Name: high-throughput cardiotoxicity screening using mature human induced pluripotent stem cell-derived cardiomyocyte monolayers
Uploaded: 2023-03-24T14:30:00
Description: Watch this Scientific Journal Video about High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers at JoVE.com

这项工作得到了NIH拨款HL148068-04和R44ES027703-02（TJH）的支持。

该协议中的hiPSC使用已获得密歇根大学HPSCO委员会（人类多能干细胞监督委员会）的批准。有关材料和设备的列表，请参阅 材料表 。有关培养基及其组成，请参见 表1 。
1. 解冻和接种市售冷冻保存的 hiPSC-CM，以便在诱导成熟的细胞外基质 （MECM） 上成熟
<ol><li>将所有试剂加热至室温，并在心肌细胞接种前用含有钙和镁的Hank平衡盐溶液?...

<ol>
	<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>Ma, J., 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+purity+human-induced+pluripotent+stem+cell-derived+cardiomyocytes:+electrophysiological+properties+of+action+potentials+and+ionic+currents.">High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents.</a> American Journal of Physiology. Heart and Circulatory Physiology. 301 (5), 2006-2017 (2011).</li><li>Lee, P., 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=Simultaneous+voltage+and+calcium+mapping+of+genetically+purified+human+induced+pluripotent+stem+cell-derived+cardiac+myocyte+monolayers.">Simultaneous voltage and calcium mapping of genetically purified human induced pluripotent stem cell-derived cardiac myocyte monolayers.</a> Circulation Research. 110 (12), 1556-1563 (2012).</li><li>Fermini, B., Coyne, S. T., Coyne, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+trials+in+a+dish:+a+perspective+on+the+coming+revolution+in+drug+development.">Clinical trials in a dish: a perspective on the coming revolution in drug development.</a> SLAS Discovery. 23 (8), 765-776 (2018).</li><li>Strauss, D. G., Blinova, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+trials+in+a+dish.">Clinical trials in a dish.</a> Trends in Pharmacological Sciences. 38 (1), 4-7 (2017).</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=Clinical+trial+in+a+dish:+personalized+stem+cell-derived+cardiomyocyte+assay+compared+with+clinical+trial+results+for+two+QT-prolonging+drugs.">Clinical trial in a dish: personalized stem cell-derived cardiomyocyte assay compared with clinical trial results for two QT-prolonging drugs.</a> Clinical and Translational Science. 12 (6), 687-697 (2019).</li><li>Blinova, ., 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=Comprehensive+translational+assessment+of+human-induced+pluripotent+stem+cell+derived+cardiomyocytes+for+evaluating+drug-induced+arrhythmias.">Comprehensive translational assessment of human-induced pluripotent stem cell derived cardiomyocytes for evaluating drug-induced arrhythmias.</a> Toxicological Sciences. 155 (1), 234-247 (2017).</li><li>da Rocha, A. 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=hiPSC-CM+monolayer+maturation+state+determines+drug+responsiveness+in+high+throughput+pro-arrhythmia+screen.">hiPSC-CM monolayer maturation state determines drug responsiveness in high throughput pro-arrhythmia screen.</a> Scientific Reports. 7 (1), 13834 (2017).</li><li>da Rocha, A. M., Creech, J., Thonn, E., Mironov, S., Herron, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+drug-induced+Torsades+de+Pointes+arrhythmia+mechanisms+using+hiPSC-CM+syncytial+monolayers+in+a+high-throughput+screening+voltage+sensitive+dye+assay.">Detection of drug-induced Torsades de Pointes arrhythmia mechanisms using hiPSC-CM syncytial monolayers in a high-throughput screening voltage sensitive dye assay.</a> Toxicological Sciences. 173 (2), 402-415 (2020).</li><li>Knollmann, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell-derived+cardiomyocytes:+boutique+science+or+valuable+arrhythmia+model.">Induced pluripotent stem cell-derived cardiomyocytes: boutique science or valuable arrhythmia model.</a> Circulation Research. 112 (6), 969-976 (2013).</li><li>Lam, C. K., 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=Disease+modelling+and+drug+discovery+for+hypertrophic+cardiomyopathy+using+pluripotent+stem+cells:+how+far+have+we+come.">Disease modelling and drug discovery for hypertrophic cardiomyopathy using pluripotent stem cells: how far have we come.</a> European Heart Journal. 39 (43), 3893-3895 (2018).</li><li>Jiang, Y., Park, P., Hong, S. M., Ban, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+cardiomyocytes+derived+from+human+pluripotent+stem+cells:+current+strategies+and+limitations.">Maturation of cardiomyocytes derived from human pluripotent stem cells: current strategies and limitations.</a> Molecules and Cells. 41 (7), 613-621 (2018).</li><li>Ahmed, R. E., Anzai, T., Chanthra, N., Uosaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+brief+review+of+current+maturation+methods+for+human+induced+pluripotent+stem+cells-derived+cardiomyocytes.">A brief review of current maturation methods for human induced pluripotent stem cells-derived cardiomyocytes.</a> Frontiers in Cell and Developmental Biology. 8, 178 (2020).</li><li>Guo, Y., Pu, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte+maturation:+new+phase+in+development.">Cardiomyocyte maturation: new phase in development.</a> Circulation Research. 126 (8), 1086-1106 (2020).</li><li>Yang, X., Pabon, L., Murry, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+adolescence:maturation+of+human+pluripotent+stem+cell-derived+cardiomyocytes.">Engineering adolescence:maturation of human pluripotent stem cell-derived cardiomyocytes.</a> Circulation Research. 114 (3), 511-523 (2014).</li><li>Karbassi, E., 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=Cardiomyocyte+maturation:+advances+in+knowledge+and+implications+for+regenerative+medicine.">Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine.</a> Nature Reviews Cardiology. 17 (6), 341-359 (2020).</li><li>Nunes, S. S., 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=Biowire:+a+platform+for+maturation+of+human+pluripotent+stem+cell-derived+cardiomyocytes.">Biowire: a platform for maturation of human pluripotent stem cell-derived cardiomyocytes.</a> Nature Methods. 10 (8), 781-787 (2013).</li><li>Herron, T. J., 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=Extracellular+matrix-mediated+maturation+of+human+pluripotent+stem+cell-derived+cardiac+monolayer+structure+and+electrophysiological+function.">Extracellular matrix-mediated maturation of human pluripotent stem cell-derived cardiac monolayer structure and electrophysiological function.</a> Circulation. Arrhythmia and Electrophysiology. 9 (4), 003638 (2016).</li><li>Block, 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=Human+perinatal+stem+cell+derived+extracellular+matrix+enables+rapid+maturation+of+hiPSC-CM+structural+and+functional+phenotypes.">Human perinatal stem cell derived extracellular matrix enables rapid maturation of hiPSC-CM structural and functional phenotypes.</a> Scientific Reports. 10 (1), 19071 (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=Repolarization+studies+using+human+stem+cell-derived+cardiomyocytes:+Validation+studies+and+best+practice+recommendations.">Repolarization studies using human stem cell-derived cardiomyocytes: Validation studies and best practice recommendations.</a> Regulatory Toxicology and Pharmacology. 117, 104756 (2020).</li><li>Cyganek, 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=Deep+phenotyping+of+human+induced+pluripotent+stem+cell-derived+atrial+and+ventricular+cardiomyocytes.">Deep phenotyping of human induced pluripotent stem cell-derived atrial and ventricular cardiomyocytes.</a> JCI Insight. 3 (12), 99941 (2018).</li><li>Tohyama, S., 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=Distinct+metabolic+flow+enables+large-scale+purification+of+mouse+and+human+pluripotent+stem+cell-derived+cardiomyocytes.">Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes.</a> Cell Stem Cell. 12 (1), 127-137 (2013).</li><li>Davis, J., 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=In+vitro+model+of+ischemic+heart+failure+using+human+induced+pluripotent+stem+cell-derived+cardiomyocytes.">In vitro model of ischemic heart failure using human induced pluripotent stem cell-derived cardiomyocytes.</a> JCI Insight. 6 (10), 134368 (2021).</li><li>Diaz, R. J., Wilson, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+ischemic+preconditioning+in+isolated+cardiomyocyte+models.">Studying ischemic preconditioning in isolated cardiomyocyte models.</a> Cardiovascular Research. 70 (2), 286-296 (2006).</li></ol>

有几种不同的方法可以使用hiPSC-CMs进行 体外 心脏毒性筛查。最近一篇关于使用 hiPSC-CMs 的“最佳实践”论文介绍了各种 体外 测定、其主要读数，以及重要的是，每种测定的粒度以量化人类心脏电生理功能20。除了使用穿膜电极外，VSDs还提供了人类心脏电生理功能的最直接测量。 VSD检测读数能够直接可视化和量化关键电生理参数，包括动作电位持续时间、动作电位...

人诱导多能干细胞来源的心肌细胞（hiPSC-CMs）已在国际范围内得到验证，可用于体外心脏毒性筛查1。高纯度的hiPSC-CMs可以产生几乎无限的数量，冷冻保存和解冻。重新镀层后，它们也会复活并开始以让人联想到人类心脏的节奏收缩2，3。值得注意的是，单个hiPSC-CMs相互偶联并形成功能性合胞体，作为单个组织跳动。如今，hiPSCs通常来自患者的血液样本，因此任何人都可以使用体外hiPSC-CM心脏毒性筛查测定4，5来代表。这为进行“培养皿中的临床试验”创造了机会，来自不同人群的重要代表性6.
与现有的动物和动物细胞心脏毒性筛选方法相比，一个关键优势是hiPSC-CMs利用完整的人类基因组，并提供与人类心脏具有遗传相似性的体外系统。这对于药物基因组学和个性化医疗特别有吸引力 - 使用hiPSC-CMs进行药物和其他治疗开发预计将提供更准确，精确和安全的药物处方。事实上，二维（2D）hiPSC-CM单层测定已被证明可以预测药物的心脏毒性，使用一组临床使用的药物，已知有引起心律失常的风险1，7，8，9。尽管hiPSC-CMs具有巨大的潜力，并且有望简化和降低药物开发成本，但人们一直不愿意使用这些新型检测方法10，11，12。
到目前为止，广泛采用和接受hiPSC-CM筛查测定的一个主要限制是它们不成熟的胎儿外观以及它们的功能。hiPSC-CM成熟的关键问题已经在科学文献中进行了回顾和辩论，并进行了恶心13，14，15，16。同样，已经采用了许多方法来促进hiPSC-CM成熟，包括2D单层中的细胞外基质（ECM）操作和3D工程心脏组织（EHT）的开发17，18。目前，人们普遍认为，相对于基于2D单层的方法，使用3D EHT将提供更高的成熟度。然而，与 3D EHT 相比，2D 单层提供了更高的细胞利用效率，并提高了电镀成功率;3D EHT利用更多的细胞，并且通常需要包含可能混淆结果的其他细胞类型。因此，在本文中，重点是使用一种简单的方法来成熟培养为电和机械耦合细胞的2D单层的hiPSC-CMs。
先进的hiPSC-CM成熟可以使用ECM在2D单层中实现。hiPSC-CMs的2D单层可以使用柔软，灵活的聚二甲基硅氧烷盖玻片成熟，该盖玻片涂有由Engelbreth-Holm-Swarm小鼠肉瘤细胞（小鼠ECM）分泌的基底膜基质。2016年，报告显示，在这种软ECM条件下培养的hiPSC-CMs在功能上成熟，显示出接近成人心脏值（~50 cm / s）的动作电位传导速度18。此外，这些成熟的hiPSC-CMs显示出许多其他让人联想到成人心脏的电生理特征，包括超极化静息膜电位和K ir2.1的表达。最近，报告发现了一种人类围产期干细胞衍生的ECM涂层，可促进2D hiPSC-CMs19的结构成熟。在这里，提出了结构成熟的2D hiPSC-CM单层的易于使用的方法，用于高通量电生理筛选。此外，我们还使用电压敏感染料（VSD）和钙敏感探针和蛋白质，提供用于自动采集和分析2D hiPSC-CM单层电生理功能的光学映射仪器的验证。

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	<tbody>
		<tr>
			<td>0.25% Trypsin EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mg/mL BSA (7.5 &#181;mol/L)</td>
			<td>Millipore Sigma</td>
			<td>A3294</td>
			<td></td>
		</tr>
		<tr>
			<td>2.9788 g/500 mL HEPES (25 mmol/L)</td>
			<td>Millipore Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>AdGCaMP6m</td>
			<td>Vector biolabs</td>
			<td>1909</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin human</td>
			<td>Sigma</td>
			<td>A9731-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>alpha actinin antibody</td>
			<td>ThermoFisher</td>
			<td>MA1-22863</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Blebbistatin</td>
			<td>Sigma</td>
			<td>B0560</td>
			<td></td>
		</tr>
		<tr>
			<td>CalBryte 520AM</td>
			<td>AAT Bioquest</td>
			<td>20650</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLvo MatrixPlus 96wp</td>
			<td>StemBiosys</td>
			<td>N/A</td>
			<td>https://www.stembiosys.com/products/cellvo-matrix-plus</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>LC Laboratories</td>
			<td>c-6556</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear Assay medium (fluorobrite)</td>
			<td>ThermoFisher</td>
			<td>A1896701</td>
			<td>For adenovirus transduction</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>ThermoFisher</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM:F12</td>
			<td>Gibco</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS (Fetal Bovine Serum)</td>
			<td>Sigma</td>
			<td>F4135-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoVolt</td>
			<td>ThermoFisher</td>
			<td>F10488</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14025-092</td>
			<td></td>
		</tr>
		<tr>
			<td>iCell CM maintenance media</td>
			<td>FUJIFILM/Cellular Dynamics</td>
			<td>M1003</td>
			<td></td>
		</tr>
		<tr>
			<td>iCell2 CMs</td>
			<td>FUJIFILM</td>
			<td>1434</td>
			<td></td>
		</tr>
		<tr>
			<td>Incucyte Zoom&#160;</td>
			<td>Sartorius</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iPS DF19-9-11T.H</td>
			<td>WiCell</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoproterenol</td>
			<td>MilliporeSigma</td>
			<td>CAS-51-30-9</td>
			<td></td>
		</tr>
		<tr>
			<td>IWP4</td>
			<td>Tocris</td>
			<td>5214</td>
			<td></td>
		</tr>
		<tr>
			<td>L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate</td>
			<td>Sigma</td>
			<td>A8960-5g</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>A2916801</td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyii Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Buffer (autoMACS Running Buffer)</td>
			<td>Miltenyii Biotec</td>
			<td>130-091-221</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker Red</td>
			<td>ThermoFisher</td>
			<td>M7512</td>
			<td></td>
		</tr>
		<tr>
			<td>Nautilus HTS Optical Mapping&#160;</td>
			<td>CuriBio</td>
			<td></td>
			<td>https://www.curibio.com/products-overview</td>
		</tr>
		<tr>
			<td>Nikon A1R Confocal Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>nonessential amino acids</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>pre-separation filter</td>
			<td>Miltenyii Biotec</td>
			<td>130-041-407</td>
			<td></td>
		</tr>
		<tr>
			<td>PSC-Derived Cardiomyocyte Isolation Kit, human</td>
			<td>Miltenyii Biotec</td>
			<td>130-110-188</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulse</td>
			<td>CuriBio</td>
			<td></td>
			<td>https://www.curibio.com/products-overview</td>
		</tr>
		<tr>
			<td>Quadro MACS separator (Magnet)</td>
			<td>Miltenyii Biotec</td>
			<td>130-091-051</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640&#160;</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 (+HEPES, +L-Glutamine)</td>
			<td>Gibco</td>
			<td>22400-089</td>
			<td></td>
		</tr>
		<tr>
			<td>StemMACS iPS-Brew XF</td>
			<td>Miltenyii Biotec</td>
			<td>130-107-086</td>
			<td></td>
		</tr>
		<tr>
			<td>TnI antibody (pan TnI)</td>
			<td>Millipore Sigma</td>
			<td>MAB1691&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Versene (ethylenediaminetetraacetic acid - EDTA solution)</td>
			<td>Gibco</td>
			<td>15040-066</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>Tocris</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput cardiotoxicity screening using mature human induced pluripotent stem cell-derived cardiomyocyte monolayers

人诱导干细胞来源的心肌细胞（hiPSC-CMs）用于替代和减少对动物和动物细胞的依赖，以进行临床前心脏毒性测试。在二维单层形式中，hiPSC-CMs概括了在最佳细胞外基质（ECM）上培养的成人心肌细胞的结构和功能。人围产期干细胞衍生的ECM（成熟诱导细胞外基质-MECM）在铺板后7天内使hiPSC-CM结构、功能和代谢状态成熟。 
成熟的hiPSC-CM单层对临床相关药物也有预期的反应，已知有引起心律失常和心脏毒性的风险。到目前为止，hiPSC-CM单层的成熟是这些有价值的细胞广泛用于监管科学和安全筛选的障碍。本文介绍了用于hiPSC-CM电生理和收缩功能的电镀、成熟和高通量功能表型的验证方法。这些方法适用于市售纯化的心肌细胞，以及使用高效的腔室特异性分化方案在内部生成的干细胞来源的心肌细胞。 
使用电压敏感染料 （VSD;发射波长：488 nm）、钙敏感荧光团 （CSF） 或基因编码钙传感器 （GCaMP6） 测量高通量电生理功能。高通量光学映射设备用于每个功能参数的光学记录，并使用定制的专用软件进行电生理数据分析。MECM方案用于使用正性肌力药物（异丙肾上腺素）和人乙醚相关基因（hERG）通道特异性阻断剂进行药物筛查。这些资源将使其他研究人员能够成功地利用成熟的hiPSC-CMs进行高通量，临床前心脏毒性筛查，心脏药物疗效测试和心血管研究。

以相衬和免疫荧光共聚焦成像为特征的hiPSC-CM成熟 使用MECM包被的96孔板对市售hiPSC-CMs进行ECM介导的成熟时间表如图 1A所示。这些数据是使用市售的心肌细胞收集的，这些心肌细胞作为冷冻保存的细胞瓶到达实验室。每个小瓶含有>5×106 活心肌细胞。细胞纯度为~98%，并经过严格的质量控制测试（每个小瓶均提供分析证书）。大量的CM能够使用同一批次的?...

Watch this Scientific Journal Video about High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers at JoVE.com

High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers

人诱导多能干细胞来源的心肌细胞（hiPSC-CMs）为使用动物进行临床前心脏毒性筛查提供了一种替代方案。hiPSC-CMs在临床前毒性筛查中广泛采用的一个限制是细胞的不成熟，胎儿样表型。这里介绍的是用于hiPSC-CMs稳健和快速成熟的方案。

使用成熟的人诱导多能干细胞来源心肌细胞单层进行高通量心脏毒性筛选

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical ...

该协议意义重大，因为它使研究人员能够将商业或内部制造的hiPSC-CMs成熟为成人样表型，从而显着提高hiPSC-CMs在心脏毒性筛查中的预测价值。该协议的主要优点是它以高通量形式提供成熟的hiPSC-CM，用于钙或电压变化的光学映射。该方案可用于研究疾病机制或筛查药物的心脏毒性。演示该程序的将是我实验室的研究助理Jeffrey Creech。首先在心肌细胞铺板前 1 小时用 200 微升汉克平衡盐溶液或 HBSS 洗涤成熟诱导细胞外基质或 MECM 板两次。保持井水充足。将心肌细胞管从液氮罐转移到干冰上，并通过稍微打开管盖来释放压力。接下来，重新密封管盖并在水浴中解冻 4 分钟。细胞解冻后，在打开试管之前用70%乙醇喷洒试管。用1毫升移液管将细胞转移到15毫升锥形管中。向冷冻管中加入 1 毫升电镀介质，并将洗涤液转移到 15 毫升锥形管中。然后，慢慢滴入1毫升电镀介质并搅拌管子。重复此操作，直到转移8毫升电镀介质。将15毫升管以300×g离心5分钟，然后将沉淀重悬于1毫升电镀介质中。取出等分试样，在加入电镀培养基之前用血细胞计数器进行活细胞计数，以获得每毫升第 5 个细胞的 7.5 x 10。使用多通道移液器在MECM包被的96孔板的每孔中分配100微升细胞悬液，并将细胞孵育2天。接下来，用200微升维持培养基更换培养基，并保持板7天，第5天更换培养基。在第7天，如文本中所述进行EP测定。确保在选择延长细胞培养物时每隔一天更换一次培养基。为了通过磁性激活细胞分选或MACS纯化hiPSC-CM，吸出细胞培养基并用1毫升HBSS洗涤细胞，然后通过加入1毫升0.25%胰蛋白酶-乙二胺四乙酸或EDTA来解离细胞，并将细胞孵育10分钟。然后，通过用2毫升电镀培养基重新发送和单调化细胞来灭活胰蛋白酶/ EDTA。从六个孔中，使用70微米过滤器将细胞收集到50毫升锥形管中。用3毫升电镀介质洗涤过滤器后，计数细胞。计数后，离心并用2毫升冰冷的MACS分离缓冲液洗涤细胞沉淀，然后沉淀并将细胞重新发送到每5 x 10的80微升冷MACS分离缓冲液中至第6个细胞。加入20微升冷的非心肌细胞消耗鸡尾酒，混合悬浮液，然后在冰上孵育10分钟。用4毫升冷MACS分离缓冲液以300×g洗涤细胞5分钟后，将沉淀重悬于80微升冷MACS分离缓冲液中。向第 6 个细胞中每 5 x 10 加入 20 μL 冷抗生素素微珠，并在冰上孵育 10 分钟之前轻轻混合细胞悬液。在样品孵育时，将装有30微米预分离过滤器的正去痕柱放在MACS分离器上，并将标记的15毫升收集管放在柱下方。接下来，用 3 毫升冷 MACS 分离缓冲液灌注每根色谱柱。将抗体处理的细胞悬液与2毫升MACS分离缓冲液混合，并将其添加到色谱柱中。向每根色谱柱中加入2毫升MACS分离缓冲液，并收集12毫升流通心肌细胞悬浮液。离心心肌细胞并弃去上清液，然后将心肌细胞重悬于1毫升电镀介质中。为了确定浓度，在将纯化的心肌细胞接种在MECM 96孔板上之前，对细胞计数并将体积调节到所需的接种密度。吸出并用 100 μL 电压敏感染料或 VSD、钙敏感荧光团或 CSF 替换心肌细胞维持培养基，每孔 96 孔板。孵育30分钟后，用测定培养基或HBSS替换染料。在使用高通量光学映射设备获取基线数据光学映射之前，在 37 摄氏度下平衡细胞。为了用用于急性暴露测试的药物处理细胞或绘制长期暴露于目标药物的细胞，请将药物稀释在二甲基亚砜或DMSO中，并将它们作为储备溶液储存在20摄氏度下，然后再将它们稀释到所需的浓度在HBSS中。对于 96 孔板的心脏毒性测试，添加四剂化合物，每剂至少八孔。使用从低于到高于临床有效治疗血浆浓度的剂量，包括有效剂量。与药物应用前的基线电生理学测量类似，在药物治疗后至少 30 分钟捕获电生理学记录以进行慢性研究。基线记录后，将每种药物至少四剂施用于表达GCaMP6m的成熟hiPSC-CMs单层，每剂至少8个孔。平衡细胞上的药物30分钟，并在数据采集之前和期间将细胞加热至37摄氏度。在整个板的基线记录之后，向每个孔中加入500纳摩尔异丙肾上腺素，以获得可靠的药物反应数据，并量化异丙肾上腺素对单层跳动率，收缩幅度和钙瞬时持续时间的影响。要获取光学映射数据，请打开前抽屉并将板放在板加热器上。打开采集软件并确定软件中的文件保存位置后，选择10至30秒的采集时长和帧率以获得更高的时间分辨率，然后单击开始采集。打开分析软件，在“导入过滤器”选项卡中，选择“浏览单个文件”或“平铺多个”以重建板。选择文件并输入行数和列数，然后选择“自动”。接下来，选择更新像素大小，输入每像素的距离，然后使用孔向导确定孔在图像中的位置，然后单击处理保存并移动到下一个选项卡。打开ROI或感兴趣区域选项卡，然后选择手动，自动或根本不使用ROI绘制ROI，这将考虑用于分析整个板。选择井中的区域后，单击“处理保存”按钮进入下一步。选中隐藏井和仅显示筛选框以可视化投资回报率。打开“分析”选项卡，选择每个孔或ROI以确认自动拍频检测的准确性。通过选择单个节拍并按键盘上的 Delete 键，在轨迹中添加或删除节拍。完成后，单击保存。接下来，单击分析选项卡中的时空图按钮并添加线以确定时空图，以可视化每个井的数据或ROI。选择水平线或垂直线后，单击“生成绘图”。确保准确的节拍检测后，进入 出口 选项卡并选择 文件格式，输入 节拍统计 选项，然后单击 出口.导出后，打开数据文件并运行所选的统计分析例程。相衬成像显示，接种在MECM上的hiPSC-CMs成熟，并且在结构上与重新铺在小鼠ECM上的同一批hiPSC-CMs不同。成熟细胞变成杆状，而未成熟细胞保持圆形。用α-肌动蛋白抗体染色的心肌细胞显示出在每个ECM条件下培养的细胞的典型形状。与TnI染色一致，α-肌动蛋白染色表明，在MECM上成熟的hiPSC-CMs促进了杆状成熟表型并诱导了更大的肌节组织。在小鼠ECM和MECM上培养的细胞之间的线粒体含量和活性是不同的。使用VSD和光学映射系统记录的动作电位的电生理数据显示，心房特异性细胞的自发搏动率明显更快，动作电位持续时间（APD80）比心室特异性细胞短。APD80等参数的全板热图揭示了板内给定参数的重现性。成熟hiPSC-CM单层的典型动作电位表明分离和测试的成年心肌细胞的动作电位形态。还显示了典型的动作电位自发节律。响应异丙肾上腺素，β-1-肾上腺素能受体的激活引起正变时性、正性肌力和正光性。使用 GCaMP6m 钙荧光监测节律，研究了 HiPSC-CM 对人 Ether-a-go-go 相关基因或 hERG 通道阻滞剂（包括 E-4031、多潘立酮、凡德他尼和索他洛尔）的反应。该研究表明，检测出由E-4031 hERG通道阻断引起的早期后去极化。快速处理悬浮细胞，并通过轻轻移液细胞来避免剪切应力。此外，请仔细更换培养基，以免损坏 hiPS 合胞体。按照这一程序，细胞适合收集蛋白质、RNA、DNA 和脂质，以便使用研究人员最喜欢的技术进行分析。该技术使研究人员能够使用成人样心肌细胞研究疾病并测试新药的心脏毒性。

Research

JoVE Journal

Bioengineering

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

白色念珠菌生物膜芯片（CA BChip）为高通量的抗真菌药物筛选

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

科研

生物工程

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

开发，扩展，<em&gt;在体内</em&gt;从人类胚胎干细胞（hESCs）监测人类NK细胞和诱导多能干细胞（iPS细胞）

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

3D Microtissues 用于可注射再生疗法和高通量药物筛选

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

人类多能干细胞和免疫损伤的成熟小脑有机体可伸缩生成

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

在化学定义条件下，成熟肾细胞与人类诱导多能干细胞的引导分化

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

由人诱导多能干细胞改造的同基因肾小球芯片

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

2D人干细胞来源心肌细胞中心脏收缩力调节疗法的评估

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...