Name: preclinical cardiac electrophysiology assessment by dual voltage and calcium optical mapping of human organotypic cardiac slices
Uploaded: 2020-06-16T13:30:00
Description: Watch this Scientific Journal Video about Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices at JoVE.com

由NIH资助（资助R21 EB023106，R44 HL139248和R01 HL126802），由利杜克基金会（项目RHYTHM）和美国心脏协会博士后奖学金（19POST34370122）感谢。

所述的所有方法都符合所有机构、国家和国际人类福利准则。研究得到了乔治华盛顿大学机构审查委员会（IRB）的批准。
注：经乔治华盛顿大学IRB批准，从华盛顿地区移植社区获得捐赠人的心脏作为被解认的废弃组织。通过用冰冷心性心肌痛溶液冲洗心脏（在此过程中血液从心脏被清除），并在标准器官移植条件下转移到实验室，使心脏被心肺复苏。
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<ol>
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在这里，我们提出了分步的方法，从心肌梗活的人类心脏获得可行的心脏切片，并使用跨膜电位和细胞内钙的双重光学映射功能地描述切片。通过保存的细胞外环境和原生细胞耦合，人类心脏切片可用作人类心脏的准确模型，用于基本科学发现，以及药理剂和基因疗法的疗效和心毒性测试。该技术还允许对心脏的特定区域进行结构功能映射，如心体节点、三角结和Purkinje纤维。此处描述的协议列?...

动物模型是了解人类生理学和病理生理学基本机制的宝贵工具，也是初步测试治疗各种疾病疗法的平台。基于这些动物研究的生物医学研究领域取得了长足的进步。然而，人类和动物生理之间存在着显著的物种间差异，包括小鼠、大鼠、豚鼠、兔子、绵羊、猪和狗33、4。4因此，在动物试验阶段，有许多药物、基因和细胞疗法显示出希望，但未能达到临床试验结果5。为了弥补这一差距，分离的心肌细胞和人类诱导多能干细胞（iPSCs）被开发为模型，以测试人类生理对各种药物和疾病的反应6。干细胞衍生心肌细胞作为心脏66、7、87,8的代用品，已广泛应用于芯片器官系统。然而，iPSC衍生的心肌细胞（iPSC-CMs）的效用因其相对不成熟的表型和心肌细胞亚群的代表性而受到阻碍;成熟的心肌是一种复杂的结构，由多种共存的细胞类型组成，如成纤维细胞、神经元、巨噬细胞和内皮细胞。另一方面，分离的人类心肌细胞在电成熟，通过改变培养参数9，可以获得不同的心肌细胞亚群。然而，由于缺乏细胞-细胞耦合、快速去分化以及体外10、11,11内出现多节律行为，这些肌细胞通常表现出改变的动作潜力形态。iPSC-CM 和心形肌细胞的 3D 细胞培养模型解决了一些限制。这些模型包括球菌、水凝胶支架封装的 3D 培养物、工程心脏组织 （EHT） 和片上心脏系统，使用多个心脏细胞群，如心肌细胞、成纤维细胞和内皮细胞。它们要么自组装，要么沿着脚手架组装形成3D结构，有些甚至重现了心肌的复杂各向异性。据报道，这些模型具有与心脏组织相似的成熟表型、收缩特性和分子轮廓的细胞。片上心脏系统还允许研究药物测试和疾病模型中的系统效应。然而，体外细胞基模型缺乏原生细胞外基质，因此不能准确模拟器官水平电生理学。相比之下，人类心脏切片具有完整的细胞外基质和原生细胞对细胞的接触，因此有助于更准确地检查人类心肌的心律失常特性。
研究人员已开发出人心形器官切片，作为急性和慢性药物测试的生理前临床平台，并研究心脏电生理学和心脏病进展12、13、14、15、16、17、18、19。12,13,14,15,16,17,18,19与 iPSC 衍生的心肌细胞相比，人类心脏切片更忠实地复制了成熟的心肌细胞表型成人心脏电生理学。与分离的人类心肌细胞相比，心脏切片表现出生理作用潜力持续时间，因为细胞耦合保存完好，并且其原生细胞内和细胞外环境存在。
该协议描述了从整个供体心脏生成人类心脏切片的过程，进行急性（即数小时）和慢性（即数天）研究，通过光学映射测试心脏电生理学参数的过程。虽然该协议只描述了左心室（LV）组织的使用，但它已成功应用于心脏的其他地区以及其他物种，如小鼠、大鼠、豚鼠和猪14、20、21、22。14,20,21,22我们的实验室使用过去5年被拒绝移植的全身供体心脏，但只要组织能够分割成立方体，这些相同的程序可以用于通过其他手段获得的任何供体心脏样本组织（例如左心室辅助装置[LVAD]植入、活检、骨髓切除术）。光学映射用于本研究的分析，因为光学映射具有高空间（100 x 100 像素）和时间（>1，000 帧/秒）分辨率的光学动作电位和钙瞬变。也可以使用替代方法，例如多电极阵列 （MEA） 或微电极，但这些技术受到空间分辨率相对较低的限制。此外，MEA 被设计为用于细胞培养物，锋利的微电极更易于管理，用于整个心脏或大型组织楔块。
本文的目标是让更多的研究人员使用人类心脏组织进行心脏电生理学研究。需要注意的是，本文中描述的技术相对简单，对短期研究（按几个小时到几天的顺序）有益。其他一些研究12、18、23,18,23已经讨论和描述了用于长期研究的更多生理生物仿生培养（周数）。电刺激、机械加载和组织拉伸是有利的调节机制，可以帮助限制体外组织重塑12、18、2318,23的12发病。

<table>
	<tbody>
		<tr>
			<td>1mL BD Syringe</td>
			<td>Thomas Scientific</td>
			<td>309597</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3-butanedione monoxime</td>
			<td>Sigma-Aldrich</td>
			<td>B0753</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well culture plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet</td>
			<td>ThermoFisher Scientific</td>
			<td>1377</td>
			<td></td>
		</tr>
		<tr>
			<td>Blebbistatin</td>
			<td>Cayman</td>
			<td>13186</td>
			<td></td>
		</tr>
		<tr>
			<td>Bubble Trap</td>
			<td>Radnoti</td>
			<td>130149</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Cell Strainers</td>
			<td>Fisher Scientific</td>
			<td>07-201-432</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-4-ANEPPS</td>
			<td>Biotium</td>
			<td></td>
			<td>stock solution at 1.25 mg/mL in DMSO</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #3c Forceps</td>
			<td>Fine Science Tools</td>
			<td>11231-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Emission dichroic mirror</td>
			<td>Chroma</td>
			<td>T630LPXR-UF1</td>
			<td></td>
		</tr>
		<tr>
			<td>Emission filter (RH237)</td>
			<td>Chroma</td>
			<td>ET690/50m</td>
			<td></td>
		</tr>
		<tr>
			<td>Emission Filter (Rhod2AM)</td>
			<td>Chroma</td>
			<td>ET590/33m</td>
			<td></td>
		</tr>
		<tr>
			<td>Excitation dichroic mirror</td>
			<td>Chroma</td>
			<td>T550LPXR-UF1</td>
			<td></td>
		</tr>
		<tr>
			<td>Excitation Filter</td>
			<td>Chroma</td>
			<td>ET500/40x</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50mL Conical Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-49A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat exchanger</td>
			<td>Radnoti</td>
			<td>158821</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>ThermoFisher Scientific</td>
			<td>50145502</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Transferrin Selenium (ITS)</td>
			<td>Sigma-Aldrich</td>
			<td>I3146</td>
			<td></td>
		</tr>
		<tr>
			<td>LED excitation light source</td>
			<td>Prizmatix</td>
			<td>UHP-Mic-LED-520</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnessium chloride hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M9272</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>ThermoFisher Scientific</td>
			<td>11150059</td>
			<td></td>
		</tr>
		<tr>
			<td>Micam Ultima L type CMOS camera</td>
			<td>Scimedia</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Minutien Pins</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Pennicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic Pump</td>
			<td>Cole Parmer</td>
			<td>EW-07522-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum pacing wire</td>
			<td>Alfa Aesar</td>
			<td>43275</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F127</td>
			<td>ThermoFisher Scientific</td>
			<td>P6867</td>
			<td>nonionic, surfactant polyol</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Powerlab data acquisition and stimulator</td>
			<td>AD Instruments</td>
			<td>Powerlab 4/26</td>
			<td></td>
		</tr>
		<tr>
			<td>RH237</td>
			<td>Biotium</td>
			<td>61018</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhod2AM</td>
			<td>ThermoFisher Scientific</td>
			<td>R1245MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhod-2AM</td>
			<td>Invitrogen, Carlsbad, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9625</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilizer, dry bead</td>
			<td>Sigma-Aldrich</td>
			<td>Z378550</td>
			<td></td>
		</tr>
		<tr>
			<td>Stone Oxygen Diffuser</td>
			<td>Waterwood</td>
			<td>B00O0NUVM0</td>
			<td></td>
		</tr>
		<tr>
			<td>TissueSeal - Histoacryl Topical Skin Adhesive</td>
			<td>gobiomed</td>
			<td>AESCULAP</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Low Melting Point Agarose</td>
			<td>Thermo Fisher Scientific</td>
			<td>16520100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound sonicator</td>
			<td></td>
			<td>Branson 1800</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Campden Instruments</td>
			<td>7000 smz</td>
			<td></td>
		</tr>
	</tbody>
</table>

preclinical cardiac electrophysiology assessment by dual voltage and calcium optical mapping of human organotypic cardiac slices

人类心脏切片制剂最近被开发为人类生理学研究和治疗测试的平台，以弥合动物和临床试验之间的差距。许多动物和细胞模型被用来检查药物的作用，但这些反应在人类中往往不同。人类心脏切片为药物测试提供了一个优势，因为它们直接来自可行的人类心脏。除了保留了多细胞结构、细胞耦合和细胞外基质环境外，人类心脏组织切片还可用于直接测试无数药物对成人心脏生理学的影响。这种模式与其他心脏制剂（如整个心脏或楔形）的区别，是切片可以受长期文化的影响。因此，心脏切片允许研究药物的急性和慢性影响。此外，从一颗心脏收集几百到一千片切片的能力使得这是一种高通量模型，可以同时测试不同浓度和与其他药物组合的几种药物。切片可以从心脏的任何给定区域制备。在此协议中，我们描述了左心室切片的制备，通过将组织立方体从左心室自由壁中分离，并使用高精度振动微孔将它们分割成切片。然后，这些切片可以进行急性实验，以测量基线心脏电生理功能，或培养用于慢性药物研究。该协议还描述了心脏切片的双光学映射，用于同时记录跨膜电位和细胞内钙动力学，以确定被调查药物的影响。

根据上面详述的规程，从供体人类心脏的左心室收集人体器官切片，并图中所示图 1.双摄像头光学映射系统，如图 2用于直立成像配置，在切片协议完成后约 1 小时对电压和钙进行同步光学映射。使用 RHYTHM1.2 对数据进行分析（图 3），一个开源光学映射数据分析工具，以前由我们的实验室发布，可在 Github （https://github.com/optocardio...

Watch this Scientific Journal Video about Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices at JoVE.com

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

该协议描述了用于临床前药物测试的人体心脏切片切片和培养过程，并详细介绍了光学映射用于同时记录这些切片的跨膜电压和细胞内钙信号的过程。

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 ...

该协议意义重大，因为它为研究人员提供了利用捐赠者心脏进行临床前生理药物测试的技能。此组织利用电压和钙染料同时表征人体组织切片中的心脏作用电位和细胞内钙瞬变。要建立光学映射系统，将底部聚二甲基硅氧烷凝胶层的组织浴连接到灌注系统中，并在 37 摄氏度下循环一升回收溶液，用 100% 氧气通过灌注系统，以足够快的速度保持温度并防止沐浴灌注的积累。然后使用目标调整两个 CMOS 摄像机的焦点和对齐方式，并使用波长为 520 正负 5 纳米的绿色 LED 光源同时激发电压敏感度和钙指示器染料。在获得组织部分之前，混合冷冻和液体心痛溶液，将心脏组织放入冰冷的心痛浴中，并识别左心室自由壁。将预制的阿加罗斯凝胶粘附到振动器的金属组织支架的背面。在冷心肺溶液中切割一厘米的块状组织，并使用局部皮肤粘合剂快速将组织块安装到金属组织支架上，心内表面朝上。将金属支架转移到冰冷氧化切片溶液的振动池中，使组织完全被淹没，将刀片移动到组织的前边缘。打开振动器，开始用预设参数切片，丢弃前几个切片，直到刀片到达超过转形体进入光滑的心内组织。当达到实验组织时，小心地将每片转移到单独的100微米尼龙网状细胞过滤器中，在培养皿中含有室温氧化Tyrode的恢复溶液，并用网垫圈覆盖切片，防止组织切片卷曲。收集所有切片后，小心地将每片转移到六孔板的单个孔中，每孔含有三毫升 PBS。用轻轻摇动冲洗切片，然后用新鲜无菌 PBS 重复此过程三次，然后将样品转移到六孔板的单个孔中，每孔含有 3 毫升 37 摄氏度培养介质。然后将板以每分钟20转的速度放在一个加湿的培养箱中，温度为37度、30%氧和5%二氧化碳。在回收溶液中孵育20分钟后，小心地将感兴趣的切片转移到灌注系统组织浴，将四角固定到凝胶层，同时对组织进行最小的拉伸。让切片在此浴中休息约 10 分钟，然后向回收溶液储液中添加 0.3 至 0.5 微升库存 Blebbistatin。当切片在 Blebbistatin 中孵育时，在 37 度的恢复溶液中，将 30 微升的库存电压敏感染料重新组成。经过 10 分钟的 Blebbistatin 处理后，关闭泵，并在 30 秒内缓慢地将 0.2 至 0.3 毫升的工作染料溶液加载到切片表面。90 秒后，打开泵以洗掉多余的染料。在使用库存钙指示器的切片死亡后，将摄像机聚焦到切片上，以两毫秒脉冲宽度持续时间以 1.5 倍于预定起搏阈值的振幅的速度将切片以 1 赫兹的速度移动。在组织切片上放置盖玻片，使用 LED 激励光源照亮切片。然后以每秒 1，000 帧的速度记录摄像机发出的电压和钙信号。要条件电压和钙光学映射数据，在相应的分析软件中，单击条件参数并删除背景。然后调整信号调节参数，以获得最佳作用电位和钙瞬态痕迹。要计算传导速度，请选择激活图并输入开始和结束时间，以包含跟踪中的单个操作电位。然后单击区域地图并选择感兴趣的区域以显示所选区域的激活映射。接下来，选择传导速度图，然后选择开始和结束时间。根据需要根据设置调整像素间分辨率值。单击生成矢量贴图以选择感兴趣的区域并显示该区域内的传导速度向量。将显示分析中包含的均值、中位数、标准差和矢量数，以及选定区域传导速度矢量的平均传播角度。要计算动作潜在持续时间，请选择动作潜在持续时间、钙瞬态持续时间图并选择开始和结束时间以包含一个完整的动作潜力。然后单击区域操作潜在持续时间计算以选择感兴趣的区域并生成操作潜在持续时间图。要确定上升时间，请选择上升时间并选择开始和结束时间，以选择单个动作电位或钙瞬变的向上冲程。输入开始和结束百分比值，然后单击计算以选择感兴趣区域并确定上升时间分析中包含的平均值、中位数、标准偏差和像素数。要确定钙衰变，请选择钙衰变并输入开始和结束时间，以包含单个钙瞬态信号的整个衰变部分。然后单击计算 tau 以选择感兴趣的区域，并确定钙衰减时间常数分析中包含的平均值、中位数、标准偏差和像素数。在这个具有代表性的实验中，作用潜力和钙瞬态痕迹被信号调节。确定了每个像素的激活时间，并且从条件电压和钙痕迹绘制了激活时间等时映射。使用激活时间和已知的像素间分辨率计算传导速度向量。钙瞬态衰变常数是通过在钙痕迹的衰变部分上安装一个多项式来测量的。作用潜力持续时间和钙瞬态持续时间分别测量为激活时间与从细胞质中重新极化或钙去除的指定百分比之间的持续时间。还测量和映射了电压和钙痕迹的上升时间。在此代表性分析中，测试了多索鲁比辛对心脏传导的影响，从而降低横向传导速度。请务必确保切片完全在心肺恢复中，以在切片过程中最大限度地减少对组织的损害，并获得可行的切片。在协议结束时，可以冻结或固定切片进行分子评估。然后可以确定涉及电生理现象的机械通路。这种人类心脏切片模型的发展使得人类心脏组织对治疗和疾病的反应的研究填补了动物与临床研究之间的差距。在执行此技术时，当使用人体组织时，请务必佩戴适当的 PPE 并采取任何其他必要的安全预防措施。

Research

JoVE Journal

Bioengineering

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

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

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 ...

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

用超常光透射法定量测定人血清中的心脏标志物

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy

用无标签荧光显微镜评价活体、人体阻力动脉中胶原蛋白和弹性蛋白的压力依赖性 Microarchitectures

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations ...

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 ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

基于网模的无支架三维心脏组织创建方法

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Optical Imaging of Isolated Murine Ventricular Myocytes

分离的月毛心室肌细胞的光学成像

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

使用人iPSC衍生的心肌细胞，心脏成纤维细胞和内皮细胞制造3D心脏微组织阵列

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

在微流体平台内开发 3D 组织人类心脏组织

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...