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).

1. Preparation of media and solutions
<ol>
	<li>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 &#176;C and use it within a month. Equilibrate the medium to room temperature (RT) before use.</li>
	<li>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 &#176;C for...

<ol>
	<li>Wouters, O. J., McKee, M., Luyten, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimated+research+and+development+investment+needed+to+bring+a+new+medicine+to+market,+2009-2018.">Estimated research and development investment needed to bring a new medicine to market, 2009-2018.</a> Journal of the American Medical Association. 323 (9), 844-853 (2020).</li><li>Pang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Workshop+report:+FDA+workshop+on+improving+cardiotoxicity+assessment+with+human-relevant+platforms.">Workshop report: FDA workshop on improving cardiotoxicity assessment with human-relevant platforms.</a> Circulation Research. 125 (9), 855-867 (2019).</li><li>Mamoshina, P., Rodriguez, B., Bueno-Orovio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+broader+view+of+mechanisms+of+drug+cardiotoxicity.">Toward a broader view of mechanisms of drug cardiotoxicity.</a> Cell Reports Medicine. 2 (3), 100216 (2021).</li><li>Paik, D. 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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+PDGFRA+signaling+contributes+to+filamin+C-related+arrhythmogenic+cardiomyopathy.">Activation of PDGFRA signaling contributes to filamin C-related arrhythmogenic cardiomyopathy.</a> Science Advances. 8 (8), (2022).</li><li>Sharma, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+of+tyrosine+kinase+inhibitor+cardiotoxicity+with+human+induced+pluripotent+stem+cells.">High-throughput screening of tyrosine kinase inhibitor cardiotoxicity with human induced pluripotent stem cells.</a> Science Translational Medicine. 9 (377), (2017).</li><li>Burridge, P. 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Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+to+measure+contraction,+calcium,+and+action+potential+in+human-induced+pluripotent+stem+cell-derived+cardiomyocytes.">Protocol to measure contraction, calcium, and action potential in human-induced pluripotent stem cell-derived cardiomyocytes.</a> STAR Protocols. 2 (4), 100859 (2021).</li><li>Blinova, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+multisite+study+of+human-induced+pluripotent+stem+cell-derived+cardiomyocytes+for+drug+proarrhythmic+potential+assessment.">International multisite study of human-induced pluripotent stem cell-derived cardiomyocytes for drug proarrhythmic potential assessment.</a> Cell Reports. 24 (13), 3582-3592 (2018).</li><li>Greensmith, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca+analysis:+an+Excel+based+program+for+the+analysis+of+intracellular+calcium+transients+including+multiple,+simultaneous+regression+analysis.">Ca analysis: an Excel based program for the analysis of intracellular calcium transients including multiple, simultaneous regression analysis.</a> Computer Methods and Programs in Biomedicine. 113 (1), 241-250 (2014).</li><li>Ma, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+the+pathogenicity+of+a+genomic+variant+of+uncertain+significance+using+CRISPR/Cas9+and+human-induced+pluripotent+stem+cells.">Determining the pathogenicity of a genomic variant of uncertain significance using CRISPR/Cas9 and human-induced pluripotent stem cells.</a> Circulation. 138 (23), 2666-2681 (2018).</li><li>Lam, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+the+transcriptome+signatures+of+calcium+channel+blockers+in+human+induced+pluripotent+stem+cell-derived+cardiomyocytes.">Identifying the transcriptome signatures of calcium channel blockers in human induced pluripotent stem cell-derived cardiomyocytes.</a> Circulation Research. 125 (2), 212-222 (2019).</li><li>Seeger, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+premature+termination+codon+mutation+in+MYBPC3+causes+hypertrophic+cardiomyopathy+via+chronic+activation+of+nonsense-mediated+decay.">A premature termination codon mutation in MYBPC3 causes hypertrophic cardiomyopathy via chronic activation of nonsense-mediated decay.</a> Circulation. 139 (6), 799-811 (2019).</li><li>Zhang, J. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cryopreservation+on+human+induced+pluripotent+stem+cell-derived+cardiomyocytes+for+assessing+drug+safety+response+profiles.">Effects of cryopreservation on human induced pluripotent stem cell-derived cardiomyocytes for assessing drug safety response profiles.</a> Stem Cell Reports. 16 (1), 168-181 (2021).</li><li>Yu, B., Zhao, S. R., Yan, C. D., Zhang, M., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deconvoluting+the+cells+of+the+human+heart+with+iPSC+technology:+cell+types,+protocols,+and+uses.">Deconvoluting the cells of the human heart with iPSC technology: cell types, protocols, and uses.</a> Current Cardiology Reports. 24 (5), 487-496 (2022).</li><li>Thomas, D., Cunningham, N. J., Shenoy, S., Wu, J. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+maturation+media+improve+physiological+function+of+human+iPSC-derived+cardiomyocytes.">Metabolic maturation media improve physiological function of human iPSC-derived cardiomyocytes.</a> Cell Reports. 32 (3), 107925 (2020).</li><li>Gintant, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+human+induced+pluripotent+stem+cell-derived+cardiomyocytes+in+preclinical+cancer+drug+cardiotoxicity+testing:+a+scientific+statement+from+the+American+Heart+Association.">Use of human induced pluripotent stem cell-derived cardiomyocytes in preclinical cancer drug cardiotoxicity testing: a scientific statement from the American Heart Association.</a> Circulation Research. 125 (10), 75-92 (2019).</li><li>Kussauer, S., David, R., Lemcke, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hiPSCs+Derived+cardiac+cells+for+drug+and+toxicity+screening+and+disease+modeling:+what+micro-+electrode-array+analyses+can+tell+us.">hiPSCs Derived cardiac cells for drug and toxicity screening and disease modeling: what micro- electrode-array analyses can tell us.</a> Cells. 8 (11), (2019).</li><li>Ng, C. 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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</su...

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).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 mm glass bottom dish with 20 mm micro-well #1.5 cover glass</td>
			<td>Cellvis</td>
			<td>D35-20-1.5-N</td>
			<td>Patch clamp</td>
		</tr>
		<tr>
			<td>50x B27 supplements</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td>hiPSC-CM culture medium</td>
		</tr>
		<tr>
			<td>6-well culture plate</td>
			<td>E &#38; K Scientific</td>
			<td>EK-27160</td>
			<td>hiPSC-CM culture</td>
		</tr>
		<tr>
			<td>96-well flat clear bottom black polystyrene TC-treated microplates</td>
			<td>Corning</td>
			<td>3603</td>
			<td>Contraction motion measurement</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Sigma-Aldrich</td>
			<td>A6964</td>
			<td>Enzymatic dissociation</td>
		</tr>
		<tr>
			<td>Axion&#39;s Integrated Studio (AxIS)</td>
			<td>Axion Biosystems</td>
			<td></td>
			<td>navigator software</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>Harvard Apparatus</td>
			<td>BF 100-50-10,</td>
			<td>Patch clamp</td>
		</tr>
		<tr>
			<td>CaCl2 1 M in H2O</td>
			<td>Sigma-Aldrich</td>
			<td>21115</td>
			<td>Tyrode&#8217;s solution</td>
		</tr>
		<tr>
			<td>Cell counting chamber slides</td>
			<td>ThermoFisher Scientific</td>
			<td>C10228</td>
			<td>Cell counting</td>
		</tr>
		<tr>
			<td>CytoView 48-well MEA plates</td>
			<td>Axion Biosystems</td>
			<td>M768-tMEA-48B</td>
			<td>MEA</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco/Life Technologies</td>
			<td>12634028</td>
			<td>Extracellular matrix medium</td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Fisher Scientific</td>
			<td>14-190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td>Intracellular pipette solution</td>
		</tr>
		<tr>
			<td>EPC 10 USB&#160;patch clamp amplifier</td>
			<td>Warner Instruments</td>
			<td>89-5000</td>
			<td>Patch clamp</td>
		</tr>
		<tr>
			<td>Fura-2, AM, cell permeant</td>
			<td>ThermoFisher Scientific</td>
			<td>F1221</td>
			<td>Ca2+ transient measurement</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td>Tyrode&#8217;s solution</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>Tyrode&#8217;s solution</td>
		</tr>
		<tr>
			<td>hiPSCs</td>
			<td>Stanford Cardiovascular Institute iPSC Biobank</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>529552</td>
			<td>Tyrode&#8217;s solution</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>ThermoFisher Scientific</td>
			<td>10828-028</td>
			<td>hiPSC-CM seeding medium</td>
		</tr>
		<tr>
			<td>KOH 8&#160;M</td>
			<td>Sigma-Aldrich</td>
			<td>P4494</td>
			<td>Intracellular pipette solution</td>
		</tr>
		<tr>
			<td>Lambda DG 4</td>
			<td>Sutter Instrument Company</td>
			<td></td>
			<td>Ca2+ transient measurement; ultra-high-speed wavelength switching light source</td>
		</tr>
		<tr>
			<td>Luna-FL automated fluorescence cell counter</td>
			<td>WISBIOMED</td>
			<td>LB-L20001</td>
			<td>Cell counting</td>
		</tr>
		<tr>
			<td>Maestro Pro&#160;MEA system</td>
			<td>Axion Biosystems</td>
			<td></td>
			<td>MEA</td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Extracellular matrix medium</td>
		</tr>
		<tr>
			<td>MgATP</td>
			<td>Sigma-Aldrich</td>
			<td>A9187</td>
			<td>Intracellular pipette solution</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td>Tyrode&#8217;s solution</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td>Tyrode&#8217;s solution</td>
		</tr>
		<tr>
			<td>NaOH 10&#160;M</td>
			<td>Sigma-Aldrich</td>
			<td>72068</td>
			<td>Tyrode&#8217;s solution</td>
		</tr>
		<tr>
			<td>NIS Elements AR</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127 (20% Solution in DMSO)</td>
			<td>ThermoFisher Scientific</td>
			<td>P3000MP</td>
			<td>Ca2+ transient measurement</td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Life Technologies</td>
			<td>11875-119</td>
			<td>hiPSC-CM culture medium</td>
		</tr>
		<tr>
			<td>Sony SI8000 Cell Motion Imaging System</td>
			<td>Sony Biotechnology</td>
			<td></td>
			<td>Contraction motion measurement</td>
		</tr>
		<tr>
			<td>Sutter Micropipette puller</td>
			<td>Sutter Instruments</td>
			<td>P-97</td>
			<td>Patch clamp</td>
		</tr>
		<tr>
			<td>Trypan blue stain</td>
			<td>Life Technologies</td>
			<td>T10282</td>
			<td>Cell counting</td>
		</tr>
	</tbody>
</table>

technical applications of microelectrode array and patch clamp recordings on human induced pluripotent stem cell-derived cardiomyocytes

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.

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...

Watch this Scientific Journal Video about Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes at JoVE.com

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

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.

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety ...

Cardiac safety assessment is a crucial component during the drug development process. Human iPSC derived cardiomyocytes have proven to be a reliable, efficient, and human based model for pre-clinical cardiac safety assessment. We have established methodologies to comprehensively investigate the functional characteristic of human iPSC derived cardiomyocytes, which are of really important disease modeling, drug induced cardio toxicity screening and precision medicine.The growing incorporation of human iPSC derived cardiomyocytes into the standard pre-clinical safety evaluations process has the potential to improve the accuracy of toxicity prediction of a candidate's compounds. Begin by culturing the human iPSC derived cardiomyocytes for at least 10 days before taking the measurement. Turn on the temperature controller and set it at 37 degrees celsius.Turn on the microscope, camera, and carbon dioxide supply. Fill the water jacket of the plate holder with an appropriate amount of sterile deionized water. Place the 96 well plate on the plate holder, and incubate the cells for at least five minutes.Open the view software, then set the camera frame rate to 75 frames per second, and the video or image resolution to 1024 x 1024 pixels. Apply the auto focus and auto brightness functions, record videos and images of human iPSC derived cardiomyocytes. Carefully place an eight microliter droplet of extra cellular matrix coating solution over the recording electrode area of each well of the 48 well microelectrode array plates.Then add three milliliters of sterile deionized water to the area surrounding the wells to prevent coating solution evaporation. Using a 200 microliter pipette tip, remove most of the extra cellular matrix medium from the well surface within the same single row or column without touching the electrodes. Seed 50, 000 cells per well in an eight microliter droplet of seeding medium over the recording electrode area.Repeat until all wells have been plated with cells. Then incubate the microelectrode array plates in a humidified cell culture incubator. Slowly add 150 microliters of seeding medium to each well of the microelectrode array plates.On day 10 post plating, check the density of spontaneous beating human iPSC CM monolayer under a microscope. Place the 48 well microelectrode array plate in the recording instrument. Ensure that the temperature is 37 degrees celsius and carbon dioxide is 5%Open the navigator software.In the experimental setup window, select cardiac real time configuration and field potential recordings. Apply spontaneous or paste beating configurations. In beat detection parameters, set detection threshold to 300 micro volts, minimum beating period to 250 milliseconds, and maximum beating period to five seconds.Select polynomial regression for field potential duration calculation. Check whether the baseline electrical activity signal is mature and stable, and acquire the baseline cardiac activities for one to three minutes. Perform the recording 10 days post plating.Replace the human iPSC CM maintenance medium with tyrode's solution, and allow the cells to acclimate for 15 minutes. Insert the temperature sensors in the chamber, and put the 35 millimeter dish inside. Adjust the temperature to 37 degrees celsius.Pull the micro pipettes from borosilicate glass capillaries using a micro pipette puller. Fill the micro pipettes with the intracellular solution, and insert them into the holder connected to the head stage of the patch clamp amplifier. Open the acquisition software, load the action potential recording protocol, and select the voltage clamp configuration.Insert the micro pipette into the bath solution, and select appropriate single human iPSC CMs. Adjust and lower the position of the micro pipette next to the selected cell using a micromanipulator. When the pipette tip is inserted into the bath solution, check for pipette resistance on the oscilloscope monitor.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. This will form a gigaseal.Then apply the seal function. Apply additional suction to break the membrane to get a whole cell recording configuration. At this point, switch from voltage clamp mode to current clamp mode, or no current injection using the amplifier dialogue.Press the record button to generate and save the action potential recording files. Perform the calcium transient measurement at least 10 days post plating. Prepare fresh tyrode's solution and Fura-2 AM loading solution.Aspirate the human iPSC CM maintenance medium, and wash the chambers with tyrode's solution twice. Apply 100 microliters of Fura-2 AM loading solution and incubate for 10 minutes at room temperature. Replace the Fura-2 AM loading solution with tyrode's solution and incubate for at least five minutes for complete deesterification of Fura-2 AM.Add a drop of immersion oil on the 40x oil immersion objective of an inverted epifluorescence microscope.Place the cell chamber in the stage adaptor of the microscope. Install the temperature controller wires to the bath chamber, and set it to 37 degrees celsius. Install the electrical field stimulation electrodes, and set the pulses at 0.5 hertz with a 20 millisecond duration.Turn on the ultra high speed wavelength switching light source, and set it to 340 nanometers and 380 nanometers fast switching mode. Apply an exposure time of 20 milliseconds for both wavelengths, and a recording time of 20 seconds. Record the video reflecting real time changes of calcium transient, export the data to a spreadsheet for analysis.Human iPSC derived cardiomyocytes monolayer was used for the contraction motion measurement. Analysis of a trace of the contraction relaxation motion using the analyzer software detected the contractions start, contraction peak, contraction end, relaxation peak, and relaxation end. Full coverage of the human iPSC derived cardiomyocytes monolayer was observed on all the electrodes within the microelectrode array plates.The mature waveforms recorded by each electrode reflected the cardiac field potential with identifiable characteristics. After analysis, beat period, field potential duration, and spike amplitude were obtained. Whole cell patch clamp recordings in current clamping mode recorded the spontaneous action potentials of single cardiomyocytes.After analysis, several parameters, including the amplitude, max diastolic potential, and action potential duration were obtained. After the analysis of calcium imaging, F340 by F380 ratio over time was obtained, and raw traces of stimulated calcium transients were plotted. In addition, parameters such as amplitude, diastolic calcium, and calcium decay tau were obtained.It is important to use the human iPSC derived cardiomyocytes that are in good beating conditions, as well as to ensure the high purity of the human iPSC derived cardiomyocytes. This protocol includes four specific techniques to study human iPSC derived cardiomyocyte functionality, other configuration, or automatic whether to study cardiac ionic currents that play a crucial role in cardiac

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2.5K vistas.  Stanford University School of Medicine. La evaluación de la seguridad cardíaca es un componente crucial durante el proceso de desarrollo del fármaco. Los cardiomiocitos derivados de iPSC humanos han demostrado ser un modelo confiable, eficiente y basado en humanos para la evaluación preclínica de la seguridad cardíaca. Hemos establecido metodologías para investigar exhaustivamente las características funcionales de los cardiomiocitos humanos derivados de iPSC, que son de modelado de enfermedades realmente importantes, detec...