Name: hybrid cell analysis system to assess structural and contractile changes of human ipsc-derived cardiomyocytes for preclinical cardiac risk evaluation
Uploaded: 2022-10-20T13:30:00
Description: Watch this Scientific Journal Video about Hybrid Cell Analysis System to Assess Structural and Contractile Changes of Human iPSC-Derived Cardiomyocytes for Preclinical Cardiac Risk Evaluation at JoVE.

这项工作得到了德国联邦经济事务和气候行动部（ZIM）和德国联邦教育与研究部（KMUinnovativ）的资助。我们感谢FUJIFILM Cellular Dynamics，Inc（美国威斯康星州麦迪逊）慷慨地提供心肌细胞，并感谢Ncardia B.V.（荷兰莱顿）慷慨地提供本研究中使用的心肌细胞。

注意：收缩性和阻抗/EFP 测量的工作流程在 补充图 2 中给出。
1. 板材涂层
<ol><li>打开真空密封包装，取出96孔板。两个模块的96孔板的处理程序相同。让收缩板被额外提供的膜护罩覆盖，直到在收缩力模块中进行测量。</li>
	<li>涂覆用于接种心肌细胞的柔性 96 孔板。<ol><li>通过在无菌离心管中转移 2.75 mL EHS 凝胶即用型溶液来制备稀释的 EHS 凝胶包衣?...

<ol>
	<li>Weaver, R. J., Valentin, J. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Today's+challenges+to+de-risk+and+predict+drug+safety+in+human+&amp;#34;mind-the-gap&amp;#34.">Today's challenges to de-risk and predict drug safety in human &amp;#34;mind-the-gap&amp;#34.</a> Toxicological Sciences. 167 (2), 307-321 (2019).</li><li>Burnett, S. D., Blanchette, A. D., Chiu, W. 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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>Gossmann, 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=Integration+of+mechanical+conditioning+into+a+high+throughput+contractility+assay+for+cardiac+safety+assessment.">Integration of mechanical conditioning into a high throughput contractility assay for cardiac safety assessment.</a> Journal of Pharmacological and Toxicological Methods. 105, 106892 (2020).</li><li>Hansen, 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=Development+of+a+drug+screening+platform+based+on+engineered+heart+tissue.">Development of a drug screening platform based on engineered heart tissue.</a> New Methods in Cardiovascular Biology. 107 (1), 35-44 (2010).</li><li>Zuppinger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Cardiac+cell+culture:+a+critical+review+of+current+technologies+and+applications.">3D Cardiac cell culture: a critical review of current technologies and applications.</a> Frontiers in Cardiovascular Medicine. 6, 87 (2019).</li><li>Laverty, H. 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J., Hearse, 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=The+isolated+blood+and+perfusion+fluid+perfused+heart.">The isolated blood and perfusion fluid perfused heart.</a> Pharmacological Research. 41 (6), 613-627 (2000).</li><li>Brown, G. E., Khetani, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabrication+of+liver+and+heart+tissues+for+drug+development.">Microfabrication of liver and heart tissues for drug development.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 373 (1750), 20170225 (2018).</li><li>Scott, C. W., 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=An+impedance-based+cellular+assay+using+human+iPSC-derived+cardiomyocytes+to+quantify+modulators+of+cardiac+contractility.">An impedance-based cellular assay using human iPSC-derived cardiomyocytes to quantify modulators of cardiac contractility.</a> Toxicological Sciences. 142 (2), 313-338 (2014).</li><li>Gossmann, 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=Mechano-pharmacological+characterization+of+cardiomyocytes+derived+from+human+induced+pluripotent+stem+cells.">Mechano-pharmacological characterization of cardiomyocytes derived from human induced pluripotent stem cells.</a> Cellular Physiology and Biochemistry. 38 (3), 1182-1198 (2016).</li><li>Rappaz, B., 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=Automated+multi-parameter+measurement+of+cardiomyocytes+dynamics+with+digital+holographic+microscopy.">Automated multi-parameter measurement of cardiomyocytes dynamics with digital holographic microscopy.</a> Optics Express. 23 (10), 13333-13347 (2015).</li><li>Doerr, 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=New+easy-to-use+hybrid+system+for+extracellular+potential+and+impedance+recordings.">New easy-to-use hybrid system for extracellular potential and impedance recordings.</a> Journal of Laboratory Automation. 20 (2), 175-188 (2014).</li><li>Obergrussberger, 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=Safety+pharmacology+studies+using+EFP+and+impedance.">Safety pharmacology studies using EFP and impedance.</a> Journal of Pharmacological and Toxicological Methods. 81, 223-232 (2016).</li><li>Bot, C., 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=Cross-site+comparison+of+excitation-contraction+coupling+using+impedance+and+field+potential+recordings+in+hiPSC+cardiomyocytes.">Cross-site comparison of excitation-contraction coupling using impedance and field potential recordings in hiPSC cardiomyocytes.</a> Journal of Pharmacological and Toxicological Methods. 93, 46-58 (2018).</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>Edwards, S. 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=A+multiwell+cardiac+&#181;GMEA+platform+for+action+potential+recordings+from+human+iPSC-derived+cardiomyocyte+constructs.">A multiwell cardiac &#181;GMEA platform for action potential recordings from human iPSC-derived cardiomyocyte constructs.</a> Stem Cell Reports. 11 (2), 522-536 (2018).</li><li>Zlochiver, V., Kroboth, S., Beal, C. R., Cook, J. A., Joshi-Mukherjee, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R.Human+iPSC-derived+cardiomyocyte+networks+on+multiwell+micro-electrode+arrays+for+recurrent+action+potential+recordings.">R.Human iPSC-derived cardiomyocyte networks on multiwell micro-electrode arrays for recurrent action potential recordings.</a> Journal of Visualized Experiments. (149), e59906 (2019).</li></ol>

阻抗/EFP/收缩力混合系统是一种综合方法，用于临床前药物开发的心脏负债的高通量安全性药理学和毒理学评估。它提供了一种现代的临床前安全性测试方法，无需使用动物模型，但具有更高的通量能力，可显着减少时间和成本。该系统有可能用作Langendorff心脏和其他动物模型的补充方法，用于临床前功能和结构毒性评估。
作为一种无动物的方法，人类iPSC-CMs被用于杂交系统<s...

现代药物开发的主要目标之一是提高药物发现管道中新疗法从实验室到床边的成功率。这些新药的安全性药理学测试通常揭示心血管系统的药物不良反应，几乎占临床前阶段药物损耗率的四分之一1。新方法（NAM）的开发和整合在临床前评估的现代化中发挥着关键作用，特别是心脏等核心电池器官。由于这些方法是无动物方法，因此在过去十年中，使用基于人类的细胞模型（如诱导多能干细胞（iPSC）来源的心肌细胞（CMs））成为现代评估安全性药理学和毒理学问题的主力2。用于此类研究的广泛使用的测定系统是微电极阵列（MEA）和基于电压敏感染料的实验方法3。
然而，这种细胞类型声称的表型和功能不成熟性为理想的基于人类的细胞模型设置了障碍，有可能减少非临床和临床研究之间的翻译差距4。
多年来进行了大量研究，以了解隐含的未成熟表型的原因，并找到在 体外推动人类iPSC-CMs成熟过程的方法。
缺乏心脏成熟线索，例如延长的细胞培养时间，附近没有其他细胞类型或缺乏激素刺激，已被证明会影响成熟过程5。此外，由于天然人心脏缺少生理底物硬度，常规细胞培养板的非生理环境被确定为阻碍人类iPSC-CMs成熟的重要原因5，6。
为了解决这个问题，开发了专注于天然生理条件的不同测定系统，包括3D细胞培养系统，其中细胞在三维上排列以类似于天然心脏结构而不是典型的二维细胞培养物7。尽管通过3D测定可以提高成熟度，但对熟练劳动力的需求和这些系统的低通量阻碍了其在药物开发过程中的大量使用，因为时间和成本在财务层面评估新疗法中起着根本作用8。
新疗法的安全性药理学和毒理学评估的重要读数是人iPSC-CMs的功能和结构特征的变化，因为化合物诱导的心血管系统不良药物反应通常会影响这些特性中的一个或两个1，9。这种广泛不良反应的著名例子是蒽环类药物家族的抗癌药物。在这里，在患者癌症治疗期间和之后以及体外基于细胞的测定中，对心血管系统的危险功能和不良结构影响被广泛报道10，11。
在本研究中，我们描述了一种评估hiPSC-CMs的功能和结构化合物副作用的综合方法。该方法包括心肌细胞收缩力和阻抗/细胞外场电位（EFP）的分析。收缩力是在生理机械条件下测量的，细胞培养在柔软（33kPa）硅胶基质上，反映了天然人类心脏组织的机械环境。
该系统配备了 96 孔板，用于对人 iPSC-CM 进行高通量分析，用于临床前心脏安全性药理学和毒理学研究，因此为目前使用的 3D 方法（如 Langendorff 心脏或心脏切片12，13）提供了优势。
详细地说，混合系统由两个模块组成，用于评估生理条件下的心脏收缩力或实时细胞结构毒性分析6，14。两个模块均与专用的高通量 96 孔板配合使用，可实现快速且经济高效的数据采集。
无需3D结构，收缩性模块采用包含柔性有机硅膜的特殊板作为细胞基板，而不是常规细胞培养板通常由硬玻璃或塑料组成。这些膜反映了典型的人类生物力学心脏特性，因此以高通量的方式模拟 体内 条件。虽然人iPSC-CMs在其他基于细胞的测定中通常无法显示成人心肌细胞对化合物诱导的正性肌力的行为14，但当细胞在收缩模块的平板上培养时，可以评估更像成人的反应。在以前的研究中，已经证明iPSC-CMs在使用异丙肾上腺素，S-Bay K8644或奥卡替夫美卡比6，15等化合物处理时表现出积极的正性肌力作用。在这里，可以评估多个收缩力参数，例如收缩力振幅 （mN/mm 2）、搏动持续时间和搏动率等主要参数，以及收缩周期的次要参数，如曲线下面积、收缩和松弛斜率、搏动率变化和心律失常（补充图 1）16.通过电容式距离传感非侵入性评估药物诱导的所有参数的变化。原始数据随后由专门的软件进行分析。
结构毒性模块添加了其独特的阻抗和EFP参数，作为结构细胞毒性和电生理特性分析的读数17，18。电阻抗谱技术揭示了化合物诱导的细胞密度变化或实时监测的细胞和单层完整性，如用已知心脏毒性化合物处理的人 iPSC-CM 所示13。通过不同频率（1-100 kHz）的阻抗读数，可以进一步剖析生理反应，从而揭示膜形貌、细胞间或细胞-基质连接的变化。正如CiPA研究17，19所示，人iPSC-CMs的额外EFP记录进一步能够分析复合治疗引起的电生理效应。
在本研究中，使用人iPSC-CMs，用表柔比星和多柔比星（两者都被描述为心脏毒性蒽环类药物）和厄洛替尼（一种酪氨酸激酶抑制剂（TKI））治疗，心血管毒性风险相当低。使用表柔比星、多柔比星和厄洛替尼进行 5 天的慢性评估。结果显示，当细胞用厄洛替尼处理时，收缩力和碱基阻抗有微小变化，但分别用表柔比星和多柔比星处理时，收缩幅度和碱基阻抗的时间和剂量依赖性毒性降低。使用钙通道阻滞剂硝苯地平进行急性测量，显示收缩幅度、场电位持续时间和碱基阻抗降低，表明该化合物对功能和结构水平的心脏毒性副作用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Commercial human iPSC-derived cardiomyocytes&#160;</td>
			<td>Fujifilm Cellular Dynamics International (FCDI)</td>
			<td>R1059</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge (50 mL tubes)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15878722</td>
			<td></td>
		</tr>
		<tr>
			<td>12-channel adjustable pipette (100-1250 &#956;L)</td>
			<td>Integra Biosciences</td>
			<td>4634</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS with Ca2+ and Mg2+</td>
			<td>GE Healthcare HyClone</td>
			<td>SH304264.01</td>
			<td></td>
		</tr>
		<tr>
			<td>96 deep well plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>A43075</td>
			<td></td>
		</tr>
		<tr>
			<td>EHS gel</td>
			<td></td>
			<td></td>
			<td>Extracellular Matrix Gel</td>
		</tr>
		<tr>
			<td>FLEXcyte 96/CardioExcyte hybrid device</td>
			<td>Nanion Technologies&#160;</td>
			<td>19 1004 1005</td>
			<td>Hybrid cell analysis system&#160;</td>
		</tr>
		<tr>
			<td>FLX-96 FLEXcyte Sensor Plates</td>
			<td>Nanion Technologies</td>
			<td>20 1010</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Fibronectin stock solution (Optional to Geltrex)</td>
			<td>Sigma Aldrich</td>
			<td>F1141</td>
			<td></td>
		</tr>
		<tr>
			<td>Geltrex hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane Matrix</td>
			<td>ThermoFischer Scientific</td>
			<td>A1569601</td>
			<td></td>
		</tr>
		<tr>
			<td>Human iPSC-derived cardiomyocytes plating and maintenance medium</td>
			<td>FCDI</td>
			<td>R1059</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator (37 &#176;C, 5% CO2)</td>
			<td>Thermo Fisher Scientific</td>
			<td>51023121</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar Flow Hood</td>
			<td>Thermo Fisher Scientific</td>
			<td>51032678</td>
			<td></td>
		</tr>
		<tr>
			<td>NSP-96 CardioExcyte 96 Sensor Plates 2.0 mm transparent</td>
			<td>Nanion Technologies</td>
			<td>20 1011</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips (1250&#181;L)</td>
			<td>Integra Biosciences</td>
			<td>94420813</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent Reservoir</td>
			<td>Integra Biosciences</td>
			<td>8096-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette (e.g. 25 mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>16440901</td>
			<td></td>
		</tr>
		<tr>
			<td>Single channel adjustable pipette (e.g. 100-1000 &#956;L)</td>
			<td>Eppendorf</td>
			<td>3123000063</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum aspiration system</td>
			<td>Thermo Fisher Scientific</td>
			<td>15567479</td>
			<td></td>
		</tr>
		<tr>
			<td>Optional: VIAFLO ASSIST</td>
			<td>Integra Biosciences</td>
			<td>4500</td>
			<td>Lab automation Robot</td>
		</tr>
		<tr>
			<td>Water bath (37 &#176;C)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15365877</td>
			<td></td>
		</tr>
	</tbody>
</table>

hybrid cell analysis system to assess structural and contractile changes of human ipsc-derived cardiomyocytes for preclinical cardiac risk evaluation

心脏收缩力评估对于新疗法的开发及其安全过渡到临床阶段非常重要。虽然人类诱导的多能干细胞衍生心肌细胞（hiPSC-CMs）有望在药物发现和安全药理学的临床前阶段作为人类相关模型，但它们的成熟度在科学界仍然存在争议，并且正在不断发展。我们提出了一种混合收缩性和阻抗/细胞外场电位（EFP）技术，为行业标准的96孔平台增加了重要的促成熟特征。
阻抗/EFP 系统实时监控蜂窝功能。除了收缩细胞的搏动率外，电阻抗谱读数还可以检测化合物诱导的形态变化，如细胞密度和细胞单层的完整性。在杂交细胞分析系统的另一个组件中，细胞在模拟真实心脏组织的机械环境的生物顺应性膜上培养。这种生理环境支持hiPSC-CMs在 体外成熟，导致更多的成人样收缩反应，包括用异丙肾上腺素，S-Bay K8644或奥卡替夫美卡比治疗后的正性肌力作用。收缩力振幅（mN/mm 2）和搏动持续时间等参数也揭示了化合物对电生理性能和钙处理影响的下游效应。
该混合系统为整体细胞分析提供了理想的工具，允许临床前心脏风险评估超越当前人类相关细胞测定的视角。

激酶抑制剂厄洛替尼对hiPSC-CMs收缩力的影响如图 1所示。用浓度范围为10nM至10μM的细胞处理5天，并每天记录搏动参数。厄洛替尼是一种EGFR（表皮生长因子受体）和酪氨酸激酶抑制剂，具有相对较低的心脏毒性风险，仅在微摩尔范围内的浓度下对hiPSC-CMs具有小剂量和时间依赖性影响。在最低浓度（10nM）下，厄洛替尼在化合物施用后（1小时）的第一次测量中诱导了统计学上不?...

Watch this Scientific Journal Video about Hybrid Cell Analysis System to Assess Structural and Contractile Changes of Human iPSC-Derived Cardiomyocytes for Preclinical Cardiac Risk Evaluation at JoVE.

Hybrid Cell Analysis System to Assess Structural and Contractile Changes of Human iPSC-Derived Cardiomyocytes for Preclinical Cardiac Risk Evaluation

分析人iPSC来源的心肌细胞的收缩功能和细胞完整性的变化对于非临床药物开发具有重要意义。混合 96 孔细胞分析系统以实时和生理方式处理这两个参数，以获得可靠的、与人类相关的结果，这是安全过渡到临床阶段所必需的。

用于评估人iPSC来源心肌细胞结构和收缩变化的杂交细胞分析系统，用于临床前心脏风险评估

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While ...

该方法可以使用真实的人类数据帮助回答临床前心脏安全性和毒性相关问题，以便将候选新药安全过渡到临床阶段。这种混合系统的主要优点不是使用一组不同的体外和体内技术，而是同时评估人iPC来源的心肌细胞，电生理和活力参数，以及分析生理条件下的收缩特性。该方法可用于药物发现，因为它涵盖了心肌细胞的电生理、结构和收缩变化，以及允许同时测试 69 个孔的更高通量系统。要开始板涂层，请将收缩板的底部由额外提供的膜护罩覆盖，直到在收缩力模块中进行测量。为了接种心肌细胞，通过在无菌离心管中转移2.75毫升EHS凝胶即用型溶液和8.25毫升DPBS与钙和镁来制备稀释的EHS凝胶包被溶液。然后混合溶液。从收缩板上拆下膜护罩。将制备好的涂层溶液转移到实验室自动化机器人中的无菌试剂储液器中。使用该程序，使用实验室自动化机器人添加100微升，每孔添加100微升涂层溶液。然后将盖子放回 96 孔板上，并在 37 摄氏度下孵育三小时。解冻并计数细胞后，根据细胞制造商的说明在推荐的电镀介质中调整细胞，产生每11毫升11倍10至第六个细胞，用于接种整个96孔板。使用程序去除100微升，通过实验室自动化机器人从孔中去除EHS凝胶溶液。接下来，将11毫升细胞悬液转移到放置在实验室自动化机器人中的无菌试剂储液器中，并在每孔中接种100微升悬浮液的细胞。使用该程序，细胞增加100微升。细胞接种后，立即将灵活的96孔板转移到37摄氏度，5%二氧化碳的湿度控制培养箱中，并使细胞沉降过夜。将22毫升心肌细胞维持培养基温热至37摄氏度加入到每个板的50毫升离心管中。接种板后 18 至 24 小时，将新鲜培养基转移到无菌试剂储液槽中，并将其放在实验室自动化机器人旁边。然后使用该程序进行培养基去除，去除 100 微升。接下来将含有新鲜培养基的试剂储液槽放入实验室自动化机器人中，并用程序每孔分配200微升新鲜培养基，加入100微升。执行此步骤两次以达到每孔200微升。培养基交换后，立即将板转移到培养箱中。每隔一天进行一次培养基更换，直到加入化合物。在化合物添加前四到六个小时，通过将22毫升新鲜和温暖的测定缓冲液转移到无菌试剂储液槽中并将其放在实验室自动化机器人旁边，进行最终的培养基更换。培养基交换后立即将柔性板转移回培养箱中。基线测量前一小时将板转移到相应的测量设备上。在控制软件中，打开编辑协议并选择相应的测量模式、弹性站点或阻抗 EFP。在保存协议编号之前，将一次测量的扫描持续时间或长度定义为 30 秒，将重复间隔定义为 10 分钟。然后选择“启动协议”并继续填写请求的字段。设置参数后，选择开始测量。在化合物添加前不久，每隔五分钟至少进行三次基线测量。从每个孔中取出50微升测定缓冲液，而不从测量装置上取下柔性96孔板，然后根据测量计划向板的每个孔中加入50微升四倍浓缩的化合物溶液。添加化合物后，选择添加试剂标记以定义化合物板布局和化合物溶液的体积。然后根据实验计划选择“继续标准测量”或“继续测量系列”。代表性分析显示了激酶抑制剂埃洛替尼对人iPSC来源心肌细胞收缩力的影响。在最低浓度下，埃罗替尼诱导振幅在统计学上不显着降低，而埃罗替尼的微摩尔浓度显示出时间和剂量依赖性心脏毒性作用。使用1微摩尔厄洛替尼从96小时开始见效果，而用10微摩尔埃洛替尼治疗导致化合物添加后24小时显着降低。施用10微摩尔表柔比星后，细胞在24小时内停止跳动。应用一微摩尔表柔比星后，振幅在24小时后急剧下降，然后完全停止搏动，直到48小时。在100纳摩尔时，观察到搏动幅度随时间变化。在10纳摩尔的最低浓度下，振幅稳定波动，证实表柔比星的作用仅是剂量依赖性的。用阿霉素和硝苯地平处理24小时以浓度和时间依赖性方式降低细胞活力。在应用增加硝苯地平浓度时，细胞上的场电位记录显示归一化的场持续时间的浓度依赖性缩短。硝苯地平关于心脏收缩力的评估也显示，在10纳摩尔和13纳摩尔浓度的急性测量中，浓度依赖性振幅显着降低。重要的是要记住，细胞通常对环境参数敏感，如温度和pH变化以及机械模拟，这在收缩板上特别支持。

Research

JoVE Journal

Bioengineering

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

心肌在心脏组织工程中的纤维蛋白凝胶的封装

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

科研

生物工程

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

微型硅悬臂梁的利用率，评估细胞收缩功能<em&gt;在体外</em

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

Hybrid &#181;CT-FMT imaging and image analysis

Hybrid  &#181;CT-FMT imaging and image analysis

混合μCT-FMT成像和图像分析

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

定义人工程心脏组织建设探讨心肌细胞疗法的机理

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

人工程心脏组织的收缩自动分析心脏药物安全筛选

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

MicroRNA 移植成人骨髓造血干细胞促进细胞工程与磁性靶向结合的协议

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

标准化和可扩展的测定，以研究iPSC衍生内皮细胞在Vitro的渗透3D血管生成发芽

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current ...

Human iPSC-Derived Cardiomyocyte Networks on Multiwell Micro-electrode Arrays for Recurrent Action Potential Recordings

用于循环作用电位记录的多井微电极阵列上的人类 iPSC 衍生心肌细胞网络

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

人体器官性心脏切片双电压和钙光学映射的临床前心脏电生理评估

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...