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

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 species^6,7. 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 established^8,9. 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 cardiomyopathy^11,12, and dilated cardiomyopathy^13,14,15. 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 inhibitors^16,17,18; 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 levels^19,20. 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).

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 publications^8,9. 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 cardiomyocytes^8,9.
   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.

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 i...

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 group^12,13,18

Materials

List of materials used in this article

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