMs. (D) Schematic diagram of a Ca2+ transient trace showing how the parameters were analyzed. Please click here to view a larger version of this figure.

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Discussion

Human iPSC technology has emerged as a powerful platform for disease modeling and drug screening. Here, we describe a detailed protocol for measuring hiPSC-CM contractility, field potential, action potential, and Ca2+ transient. This protocol provides a comprehensive characterization of hiPSC-CM contractility and electrophysiology. These functional assays have been applied in multiple publications from our group12,13,18,24,25,26,27.

It is essential to use the hiPSC-CMs that are in good beating conditions, otherwise, the signal-to-noise ratio of the readouts will drop dramatically. In addition, it is desirable to ensure the high purity of hiPSC-CMs before the measurements. The existence of non-cardiomyocytes could particularly compromise data accuracy of contraction motion and field potential measurements, both of which require the formation of hiPSC-CM monolayer. Furthermore, the immaturity of hiPSC-CMs remains a major concern that hinders the full application of hiPSC-CMs in various fields28. It is known that hiPSC-CMs are functionally immature in multiple aspects, including morphology, contractility, electrophysiology, Ca2+ handling, and metabolism29. Therefore, it is recommended to keep the hiPSC-CMs in the culture at least until day 30 before performing functional assays. Alternatively, various strategies that have been established to improve the maturity of hiPSC-CMs can be adopted before conducting the functional assays30. For example, the hiPSC-CMs cultured in metabolic maturation media exhibited highly negative MDP of action potentials and significantly faster decay of Ca2+ transients when compared with the hiPSC-CMs cultured in normal RPMI media31. Finally, there are batch-to-batch variations of hiPSC-CMs32. Thus, data should be collected on hiPSC-CMs generated from at least three independent differentiations of the same iPSC lines.

The cell motion imaging system provides a high throughput and efficient platform to measure the contraction and relaxation motion of hiPSC-CMs. However, a major disadvantage is that it does not measure the direct contractile force but rather the contraction and relaxation velocity instead. In addition, the velocities vary when the number of hiPSC-CMs to form a monolayer is different. Therefore, it is crucial to seed precisely 50,000 cells in each well of the 96-well plates.

FPD measured by MEA has been used as a surrogate for QT duration and thus is widely used in assessing drug-induced QT prolongation33. Similar to QT duration, FPD is beating rate dependent. Therefore, the correction in beating rate is necessary to precisely interpret the FPD data. In this protocol, it is recommended that MEA measurement is performed at least 10 days post-plating. However, sometimes it is possible that the field potential signal is still unstable after 10 days post-plating. If this happens, prolong the hiPSC-CM culture for another 2-3 days.

The patch-clamp technique is a versatile tool for assessing the electrophysiology of hiPSC-CMs and assaying ion channels. The major disadvantage of the conventional patch-clamp is its low throughput property, which hinders its application in high throughput studies. In addition, it requires a long learning curve and extensive practice before one can become experienced in this technique. However, the conventional patch-clamp technique is still considered the gold standard to understand the electrophysiology of hiPSC-CMs. If the APD of hiPSC-CMs needs to be evaluated in a high throughput manner, it is also suggested to start with optical imaging of hiPSC-CM action potential using the accelerated sensor of action potentials 2 (ASAP2)21. After primary targets are obtained, a conventional action potential can be used in further studies for more accurate phenotyping and validation. In addition, automated patch clamp has been increasingly employed in various fields, especially channelopathy. For instance, the ion channel currents carried by multiple variants of hERG channels and voltage-gated sodium channels have been characterized by automated patch clamp34,35,36,37. Moreover, the usage of automated patch clamp in hiPSC-CMs has been established recently38,39. For the execution of automated patch clamp, readers can refer to previously published protocols40. With the advantages of being high throughput and the simplicity in machine handling, there is no doubt that automated patch clamps will play more and more important roles in drug discovery and drug development.

The usage of a ratiometric Ca2+-sensitive dye, Fura-2, allows the measurement of diastolic Ca2+ of hiPSC-CMs to be much less affected by the experimental bias factors such as dye loading time, uneven dye distribution, and photo-bleaching. This is important to recapitulate the diastolic dysfunction of particular heart diseases. The main limitation is that it requires a UV excitation light source, which may be incompatible with confocal microscopy.

In summary, despite recent progress in cardiotoxicity assessment in the preclinical phase of drug development, cardiotoxicity remains one of the leading safety concerns. The growing incorporation of hiPSC-CMs into the standard preclinical safety evaluation process has the potential to improve the accuracy of toxicity prediction of candidate compounds. In addition, patient-specific hiPSC-CMs enable personalized cardiac drug selection and adverse drug response prediction. The protocol using various functional assays described here will help expand the applications of hiPSC-CMs in drug screening and disease modeling.

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Disclosures

J.C.W. is a co-founder of Greenstone Biosciences but has no competing interests, as the work presented here is completely independent. The other authors declare no competing interests.

Acknowledgments

We thank Blake Wu for proofreading the manuscript. This work was supported by the National Institutes of Health (NIH) R01 HL113006, R01 HL141371, R01 HL163680, R01 HL141851, U01FD005978, and NASA NNX16A069A (JCW), and AHA Postdoctoral Fellowship 872244 (GMP).

Materials

Name Company Catalog Number Comments
35 mm glass bottom dish with 20 mm micro-well #1.5 cover glass Cellvis D35-20-1.5-N Patch clamp
50x B27 supplements Life Technologies 17504-044 hiPSC-CM culture medium
6-well culture plate E & K Scientific EK-27160 hiPSC-CM culture
96-well flat clear bottom black polystyrene TC-treated microplates Corning 3603 Contraction motion measurement
Accutase Sigma-Aldrich A6964 Enzymatic dissociation
Axion's Integrated Studio (AxIS) Axion Biosystems navigator software
Borosilicate glass capillaries Harvard Apparatus BF 100-50-10, Patch clamp
CaCl2 1 M in H2O Sigma-Aldrich 21115 Tyrode’s solution
Cell counting chamber slides ThermoFisher Scientific C10228 Cell counting
CytoView 48-well MEA plates Axion Biosystems M768-tMEA-48B MEA
DMEM/F12 Gibco/Life Technologies 12634028 Extracellular matrix medium
DPBS, no calcium, no magnesium Fisher Scientific 14-190-250
EGTA Sigma-Aldrich E3889 Intracellular pipette solution
EPC 10 USB patch clamp amplifier Warner Instruments 89-5000 Patch clamp
Fura-2, AM, cell permeant ThermoFisher Scientific F1221 Ca2+ transient measurement
Glucose Sigma-Aldrich G8270 Tyrode’s solution
HEPES Sigma-Aldrich H3375 Tyrode’s solution
hiPSCs Stanford Cardiovascular Institute iPSC Biobank
KCl Sigma-Aldrich 529552 Tyrode’s solution
KnockOut Serum Replacement ThermoFisher Scientific 10828-028 hiPSC-CM seeding medium
KOH 8 M Sigma-Aldrich P4494 Intracellular pipette solution
Lambda DG 4 Sutter Instrument Company Ca2+ transient measurement; ultra-high-speed wavelength switching light source
Luna-FL automated fluorescence cell counter WISBIOMED LB-L20001 Cell counting
Maestro Pro MEA system Axion Biosystems MEA
Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix Corning 356231 Extracellular matrix medium
MgATP Sigma-Aldrich A9187 Intracellular pipette solution
MgCl2 Sigma-Aldrich M8266 Tyrode’s solution
NaCl Sigma-Aldrich S9888 Tyrode’s solution
NaOH 10 M Sigma-Aldrich 72068 Tyrode’s solution
NIS Elements AR
Pluronic F-127 (20% Solution in DMSO) ThermoFisher Scientific P3000MP Ca2+ transient measurement
RPMI 1640 medium Life Technologies 11875-119 hiPSC-CM culture medium
Sony SI8000 Cell Motion Imaging System Sony Biotechnology Contraction motion measurement
Sutter Micropipette puller Sutter Instruments P-97 Patch clamp
Trypan blue stain Life Technologies T10282 Cell counting

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References

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Microelectrode Array Patch Clamp Recordings Human Induced Pluripotent Stem Cells Cardiomyocytes Cardiac Safety Assessment Drug Development Disease Modeling Drug-induced Cardiotoxicity Screening Precision Medicine Toxicity Prediction Culturing Temperature Controller Microscope Camera Carbon Dioxide Supply Water Jacket 96-well Plate Incubate Cells View Software Camera Frame Rate Video/image Resolution
Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes
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Zhao, S. R.,… More

Zhao, S. R., Mondéjar-Parreño, G., Li, D., Shen, M., Wu, J. C. Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes. J. Vis. Exp. (186), e64265, doi:10.3791/64265 (2022).

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