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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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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凯恩研究有限公司通过支付视频文件的制作成本来支持该出版物。

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.25 Trypsin EDTA&#160;</td>
			<td>Gibco&#160;</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement&#160;</td>
			<td>Gibco&#160;</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Carl Roth&#160;</td>
			<td>HN04.2</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)-Glucose anhydrous&#160;BioChemica</td>
			<td>ITW Reagents</td>
			<td>A1422</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</td>
			<td>Gibco&#160;</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoVolt Membrane Potential Kit&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>F10488</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Carl Roth&#160;</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&#160;</td>
			<td>BD</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>9265.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Nifedipine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>21829-25-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Invitrogen&#160;</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&#160;</td>
			<td>Gibco&#160;</td>
			<td>61870010</td>
			<td></td>
		</tr>
		<tr>
			<td>Versene EDTA&#160;</td>
			<td>Gibco&#160;</td>
			<td>15040033</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>495LP Dichroic Beamsplitter&#160;</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axopatch 200B Amplifier&#160;</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&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ET470/40x Excitation Filter&#160;</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&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IX73 Inverted Microscope&#160;</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MonoLED</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multiport Adaptors&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Myopacer Cell Stimulator&#160;</td>
			<td>IonOptix</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optomask Shutter&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optoscan System Controller</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PH-1 Temperature Controlled Platform&#160;&#160;</td>
			<td>Warner Instruments&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Photomultiplier Detector&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PMT Amplifier Insert</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PMT Supply Insert</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RC-26G Open Bath Chamber&#160;</td>
			<td>Warner Instruments&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SA-OLY/2AL Stage Adaptor&#160;</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T565lpxr Dichroic Beamsplitter&#160;</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&#160;</td>
			<td>npi Electronic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UPLFLN 40X Objective</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>USB 3.0 Colour Camera&#160;</td>
			<td>Imaging Source</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clampex 11.1</td>
			<td>Molecular Devices&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clampfit 11.1</td>
			<td>Molecular Devices&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IC Capture 2.4&#160;</td>
			<td>Imaging Source&#160;</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± AP...

Watch this Scientific Journal Video about Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes at JoVE.com

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 ...

single-cell-optical-action-potential-measurement-human-induced

Research

JoVE Journal

Bioengineering

4.0K 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 ...

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

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic 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 ...

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 ...

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

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney disease and eventual end-stage renal failure are often caused by the damage to the glomerular podocytes, which are the highly specialized epithelial cells that function together with endothelial cells and the glomerular basement membrane to form the kidney&#8217;s filtration barrier. Advances in renal medicine have been hindered by the limited availability of primary tissues and the lack of robust methods for the derivation of functional human kidney cells, such as podocytes. The ability to derive podocytes from renewable sources, such as stem cells, could help advance current understanding of the mechanisms of human kidney development and disease, as well as provide new tools for therapeutic discovery. The goal of this protocol was to develop a method to derive mature, post-mitotic podocytes from human induced pluripotent stem (hiPS) cells with high efficiency and specificity, and under chemically defined conditions. The hiPS cell-derived podocytes produced by this method express lineage-specific markers (including nephrin, podocin, and Wilm&#8217;s Tumor 1) and exhibit the specialized morphological characteristics (including primary and secondary foot processes) associated with mature and functional podocytes. Intriguingly, these specialized features are notably absent in the immortalized podocyte cell line widely used in the field, which suggests that the protocol described herein produces human kidney podocytes that have a developmentally more mature phenotype than the existing podocyte cell lines typically used to study human kidney biology.

Presented here is a chemically defined protocol for the derivation of human kidney podocytes from induced pluripotent stem cells with high efficiency (&#62;90%) and independent of genetic manipulations or subpopulation selection. This protocol produces the desired cell type within 26 days and could be useful for nephrotoxicity testing and disease modeling.

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 ...

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

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 functional models that can accurately predict human biological responses and nephrotoxicity. Advancements in kidney precision medicine could help overcome these limitations. However, previously established in vitro models of the human kidney glomerulus-the primary site for blood filtration and a key target of many diseases and drug toxicities-typically employ heterogeneous cell populations with limited functional characteristics and unmatched genetic backgrounds. These characteristics significantly limit their application for patient-specific disease modeling and therapeutic discovery. 
This paper presents a protocol that integrates human induced pluripotent stem (iPS) cell-derived glomerular epithelium (podocytes) and vascular endothelium from a single patient to engineer an isogenic and vascularized microfluidic kidney glomerulus chip. The resulting glomerulus chip is comprised of stem cell-derived endothelial and epithelial cell layers that express lineage-specific markers, produce basement membrane proteins, and form a tissue-tissue interface resembling the kidney's glomerular filtration barrier. The engineered glomerulus chip selectively filters molecules and recapitulates drug-induced kidney injury. The ability to reconstitute the structure and function of the kidney glomerulus using isogenic cell types creates the opportunity to model kidney disease with patient specificity and advance the utility of organs-on-chips for kidney precision medicine and related applications.

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

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

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

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 ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

从多能干细胞生成人体血管类器官

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

人诱导多能干细胞来源的心脏球体的生成、高通量筛选和生物样本库

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

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 ...