Name: single-cell optical action potential measurement in human induced pluripotent stem cell-derived cardiomyocytes
Uploaded: 2020-12-22T13:30:00.000+00:00
Duration: 8 min 39 s
Description: Watch this Scientific Journal Video about Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes at JoVE.com

作者要感谢凯恩研究有限公司为支付本出版物制作成本而作出的善意财政贡献。此外，我们感谢伊内斯·穆勒女士和斯特凡妮·凯斯特尔女士给予的出色技术支持。
作者的研究得到了德国心血管研究中心（DZHK）的支持，德国福松斯格梅因沙夫特（DFG， 德国研究基金会，VO 1568/3-1，IRTG1816 RP12，SFB1002 TPA13和德国卓越战略 - EXC 2067/1-390729940）和埃尔塞-克雷纳-弗雷塞纽斯·斯蒂夫通（EKFS 2016_A20）。

1. 细胞制剂
注:本协议中使用的人类iPSC来自健康的捐赠者，并采用完全定义的小分子调制WNT信号和乳酸纯化技术，如前所述12，13，14。iPSC-CM 每 2-3 天维护一次，下面概述了文化媒介。
<ol><li>准备基础介质培养基（RPMI 1640）和2%补充（B27）。储存在 4 °C。 在室温下使用 （RT）。</li>
	<li>准备基底介质的电镀介质 （RPMI 1640），2% 补充 （B27） 和 1:2000 ROCK 抑制剂。储存在 4 °C。 在 RT 中使用。</li>
	<li>涂层消毒 10 毫米圆形玻璃 #0 盖片，150 μL 的 1:60 系数免费地下室膜基质，并在 4 °C 4 h 下孵育。 注意:为了确保整个玻璃被覆盖，同时保持足够的表面张力以防止溢出，有必要优化覆盖唇体积。150 μL 建议用于 10 mm 圆形覆盖唇。批量大小、覆盖式、覆盖体积、培养板型可满足实验者的需求。</li>
	<li>开始 iPSC-CM 与基于 EDTA 的细胞分离试剂分离。通过用 1，000 μL 移液器轻轻冲洗，确保单层完全分离。</li>
	<li>将细胞悬架转移到 15 mL 管中，并添加双音量电镀介质。离心机10分钟，100 x g。</li>
	<li>用电镀介质所需的体积（悬念体积）重新悬念颗粒。手动或电子计数细胞。</li>
	<li>选择每个覆盖唇的最佳密度（15，000），这将允许以后进行孤立的细胞分析。</li>
	<li>计算以此所需密度平滑所有盖片所需的"活动"细胞悬浮 （A） 体积。应用以下公式并取入单独的管子: <img alt="Equation 1" src="/files/ftp_upload/61890/61890equ01v2.jpg">
</li>
	<li>计算以所需的体积容纳每个盖片所需的额外电镀介质 （B） 的体积。应用以下公式，并将产生的体积添加到主动悬浮管中: <img alt="Equation 2" src="/files/ftp_upload/61890/61890equ02v2.jpg">
</li>
	<li>从盖唇中取出基质，将细胞悬浮 （A+B） 的"盖片体积"应用到每个盖唇上。定期在管中重复使用，以确保均匀的细胞分布。</li>
	<li>在 37 °C 下孵育 1 小时。轻轻地用电镀介质填充井。</li>
	<li>24小时后，与正常文化媒介交换媒体，每2-3天保持一次。</li>
</ol>2. 实验设置
<ol><li>为倒置的表观显微镜配备 40 倍放大倍率、高数值光圈镜头（N.A: > 0.75）进行实验。</li>
	<li>将暖白色 LED 快速切换到显微镜的传输照明端口。将简单的红色 660 nm 滤清器插入传输的光路。 注:这种光可以在光度测量实验中激活，以观察样品，而不会污染绿色荧光信号。</li>
	<li>安装快速切换 470 nm LED 头进行照片测量记录。在显微镜的表光端口插入 470/40 激发滤镜，以清理 LED 产生的光。 注:为了优化信号量化，建议使用具有高速反馈控制光输出的照明系统。</li>
	<li>在显微镜内的镜片单元旋转木马中插入一个显微镜立方体，其中包含一个 495 nm 长的传递光束分离器。</li>
	<li>将装有可调现场隔膜的检测臂安装到显微镜 C-mount 端口，以便选择感兴趣的区域。</li>
	<li>另外，将一个光子探测器 （PMT） 和 USB 摄像机耦合到显微镜上。这将形成排放检测系统的基础。</li>
	<li>将包含 565 nm 长通过光束分离器和 535/50 排放过滤器的过滤器插入 PMT 端口。这将两个探测器之间的发射光分开。 注:连接到发射检测系统传输端口的摄像机可以在所有实验中检测到亮场下的传输光。</li>
	<li>将 PMT 与电源和 PMT 放大器耦合。将 PMT 放大器输出连接到数据采集系统的模拟输入引脚。</li>
	<li>以 1 kHz 或更高速度过滤来自 PMT 的模拟数据。</li>
	<li>以至少是模拟信号（2 kHz 或更高）中最高频率两倍的频率对数据进行数字化，以满足奈奎斯特标准并防止别名化。</li>
</ol>3. 带 VF2.1Cl 的细胞加载
注:涉及这种染料的所有步骤必须在低光照条件下进行。
<ol><li>准备 Tyrode 的沐浴解决方案 （在 mM）: 140 NaCl， 10 HEPES， 10 葡萄糖， 4 KCl， 1 MgCl2， 2 CaCl2， pH = 7.35 和温暖到 37 °C.</li>
	<li>通过混合 5 μL 的 1，000x VF2.1Cl 和 50 μL 的 20% 溶解，在微中心富尔管中准备装载溶液。</li>
	<li>将 5 μL 的装载溶液应用于 20 mm Petri 盘中 5 mL 加热 Tyrode 溶液（总染料浓度为 0.1 倍）。 注:最终染料浓度为0.1倍。这是制造商建议的 1/10。这节约了资源，确保了可忽略不清的细胞毒性，而且重要的是，仍然保留来自具有高信号与噪声比的加载电池的清晰光学信号。</li>
	<li>在菜中加入一个 iPSC-CM 盖片，在 37 °C 下孵育 20 分钟。</li>
	<li>组装加热的活细胞成像室。登上显微镜舞台，装满 500 μL 的新鲜 Tyrode 溶液。</li>
	<li>在 37 °C 下用新鲜的 Tyrode 溶液清洗盖唇。</li>
	<li>使用细点钳小心地将 iPSC-CM 盖唇涂抹到预热浴室。 注意:确保房间及其内容始终在生理温度下加热。如果需要，从持续灌注加热泰罗德的解决方案开始。</li>
</ol>4. 电场刺激
注:iPSC-CM的外部触发是可选的，但可用于细胞动力学和实验参数的标准化。它增加了分析的易用性，并允许对频率依赖效应进行调查。
<ol><li>将两个相距 5 mm 的铂电极连接到录音室中，并附上刺入。</li>
	<li>将外部刺激器连接到刺入件。设置为 5 ms 双极场脉冲在 0.5 Hz.</li>
	<li>通过将刺激从 1 V 向上增加，确定最佳刺激电压。阈值电刺激被定义为单元格开始收缩的最低电压。施加电压大约比此阈值高 25%。正常范围在 1 V 和 30 V 之间。</li>
	<li>用外部刺激器修复刺激频率，或用采集软件触发刺激频率。</li>
</ol>5. 光学动作潜在收购
注:此协议使用商业软件进行获取和分析。
<ol><li>使用传输的光路径和 USB 摄像机在明亮的视野中可视化肌细胞。</li>
	<li>选择一个孤立的细胞，并紧紧裁剪其光学路径与现场隔膜，确保只有光从感兴趣的细胞被监测。</li>
	<li>激活 PMT 放大器，将 PMT 电源设置为 750 V。</li>
	<li>运行刺激协议（参见步骤 4）以及采集软件，同时激活 470 nm 激发灯。后者可以通过远程面板完成，也可以在固定强度（TTL 信号）下自动完成。</li>
	<li>调整 PMT 放大器的增益和偏移，以确保信号不饱和，并针对录制系统的检测范围进行优化。</li>
	<li>记录 10 次扫描，确保检测到稳定的行动潜力。</li>
	<li>继续录制并立即移动显微镜阶段，以便从没有细胞的区域短暂获取背景信号。关掉激发灯。 注:此背景值（F偏移 ）将用于计算任何背景荧光。</li>
	<li>如果需要，本地香水参考药物，如1μM尼菲丁，以确定细胞对药理操作的反应。</li>
	<li>以顺序方式，每次选择新单元格时重复步骤 5.2-5.7。如果需要，则更换盖片，以确保单次试用率高。 注: 图2中描述了加载和图像采集协议。</li>
</ol>6. 数据分析
<ol><li>使用分析软件打开保存的记录，平均 10 次扫描，其中包含来自单个单元格的刺激作用电位。</li>
	<li>以代表 F偏移 的基线信号的表示值为例，并从平均跟踪中减去此值。</li>
	<li>使用以下公式计算∆F/F0（其中F测量荧光，F0测量舒张荧光）: <img alt="Equation 3" src="/files/ftp_upload/61890/61890equ03v2.jpg">
</li>
	<li>识别微量基线（舒张）和兴趣区域（AP），并测量所需的心脏动作潜在参数。这包括但不限于50%（APD50）和90%（APD90）再极化的衰变时间。</li>
	<li>将来自该单个单元格的数据导出到电子表格软件。</li>
	<li>所有录音重复步骤 6.1 或 6.5。通过适当的未修复测试或差异分析来评估结果。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61890/61890fig2v2.jpg"> 图 2:加载和图像采集协议。（A） iPSC-CMs 和原生心肌细胞完整 VF2.1Cl 加载协议的流程图。（B） 本协议中用于激发和检测 VF2.1Cl 发射的光束分离器 （BS） 和过滤器配置的简化示意图，以响应跨膜电压的变化。 <a href="https://www.jove.com/files/ftp_upload/61890/61890fig2v2large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

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R., 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=All-optical+electrophysiology+in+mammalian+neurons+using+engineered+microbial+rhodopsins.">All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins.</a> Nature Methods. 11 (8), 825-833 (2014).</li><li>Huang, Y. L., Walker, A. S., Miller, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photostable+silicon+rhodamine+platform+for+optical+voltage+sensing.">A photostable silicon rhodamine platform for optical voltage sensing.</a> Journal of the American Chemical Society. 137 (33), 10767-10776 (2015).</li><li>Deal, P. E., Kulkarni, R. U., Al-Abdullatif, S. H., Miller, E. 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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-sensitive+fluorescence+of+amphiphilic+hemicyanine+dyes+in+neuron+membrane.">Voltage-sensitive fluorescence of amphiphilic hemicyanine dyes in neuron membrane.</a> Biochimica et Biophysica Acta. 1150 (2), 111-122 (1993).</li><li>Salama, 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=Properties+of+new,+long-wavelength,+voltage-sensitive+dyes+in+the+heart.">Properties of new, long-wavelength, voltage-sensitive dyes in the heart.</a> Journal of Membrane Biology. 208 (2), 125-140 (2005).</li><li>Jin, 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=Single+action+potentials+and+subthreshold+electrical+events+imaged+in+neurons+with+a+fluorescent+protein+voltage+probe.">Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe.</a> Neuron. 75 (5), 779-785 (2012).</li><li>Kralj, J. M., Douglass, A. D., Hochbaum, D. R., MacLaurin, D., Cohen, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+recording+of+action+potentials+in+mammalian+neurons+using+a+microbial+rhodopsin.">Optical recording of action potentials in mammalian neurons using a microbial rhodopsin.</a> Nature Methods. 9 (1), 90-95 (2012).</li><li>Tsutsui, H., Karasawa, S., Okamura, Y., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+membrane+voltage+measurements+using+FRET+with+new+fluorescent+proteins.">Improving membrane voltage measurements using FRET with new fluorescent proteins.</a> Nature Methods. 5 (8), 683-685 (2008).</li><li>Lundby, A., Mutoh, H., Dimitrov, D., Akemann, W., Kn&#246;pfel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+a+genetically+encodable+fluorescent+voltage+sensor+exploiting+fast+Ci-VSP+voltage-sensing+movements.">Engineering of a genetically encodable fluorescent voltage sensor exploiting fast Ci-VSP voltage-sensing movements.</a> PLoS One. 3 (6), 2514 (2008).</li><li>Bradley, J., Luo, R., Otis, T. S., DiGregorio, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submillisecond+optical+reporting+of+membrane+potential+in+situ+using+a+neuronal+tracer+dye.">Submillisecond optical reporting of membrane potential in situ using a neuronal tracer dye.</a> The Journal of neuroscience. 29 (29), 9197-9209 (2009).</li><li>Herron, T. J., Lee, P., Jalife, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+voltage+and+calcium+in+cardiac+cells+&amp;+tissues.">Optical imaging of voltage and calcium in cardiac cells &amp; tissues.</a> Circulation Research. 110 (4), 609-623 (2012).</li><li>Kappadan, V., 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-resolution+optical+measurement+of+cardiac+restitution,+contraction,+and+fibrillation+dynamics+in+beating+vs.+blebbistatin-uncoupled+isolated+rabbit+hearts.">High-resolution optical measurement of cardiac restitution, contraction, and fibrillation dynamics in beating vs. blebbistatin-uncoupled isolated rabbit hearts.</a> Frontiers in Physiology. 11, 464 (2020).</li><li>Kettlewell, S., Walker, N. L., Cobbe, S. M., Burton, F. L., Smith, G. 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在这里，我们描述了一个基本协议，以轻松获得详细的AP配置文件从孤立的iPSC-CMs适合电生理建模和心脏药物筛查。我们从我们稀疏的种子 iPSC-CM 中检测出定期的、坚固的 APs，这表明指标功能和方法保真度。
由于iPSC重新编程的商业方法范围广泛，心脏分化协议缺乏标准化，基于iPSC的模型在功能和形态16方面具有巨大的变异性。这也会妨碍心毒性研究的有效?...

心肌细胞的电生理建模和心脏药物筛查高效平台的建设对于制定各种心律失常的治疗策略至关重要。诱导多能干细胞（iPSC）技术的迅速扩展，利用分离的患者衍生心肌细胞（iPSC-CM）在人类疾病建模和药理学研究方面取得了可喜的成效。通过贴片夹（电流夹）对这些细胞进行电生理特征的"黄金标准"技术可以量化作用潜力（AP）的形态和持续时间，然而，这种方法极其复杂和缓慢，不适合高吞吐量数据采集1。iPSC-CMs 经常被报告有增加的舒张膜潜力和增加泄漏电流相比，成人本地心肌细胞2。有人建议，在iPSC-CMs中观察到的更小的细胞大小和更小的膜电容在使用电流夹技术时可能会产生一些系统性错误，或许可以解释这些偏差3。为了最大限度地提高 iPSC-CM 平台的效用，在 iPSC-CM 中单个单元格级别上对跨膜电压变化进行描述时，采用另一种方法具有提高吞吐量和确保数据准确性的宝贵价值。
电压敏感染料 （VSD） 长期以来一直是一种建议的方法，提供更快，非侵入性和等效的心脏AP动能分析相比，传统技术4。最近的一项研究已经证明了比率电压敏感探针光度测量是否适合精确量化心脏AP5。此外，能够轻松扩展光学光度测量方法，使这项技术能够扩展到在治疗药物开发中至关重要的大型心毒性屏幕（例如 CiPA）。利用微电极阵列和电压感应光学技术进行盲目多现场研究，开发标准化心毒性协议，证明了这种方法的关键价值。
许多强力计量染料在商业上都有售，而新探针的不断合成开发显示出在各种心脏和神经结构中简化其有效性令人振奋的潜力。理想的VSD将具有增强的动能和灵敏度，同时显示电容负荷降低、光出血和细胞毒性。最近合成的VF2.1Cl（FluoVolt）表达了许多这些有益的属性，这主要是因为其新的基于电线的分子结构，由新的电压流体（VF）家族7的其他成员共享。与普通电色VSD（在这种电色中，简单的探头在分子和电学上与等离子膜结合）不同，这种染料由一条被动插入的膜跨膜合成线组成，该线将富含电子的捐赠者与经过改良的荧光素氟磷（FITC）配对。机械细节在 图1中提供。这种染料表现出对膜电压波动的极高灵敏度，显示每100 mV的发射强度变化27%，而其他常见探针以可比速度7的±10%变化。此外，基于电线的PET系统不会直接与细胞电场相互作用，而蜂窝电场产生的电干扰最小，细胞电容负荷的变化也微乎其微。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61890/61890fig01.jpg"> 图1:VF2.1Cl染料的化学、光谱和机械参数。（A） VF2.1Cl.分子特征的化学结构需要注意，包括苯乙烯分子线内的多个烷基组，这有利于插入等离子体膜。与FITC探针结合的负电压硫酸组可确保细胞外表面的氟醇稳定，并有助于相对于脂质双层的电场接近垂直插入。（B） 垂直VF2.1Cl嵌入目标细胞等离子膜的简化示意图。（C） VF2.1Cl 染料的吸收和发射光谱。光谱与标准 FITC 和 GFP 探头相同。（D） VF2.1Cl的机械动作模式的描述。在休息条件下（超极化），负细胞内电压将自由电子驱动到荧光管。电子丰度确保光诱导电子转移（PET）被青睐为在光学激发后走出兴奋状态的途径，有效抑制荧光。相比之下，去极化膜潜在影响向下电子运动，有利于荧光对光学激发。由此产生的荧光反应与膜电压有线性关系，可精确利用它来收集有关细胞电生理动力学的详细时间信息。（E） 代表亮场（上）和荧光在470纳米（下）图像的麻风病心肌细胞加载VF2.1Cl.（F）Z堆栈的单载心肌细胞。箭头表示 VF2.1Cl 到细胞膜的清晰定位区域。图像是用一个由X光v3旋转盘共焦头和50μm针孔图案组成的旋转圆盘共焦系统获得的;LDI-7 照明器;总理95B相机和普莱纳波兰姆达100倍的目标。缩放栏:20μm。<a href="https://www.jove.com/files/ftp_upload/61890/61890fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
FITC 探头与 VF2.1Cl 结合，可确保在标准和 GFP 滤清器配置下有效使用，并且只需要一个通道采集系统，这两个系统都是荧光成像平台的共同功能。最近有报道说，这种染料的致密人类iPSC-CM单层分析有8、9、10、11。我们的协议与这些研究不同，因为我们对单个、孤立的 iPSC-CMs 进行了调查，不受密集同步单层的电气和参数影响，以及我们使用经济实惠且可定制的光度测量系统，而不是复杂的共聚焦或广域成像安排。
在这里，我们描述了我们的协议，快速获取和分析强大的光学APs从孤立的人类iPSC衍生心肌细胞和原生心肌细胞（见 补充文件）。我们使用 VF2.1Cl 以及可定制的单细胞光度测量技术平台。这些实验方案已得到哥廷根大学医学中心伦理委员会（第10/9/15号）的批准。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.25 Trypsin EDTA&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>25200056</td><td></td></tr><tr><td>B27 Supplement&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>17504044</td><td></td></tr><tr><td>CaCl2</td><td>Carl Roth&amp;nbsp;</td><td>HN04.2</td><td></td></tr><tr><td>D(+)-Glucose anhydrous&amp;nbsp;&lt;em&gt;BioChemica&lt;/em&gt;</td><td>ITW Reagents</td><td>A1422</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>10270-106</td><td></td></tr><tr><td>FluoVolt Membrane Potential Kit&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</td><td>F10488</td><td></td></tr><tr><td>HEPES</td><td>Carl Roth&amp;nbsp;</td><td>HN77.4</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>6781.1</td><td></td></tr><tr><td>Lamanin</td><td>Sigma-Aldrich</td><td>114956-81-9</td><td></td></tr><tr><td>Matrigel&amp;nbsp;</td><td>BD</td><td>354230</td><td></td></tr><tr><td>NaCl&amp;nbsp;</td><td>Sigma-Aldrich</td><td>9265.2</td><td></td></tr><tr><td>Nifedipine&amp;nbsp;</td><td>Sigma-Aldrich</td><td>21829-25-4</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Invitrogen&amp;nbsp;</td><td>15140</td><td></td></tr><tr><td>ROCK Inhibitor Y27632</td><td>Stemolecule</td><td>04-0012-10</td><td></td></tr><tr><td>RPMI 1640 Medium&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>61870010</td><td></td></tr><tr><td>Versene EDTA&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>15040033</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>495LP Dichroic Beamsplitter&amp;nbsp;</td><td>Chroma Technology</td><td></td><td></td></tr><tr><td>Axopatch 200B Amplifier&amp;nbsp;</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Circle Coverslips, Thickness 0</td><td>Thermo Scientific</td><td>CB00100RA020MNT0</td><td></td></tr><tr><td>Digidata 1550B</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Dual OptoLED Power Supply&amp;nbsp;</td><td>Cairn Research&amp;nbsp;</td><td></td><td></td></tr><tr><td>ET470/40x Excitation Filter&amp;nbsp;</td><td>Chroma Technology</td><td></td><td></td></tr><tr><td>ET535/50m</td><td>Chroma Technology</td><td></td><td></td></tr><tr><td>Etched Neubauer Hemacytometer</td><td>Hausser Scientific</td><td></td><td></td></tr><tr><td>Filter Cubes&amp;nbsp;</td><td>Cairn Research&amp;nbsp;</td><td></td><td></td></tr><tr><td>IX73 Inverted Microscope&amp;nbsp;</td><td>Olympus&amp;nbsp;</td><td></td><td></td></tr><tr><td>MonoLED</td><td>Cairn Research&amp;nbsp;</td><td></td><td></td></tr><tr><td>Multiport Adaptors&amp;nbsp;</td><td>Cairn Research&amp;nbsp;</td><td></td><td></td></tr><tr><td>Myopacer Cell Stimulator&amp;nbsp;</td><td>IonOptix</td><td></td><td></td></tr><tr><td>Optomask Shutter&amp;nbsp;</td><td>Cairn Research&amp;nbsp;</td><td></td><td></td></tr><tr><td>Optoscan System Controller</td><td>Cairn Research&amp;nbsp;</td><td></td><td></td></tr><tr><td>PH-1 Temperature Controlled Platform&amp;nbsp;&amp;nbsp;</td><td>Warner Instruments&amp;nbsp;</td><td></td><td></td></tr><tr><td>Photomultiplier Detector&amp;nbsp;</td><td>Cairn Research&amp;nbsp;</td><td></td><td></td></tr><tr><td>PMT Amplifier Insert</td><td>Cairn Research&amp;nbsp;</td><td></td><td></td></tr><tr><td>PMT Supply Insert</td><td>Cairn Research&amp;nbsp;</td><td></td><td></td></tr><tr><td>RC-26G Open Bath Chamber&amp;nbsp;</td><td>Warner Instruments&amp;nbsp;</td><td></td><td></td></tr><tr><td>SA-OLY/2AL Stage Adaptor&amp;nbsp;</td><td>Olympus&amp;nbsp;</td><td></td><td></td></tr><tr><td>T565lpxr Dichroic Beamsplitter&amp;nbsp;</td><td>Chroma Technology</td><td></td><td></td></tr><tr><td>T660lpxr Dichroic Beamsplitter</td><td>Chroma Technology</td><td></td><td></td></tr><tr><td>TC-20 Dual Channel Temperature Controller&amp;nbsp;</td><td>npi Electronic</td><td></td><td></td></tr><tr><td>UPLFLN 40X Objective</td><td>Olympus&amp;nbsp;</td><td></td><td></td></tr><tr><td>USB 3.0 Colour Camera&amp;nbsp;</td><td>Imaging Source</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Clampex 11.1</td><td>Molecular Devices&amp;nbsp;</td><td></td><td></td></tr><tr><td>Clampfit 11.1</td><td>Molecular Devices&amp;nbsp;</td><td></td><td></td></tr><tr><td>IC Capture 2.4&amp;nbsp;</td><td>Imaging Source&amp;nbsp;</td><td></td><td></td></tr><tr><td>Prism 8</td><td>Graphpad</td><td></td><td></td></tr></tbody></table>

single-cell optical action potential measurement in human induced pluripotent stem cell-derived cardiomyocytes

传统的细胞内微电极技术，量化心肌细胞电生理学是非常复杂的，劳动密集型，通常进行低吞吐量。诱导多能干细胞（iPSC）技术的快速和持续扩展为心血管研究提出了新的标准，现在需要替代方法，以提高单个细胞水平的电生理数据吞吐量。VF2.1Cl 是一种最近衍生的电压敏感染料，它提供快速的单通道，对膜电位波动的高级响应。它具有优于其他现有电压指标的动力学，并提供与传统微电极技术相当的功能数据。在这里，我们展示了简化的，非侵入性的行动潜在特征在外部节奏的人类iPSC衍生心肌细胞使用模块化和高度负担得起的光测量系统。

<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61890/61890fig03.jpg"> 图3:分离的原生心肌细胞和人类诱导的多能干细胞衍生心肌细胞（iPSC-CM）的光学作用电位（AP）图谱。（A） 单个穆林心肌细胞（中心）的代表光学 AP，平均± APD 50 和 APD90（n = 7，右）。（B） 单个人类 iPSC 的代表性光学 AP 衍生心肌细胞（中），平均± APD ...

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

在这里，我们使用高速模块化光测量系统描述从诱导多能干细胞衍生心肌细胞中获得的光学采集和作用潜力的特征。

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 ...

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JoVE Journal

Bioengineering

4.1K 次观看.  University Medical Center Goettingen, Goettingen, Germany. 在这里，我们使用高速模块化光测量系统描述从诱导多能干细胞衍生心肌细胞中获得的光学采集和作用潜力的特征。

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

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

在小鼠心脏的动作电位和钙瞬变的光学测绘

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

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)

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 high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

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

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and 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 ...

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs

Electrophysiological characterization of cardiomyocytes derived from human Pluripotent Stem Cells (hPSC-CMs) is crucial for cardiac disease modeling and for determining drug responses. This protocol provides the necessary information to dissociate and plate hPSC-CMs on multi-electrode arrays, measure their field potential, and a method for analyzing QT and RR intervals.

使用多电极阵列（MEA）的人多能干细胞来源的心肌细胞（hPSC-CM）的电生理分析

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived ...

发育生物学

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

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput electrophysiological approaches for hiPSC-derived cardiomyocyte (hiPSC-CM) preparations is much needed for efficient drug testing. Although multielectrode arrays (MEAs) are frequently employed for field potential measurements of excitable cells, a recent publication by Joshi-Mukherjee and colleagues described and validated its application for recurrent action potential (AP) recordings from the same hiPSC-CM preparation over days. The aim here is to provide detailed step-by-step methods for seeding CMs and for measuring AP waveforms via electroporation with high precision and a temporal resolution of 1 &#181;s. This approach addresses the lack of easy-to-use methodology to gain intracellular access for high-throughput AP measurements for reliable electrophysiological investigations. A detailed work flow and methods for plating of hiPSC-CMs on multiwell MEA plates are discussed emphasizing critical steps wherever relevant. In addition, a custom-built MATLAB script for rapid data handling, extraction and analysis is reported for comprehensive investigation of the waveform analysis to quantify subtle differences in morphology for various AP duration parameters implicated in arrhythmia and cardiotoxicity.

This article contains a set of protocols for the development of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) networks cultured on multiwell MEA plates to reversibly electroporate the cell membrane for action potential measurements. High-throughput recordings are obtained from the same cell sites repeatedly over days.

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 ...

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

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical cardiotoxicity testing. In two-dimensional monolayer formats, hiPSC-CMs recapitulate the structure and function of the adult human heart muscle cells when cultured on an optimal extracellular matrix (ECM). A human perinatal stem cell-derived ECM (maturation-inducing extracellular matrix-MECM) matures the hiPSC-CM structure, function, and metabolic state in 7 days after plating. 
Mature hiPSC-CM monolayers also respond as expected to clinically relevant medications, with a known risk of causing arrhythmias and cardiotoxicity. The maturation of hiPSC-CM monolayers was an obstacle to the widespread adoption of these valuable cells for regulatory science and safety screening, until now. This article presents validated methods for the plating, maturation, and high-throughput, functional phenotyping of hiPSC-CM electrophysiological and contractile function. These methods apply to commercially available purified cardiomyocytes, as well as stem cell-derived cardiomyocytes generated in-house using highly efficient, chamber-specific differentiation protocols. 
High-throughput electrophysiological function is measured using either voltage-sensitive dyes (VSDs; emission: 488 nm), calcium-sensitive fluorophores (CSFs), or genetically encoded calcium sensors (GCaMP6). A high-throughput optical mapping device is used for optical recordings of each functional parameter, and custom dedicated software is used for electrophysiological data analysis. MECM protocols are applied for medication screening using a positive inotrope (isoprenaline) and human Ether-a-go-go-related gene (hERG) channel-specific blockers. These resources will enable other investigators to successfully utilize mature hiPSC-CMs for high-throughput, preclinical cardiotoxicity screening, cardiac medication efficacy testing, and cardiovascular research.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an alternative to using animals for preclinical cardiotoxicity screening. A limitation to the widespread adoption of hiPSC-CMs in preclinical toxicity screening is the immature, fetal-like phenotype of the cells. Presented here are protocols for robust and rapid maturation of hiPSC-CMs.

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 ...

A Cardiac Microphysiological System for Studying Ca2+ Propagation via Non-genetic Optical Stimulation

In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely accepted by the scientific community, these systems are still limited in longevity due to the absence of non-invasive stimulation techniques. Phototransducers provide an efficient stimulation method, offering a wireless approach with high temporal and spatial resolution while minimizing invasiveness in stimulation processes. In this manuscript, we present a fully optical method for stimulating and detecting the activity of an in vitro cardiac microphysiological model. Specifically, we fabricated engineered laminar anisotropic tissues by seeding human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated in a 3D bioreactor suspension culture. We employed a phototransducer, an amphiphilic azobenzene derivative, named Ziapin2, for stimulation and a Ca2+ dye (X-Rhod 1) for monitoring the system&#39;s response. The results demonstrate that Ziapin2 can photomodulate Ca2+ responses in the employed system without compromising tissue integrity, viability, or behavior. Furthermore, we showed that the light-based stimulation approach offers a similar resolution compared to electrical stimulation, the current gold standard. Overall, this protocol opens promising perspectives for the application of Ziapin2 and material-based photostimulation in cardiac research.

Using light to control cardiac cells and tissue enables non-contact stimulation, thereby preserving the natural state and function of the cells, making it a valuable approach for both basic research and therapeutic applications.

一种通过非遗传光刺激研究 Ca2+ 传播的心脏微生理系统

In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely ...

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.

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.

微电极阵列和膜片钳记录在人诱导多能干细胞来源心肌细胞上的技术应用

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

医学

Laser-Induced Action Potential-Like Measurements of Cardiomyocytes on Microelectrode Arrays for Increased Predictivity of Safety Pharmacology

Life-threatening drug-induced cardiac arrhythmia is often preceded by prolonged cardiac action potentials (AP), commonly accompanied by small proarrhythmic membrane potential fluctuations. The shape and time course of the repolarizing fraction of the AP can be pivotal for the presence or absence of arrhythmia.
Microelectrode arrays (MEA) allow easy access to cardiotoxic compound effects via extracellular field potentials (FP). Although a powerful and well-established tool in research and cardiac safety pharmacology, the FP waveform does not allow to infer the original AP shape due to the extracellular recording principle and the resulting intrinsic alternating current (AC) filtering.
A novel device, described here, can repetitively open the membrane of cardiomyocytes cultivated on top of the MEA electrodes at multiple cultivation time points, using a highly focused nanosecond laser beam. The laser poration results in transforming the electrophysiological signal from FP to intracellular-like APs (laser-induced AP, liAP) and enables the recording of transcellular voltage deflections. This intracellular access allows a better description of the AP shape and a better and more sensitive classification of proarrhythmic potentials than regular MEA recordings. This system is a revolutionary extension to the existing electrophysiological methods, permitting accurate evaluation of cardiotoxic effect with all advantages of MEA-based recordings (easy, acute, and chronic experiments, signal propagation analysis, etc.).

The combination of laser poration and microelectrode arrays (MEA) allows action potential-like recordings of cultivated primary and stem cell-derived cardiomyocytes. The waveform shape provides superior insight into test compounds' mode of action than standard recordings. It links patch-clamp and MEA readout to further optimize cardio safety research in the future.

激光诱导动作电位样测量微电极阵列上的心肌细胞，以提高安全药理学的预测性

Life-threatening drug-induced cardiac arrhythmia is often preceded by prolonged cardiac action potentials (AP), commonly accompanied by small ...

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a homogenous population of cells, the iPSC-CMs generated by current differentiation protocols represent a mixture of cells with ventricular-, atrial-, and nodal-like phenotypes, which complicates phenotypic analyses. Here, a method to optically record action potentials specifically from ventricular-like iPSC-CMs is presented. This is achieved by lentiviral transduction with a construct in which a genetically-encoded voltage indicator is under the control of a ventricular-specific promoter element. When iPSC-CMs are transduced with this construct, the voltage sensor is expressed exclusively in ventricular-like cells, enabling subtype-specific optical membrane potential recordings using time-lapse fluorescence microscopy.

Here we present a method to optically image action potentials, specifically in ventricular-like induced pluripotent stem cell-derived cardiomyocytes. The method is based on the promoter-driven expression of a voltage-sensitive fluorescent protein.

人诱导的多潜能干细胞源性心室心肌细胞亚型特异光学动作电位记录

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a ...

生物学

实时测量 hiPSC-CMs 中的钙和收缩力参数

Real-Time Measurements of Calcium and Contractility Parameters in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful tool for studying mutation-mediated changes in cardiomyocyte function and defining the effects of stressors and drug interventions. In this study, it is demonstrated that this optics-based system is a powerful tool to assess the functional parameters of hiPSC-CMs in 2D. By using this platform, it is possible to perform paired measurements in a well-preserved temperature environment on different plate layouts. Moreover, this system provides researchers with instant data analysis.
This paper describes a method for measuring the contractility of unmodified hiPSC-CMs. Contraction kinetics are measured at 37 &#176;C based on pixel correlation changes relative to a reference frame taken at relaxation at a 250 Hz sampling frequency. Additionally, simultaneous measurements of intracellular calcium transients can be acquired by loading the cell with a calcium-sensitive fluorophore, such as Fura-2. Using a hyperswitch, ratiometric calcium measurements can be performed on a 50 &#181;m diameter illumination spot, corresponding to the area of the contractility measurements.

作者聚焦:实时测量人诱导多能干细胞来源的心肌细胞中的钙和收缩力参数  

Here, an established method is described to perform contractility and calcium measurements in human induced pluripotent stem cell-derived cardiomyocytes using an optics-based platform. This platform enables researchers to study the effect of mutations and the response to various stimuli in a rapid and reproducible manner.

实时测量人诱导多能干细胞来源心肌细胞中的钙和收缩力参数

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful tool for studying mutation-mediated changes in cardiomyocyte ...

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

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