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
Summary
人诱导的多能干细胞来源的心肌细胞（hiPSC-CMs）已成为药物诱导的心脏毒性筛查和疾病建模的有前途的 体外 模型。在这里，我们详细介绍了用于测量hiPSC-CMs的收缩力和电生理学的方案。
Abstract
药物引起的心脏毒性是药物损耗和退出市场的主要原因。因此，使用适当的临床前心脏安全性评估模型是药物开发过程中的关键步骤。目前，心脏安全性评估仍然高度依赖于动物研究。然而，由于物种特异性差异，特别是在心脏电生理特性方面，动物模型受到人类翻译特异性差的困扰。因此，迫切需要开发一种可靠、高效和基于人的临床前心脏安全性评估模型。人诱导的多能干细胞来源心肌细胞（hiPSC-CMs）已成为药物诱导的心脏毒性筛查和疾病建模的 宝贵体外 模型。hiPSC-CMs可以从具有不同遗传背景和各种疾病状况的个体中获得，使其成为单独评估药物诱导的心脏毒性的理想替代品。因此，需要建立全面研究hiPSC-CMs功能特征的方法。在该协议中，我们详细介绍了可以在hiPSC-CMs上评估的各种功能测定，包括收缩力，场电位，动作电位和钙处理的测量。总体而言，将hiPSC-CMs纳入临床前心脏安全性评估有可能彻底改变药物开发。
Introduction
药物开发是一个漫长而昂贵的过程。一项针对美国食品和药物管理局 （FDA） 在 2009 年至 2018 年间批准的新治疗药物的研究报告称，资本化研究和临床试验的估计成本中位数为每种产品 9.85 亿美元1。药物引起的心脏毒性是药物损耗和退出市场的主要原因2.值得注意的是，多类治疗药物中报告了心脏毒性3。因此，心脏安全性评估是药物开发过程中的关键组成部分。目前的心脏安全性评估范式仍然高度依赖于动物模型。然而，与使用动物模型的物种差异越来越被认为是人类患者药物诱导的心脏毒性预测不准确的主要原因4。例如，由于不同复极电流的贡献，人类和小鼠之间的心脏动作电位形态差异很大5。此外，可影响心脏生理学的心肌球蛋白和环状RNA的差异亚型已在物种6，7中得到充分记录。为了弥合这些差距，必须建立一个可靠、高效和基于人的临床前心脏安全性评估模型。
诱导多能干细胞（iPSC）技术的突破性发明产生了前所未有的药物筛选和疾病建模平台。在过去的十年中，产生人类诱导的多能干细胞衍生心肌细胞（hiPSC-CMs）的方法已经非常成熟8，9。hiPSC-CMs在疾病建模、药物诱导的心脏毒性筛查和精准医学中的潜在应用引起了极大的兴趣。例如，hiPSC-CMs已被用于模拟由遗传引起的心脏病的病理表型，例如长QT综合征10，肥厚型心肌病11，12和扩张型心肌病13，14，15。因此，已经确定了与心脏病发病机制有关的关键信号通路，这可以阐明有效治疗的潜在治疗策略。此外，hiPSC-CMs已被用于筛选与抗癌药物相关的药物诱导的心脏毒性，包括多柔比星，曲妥珠单抗和酪氨酸激酶抑制剂16，17，18;减轻由此产生的心脏毒性的策略正在研究中。最后，hiPSC-CMs中保留的遗传信息允许在个体和人群水平上筛选和预测药物诱导的心脏毒性19，20。总的来说，hiPSC-CMs已被证明是个性化心脏安全预测的宝贵工具。
该协议的总体目标是建立全面有效地研究hiPSC-CMs功能特征的方法，这对于将hiPSC-CMs应用于疾病建模，药物诱导的心脏毒性筛查和精准医学非常重要。在这里，我们详细介绍了一系列功能测定来评估hiPSC-CMs的功能特性，包括收缩力，场电位，动作电位和钙（Ca2+）处理的测量（图1）。
Protocol
1. 培养基和溶液的制备 通过混合 10 mL 瓶装 50x B27 补充剂和 500 mL RPMI 1640 培养基来制备 hiPSC-CM 维持培养基。将培养基储存在4°C并在一个月内使用。使用前将介质平衡至室温 （RT）。 通过混合 20 mL 血清替代物和 180 mL hiPSC-CM 维持培养基（10% 稀释度，v/v）制备 hiPSC-CM 接种培养基。虽然新鲜制备的播种培养基是优选的，但它可以在4°C下储存不超过2周。使用前将培养基平衡…
Representative Results
该协议描述了如何测量hiPSC-CM的收缩运动，场电位，动作电位和Ca2 + 瞬态。包括酶消化、细胞接种、维持和功能测定传导的示意图如图 1 所示。hiPSC-CM单层的形成对于收缩运动测量是必要的（图2B）。hiPSC-CMs收缩-松弛运动的代表性迹线如图 2C所示。分析仪软件通过使用默认设置检测收缩开始、收缩峰值、收缩结束、松弛峰?…
Discussion
人类iPSC技术已成为疾病建模和药物筛选的强大平台。在这里，我们描述了用于测量hiPSC-CM收缩力，场电位，动作电位和Ca2 +瞬态的详细协议。该协议提供了hiPSC-CM收缩力和电生理学的全面表征。这些功能测定已应用于我们组12，13，18，24，25，26，<sup cl…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢Blake Wu校对稿件。这项工作得到了美国国立卫生研究院（NIH）R01 HL113006，R01 HL141371，R01 HL163680，R01 HL141851，U01FD005978和NASA NNX16A069A（JCW）以及AHA博士后奖学金872244（GMP）的支持。
Materials
| 35 mm glass bottom dish with 20 mm micro-well #1.5 cover glass | Cellvis | D35-20-1.5-N | Patch clamp |
| 50x B27 supplements | Life Technologies | 17504-044 | hiPSC-CM culture medium |
| 6-well culture plate | E & K Scientific | EK-27160 | hiPSC-CM culture |
| 96-well flat clear bottom black polystyrene TC-treated microplates | Corning | 3603 | Contraction motion measurement |
| Accutase | Sigma-Aldrich | A6964 | Enzymatic dissociation |
| Axion's Integrated Studio (AxIS) | Axion Biosystems | navigator software | |
| Borosilicate glass capillaries | Harvard Apparatus | BF 100-50-10, | Patch clamp |
| CaCl2 1 M in H2O | Sigma-Aldrich | 21115 | Tyrode’s solution |
| Cell counting chamber slides | ThermoFisher Scientific | C10228 | Cell counting |
| CytoView 48-well MEA plates | Axion Biosystems | M768-tMEA-48B | MEA |
| DMEM/F12 | Gibco/Life Technologies | 12634028 | Extracellular matrix medium |
| DPBS, no calcium, no magnesium | Fisher Scientific | 14-190-250 | |
| EGTA | Sigma-Aldrich | E3889 | Intracellular pipette solution |
| EPC 10 USB patch clamp amplifier | Warner Instruments | 89-5000 | Patch clamp |
| Fura-2, AM, cell permeant | ThermoFisher Scientific | F1221 | Ca2+ transient measurement |
| Glucose | Sigma-Aldrich | G8270 | Tyrode’s solution |
| HEPES | Sigma-Aldrich | H3375 | Tyrode’s solution |
| hiPSCs | Stanford Cardiovascular Institute iPSC Biobank | ||
| KCl | Sigma-Aldrich | 529552 | Tyrode’s solution |
| KnockOut Serum Replacement | ThermoFisher Scientific | 10828-028 | hiPSC-CM seeding medium |
| KOH 8 M | Sigma-Aldrich | P4494 | Intracellular pipette solution |
| Lambda DG 4 | Sutter Instrument Company | Ca2+ transient measurement; ultra-high-speed wavelength switching light source | |
| Luna-FL automated fluorescence cell counter | WISBIOMED | LB-L20001 | Cell counting |
| Maestro Pro MEA system | Axion Biosystems | MEA | |
| Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix | Corning | 356231 | Extracellular matrix medium |
| MgATP | Sigma-Aldrich | A9187 | Intracellular pipette solution |
| MgCl2 | Sigma-Aldrich | M8266 | Tyrode’s solution |
| NaCl | Sigma-Aldrich | S9888 | Tyrode’s solution |
| NaOH 10 M | Sigma-Aldrich | 72068 | Tyrode’s solution |
| NIS Elements AR | |||
| Pluronic F-127 (20% Solution in DMSO) | ThermoFisher Scientific | P3000MP | Ca2+ transient measurement |
| RPMI 1640 medium | Life Technologies | 11875-119 | hiPSC-CM culture medium |
| Sony SI8000 Cell Motion Imaging System | Sony Biotechnology | Contraction motion measurement | |
| Sutter Micropipette puller | Sutter Instruments | P-97 | Patch clamp |
| Trypan blue stain | Life Technologies | T10282 | Cell counting |
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Cite This Article
Zhao, S. R., Mondéjar-Parreño, G., Li, D., Shen, M., Wu, J. C. Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes. J. Vis. Exp. (186), e64265, doi:10.3791/64265 (2022).