Name: fabrication of 3d cardiac microtissue arrays using human ipsc-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells
Uploaded: 2021-03-14T13:00:00
Description: Watch this Scientific Journal Video about Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells at JoVE.com

我们感谢Amanda Chase博士对手稿的有益反馈。资金支持由加州大学烟草相关疾病研究计划（TRDRP）T29FT0380（D.T.）和27IR-0012（J.C.W.）提供;美国心脏协会 20POST35210896 （H.K.） 和 17MERIT33610009 （J.C.W.）;和美国国立卫生研究院 （NIH） R01 HL126527、R01 HL123968、 R01 HL150693、R01 HL141851 和 NIH UH3 TR002588 （J.C.W）。

1. 培养基、试剂、培养板制备
<ol><li>用于细胞培养的细胞洗涤溶液：使用1x磷酸盐缓冲盐水（PBS）或汉克斯平衡盐溶液（HBSS），不含钙或镁。</li>
	<li>心肌细胞分化培养基<ol>
<li>通过将 10 mL 补充剂（50x B27 加胰岛素）加入 500 mL 心肌细胞基础培养基 （RPMI 1640） 来制备分化培养基 #1。</li>
		<li>通过将 10 mL 补充剂（50x B27 减去胰岛素）加入 500 mL 心肌细胞基础培养基 （RPMI 1640）， 准备分化培养基 #2?...

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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+activation+and+reduced+cell-cell+contact+synergistically+induce+massive+expansion+of+functional+human+iPSC-derived+cardiomyocytes.">Wnt activation and reduced cell-cell contact synergistically induce massive expansion of functional human iPSC-derived cardiomyocytes.</a> Cell Stem Cell. 27 (1), 50-63 (2020).</li><li>Feyen, D. A. 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为了从预分化的iPSC-CM，iPSC-EC和iPSC-CFs中产生心脏微组织，在心脏微组织内接触抑制细胞压实后，必须获得高纯度的培养物以更好地控制细胞数量。最近，贾科梅利等人。al.18 已经证明了使用iPSC-CM，iPSC-EC和iPSC-CF制造心脏微组织。使用所述方法生成的心脏微组织由约5，000个细胞组成（70%iPSC-CM，15%iPSC-EC和15%iPSC-CF）。在该方法中，心肌细胞和内皮细胞共分化，然后使用CD34 +标记物?...

由于缺乏临床相关样本和转化工具不足，心血管研究领域的药物发现和疾病建模面临多项挑战1。高度复杂的临床前模型或过度简化 的体外 单细胞模型不会以可重复的方式表现出病理生理学条件。因此，已经发展了几个小型化的组织工程平台，以帮助弥合差距，目标是在高通量方式的易用性和忠实地概括组织功能之间实现平衡2，3。随着诱导多能干细胞（iPSC）技术的出现，组织工程工具可以应用于具有或不具有潜在心血管疾病状态的患者特异性细胞，以回答研究问题4，5，6。这种具有类似于心脏组织的细胞组成的组织工程模型可用于药物开发工作，以测试由一种或多种细胞类型的行为的病理变化引起的心脏毒性和功能障碍。
来自人类iPSCs的自组装微组织或类器官是三维（3D）结构，是微型组织样组件，与 体内 对应物表现出功能相似性。有几种不同的方法允许通过iPSCs的定向分化或通过胚状体 的形成在原位 形成类器官4。由此产生的类器官是研究驱动器官发生的形态发生过程的不可或缺的工具。然而，各种细胞群的存在和自组织的差异可能导致不同类器官之间结果的差异5。或者，预分化细胞自组装成具有组织特异性细胞类型的微组织以研究局部细胞 - 细胞相互作用是极好的模型，其中分离自组装组分是可行的。特别是在人类心脏研究中，当细胞来自不同的患者品系或商业来源时，具有多细胞成分的3D心脏微组织的开发已被证明是具有挑战性的。
为了在生理相关、个性化的 体外模型中 提高我们对细胞行为的机制理解，理想情况下，所有组分细胞类型都应来自同一患者品系。在人类心脏的背景下，真正具有代表性 的心脏体外 模型将捕获主要细胞类型之间的串扰，即心肌细胞（CM），内皮细胞（EC）和心脏成纤维细胞（CFs）6，7。心肌的忠实概括不仅需要生物物理拉伸和电生理刺激，还需要来自EC和CFs8等支持细胞类型的细胞 - 细胞信号传导。CFs参与细胞外基质的合成并维持组织结构;在病理状态下，CFs可以诱导纤维化并改变CMs9中的电传导。同样，EC可以通过旁分泌信号传导和满足重要的代谢需求来调节CM的收缩特性10。因此，需要由所有三种主要细胞类型组成的人类心脏微组织，以允许进行生理相关的高通量实验。
在这里，我们描述了通过衍生人iPSC衍生的心肌细胞（iPSC-CM），iPSC衍生的内皮细胞（iPSC-EC）和iPSC衍生的心脏成纤维细胞（iPSC-CFs）及其在均匀的心脏微组织阵列中的3D培养来制造心脏微组织的方法。这种产生自发跳动的心脏微系统的简单方法可用于疾病建模和药物的快速测试，以对心脏生理学的功能和机制理解。此外，这种多细胞心脏微组织平台可以通过基因组编辑技术来模拟慢性或急性培养条件下心脏病随时间的进展。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12-well plates</td>
			<td>Fisher Scientific</td>
			<td>08-772-29</td>
			<td></td>
		</tr>
		<tr>
			<td>3D micro-molds</td>
			<td>Microtissues</td>
			<td>12-81 format</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Fisher Scientific</td>
			<td>08-772-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoMACS Rinsing Solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>NC9104697</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement minus Insulin</td>
			<td>Life Technologies</td>
			<td>A1895601</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement plus Insulin</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Cytofix</td>
			<td>BD Biosciences</td>
			<td>554655</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Matrigel, hESC-qualified matrix</td>
			<td>BD Biosciences</td>
			<td>354277</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiac Troponin T Antibody</td>
			<td>Miltenyi</td>
			<td>130-120-403</td>
			<td></td>
		</tr>
		<tr>
			<td>CD144 (VE-Cadherin) MicroBeads</td>
			<td>Miltenyi</td>
			<td>130-097-857</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31 Antibody</td>
			<td>Miltenyi</td>
			<td>130-110-670</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31 Microbeads</td>
			<td>Miltenyi</td>
			<td>130-091-935</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR-99021</td>
			<td>Selleckchem</td>
			<td>S2924</td>
			<td></td>
		</tr>
		<tr>
			<td>DDR2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-81707</td>
			<td></td>
		</tr>
		<tr>
			<td>Dead Cell Apoptosis Kit with Annexin V FITC and PI</td>
			<td>Thermo Fisher Scientific</td>
			<td>V13242</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase I</td>
			<td>Millipore Sigma</td>
			<td>4942086001</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose (4.5g/L) no glutamine medium</td>
			<td></td>
			<td>11960044</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12 basal medium</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (DPBS), no calcium, no magnesium</td>
			<td>Life Technologies</td>
			<td>14190-136</td>
			<td></td>
		</tr>
		<tr>
			<td>EGM2 BulletKit</td>
			<td>Lonza</td>
			<td>CC-3124</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Life Technologies</td>
			<td>10437</td>
			<td></td>
		</tr>
		<tr>
			<td>FibroLife Serum-Free Fibroblast LifeFactors Kit</td>
			<td>LifeLIne Cell Technology</td>
			<td>LS-1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose free RPMI medium</td>
			<td>Life Technologies</td>
			<td>11879-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Life Technologies</td>
			<td>16210-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Human FGF-basic</td>
			<td>Thermo Fisher Scientific</td>
			<td>13256029</td>
			<td></td>
		</tr>
		<tr>
			<td>Human VEGF-165</td>
			<td>PeproTech</td>
			<td>100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>IWR-1-endo</td>
			<td>Selleckchem</td>
			<td>S7086</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase TL</td>
			<td>Millipore Sigma</td>
			<td>5401020001</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Sorting Columns</td>
			<td>Miltenyi</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS BSA Stock solution</td>
			<td>Miltenyi</td>
			<td>130-091-376</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Rinsing Buffer</td>
			<td>Miltenyi</td>
			<td>130-091-222</td>
			<td></td>
		</tr>
		<tr>
			<td>MidiMACS Separator</td>
			<td>Miltenyi</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium</td>
			<td>Life Technologies</td>
			<td>11835055</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Selleckchem</td>
			<td>S1067</td>
			<td></td>
		</tr>
		<tr>
			<td>TO-PRO 3</td>
			<td>Thermo Fisher Scientific</td>
			<td>R37170</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Millipore Sigma</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Select 10X</td>
			<td>Thermo Fisher Scientific</td>
			<td>red</td>
			<td></td>
		</tr>
		<tr>
			<td>Vimentin Alexa Fluor&#174; 488-conjugated Antibody</td>
			<td>R&#38;D Systems</td>
			<td>IC2105G</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of 3d cardiac microtissue arrays using human ipsc-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells

从诱导多能干细胞（iPSCs）中产生人心肌细胞（CM），心脏成纤维细胞（CFs）和内皮细胞（EC）为研究驱动组织发育和疾病的不同心血管细胞类型之间的复杂相互作用提供了独特的机会。在心脏组织模型领域，几种复杂的三维（3D）方法使用诱导多能干细胞衍生的心肌细胞（iPSC-CM）来模拟生理相关性和天然组织环境，结合细胞外基质和交联剂。然而，这些系统在没有微细加工专业知识的情况下制造起来很复杂，并且需要数周才能自行组装。最重要的是，许多这些系统缺乏血管细胞和心脏成纤维细胞，它们占人类心脏非肌细胞的60%以上。在这里，我们描述了从iPSCs中衍生出所有三种心脏细胞类型以制造心脏微组织。这种简单的复制成型技术允许心脏微量组织在标准的多孔细胞培养板中培养数周。该平台允许用户根据初始播种密度自定义控制微量样本大小，并且自组装时间不到3天即可实现可观察的心脏微组织收缩。此外，心脏微量组织可以很容易地被消化，同时通过使用流式细胞术和单细胞RNA测序（scRNA-seq）保持高细胞活力，用于单细胞质询。我们设想，这种心脏微组织的 体外 模型将有助于加速药物发现和疾病建模的验证研究。

iPSC 衍生的 CM、EC 和 CF 的免疫染色和流式细胞术表征 为了产生由iPSC-CM，iPSC-EC和iPSC-CFs组成的心脏微组织，所有三种细胞类型都单独分化和表征。在过去几年中，iPSCs与iPSC-CM的 体外 分化有所改善。然而，iPSC-CM的产量和纯度因品系而异。目前的实验方案产生超过75%的纯iPSC-CM，这些iPSC-CM在第9天左右自发地开始跳动（图1A）。如前所述，从第9天到第14天的?...

Watch this Scientific Journal Video about Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells at JoVE.com

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

在这里，我们描述了一种易于使用的方法，用于生成由预分化的人诱导多能干细胞衍生的心肌细胞，心脏成纤维细胞和内皮细胞组成的3D自组装心脏微组织阵列。这种用户友好且需要低细胞的技术来生成心脏微组织，可用于疾病建模和药物开发的早期阶段。

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 ...

该协议结合了使用诱导多能干细胞产生人类心血管细胞类型的能力，以及一种多功能技术，可在不到72小时内产生跳动的心脏球状体。这些心球体可用于疾病建模和药物测试。这种简单技术的主要优点之一是，可以在标准的12孔板中产生近1000个跳动的心脏球体。最重要的是，每个心脏球体都可以使用基于成像的技术来检测其收缩功能。在微波炉中融化琼脂糖后，将其放入生物安全柜中，让它冷却三分钟。移取700微升熔融琼脂糖，并将其添加到9×9阵列的有机硅微模中，注意避免产生气泡。小心地将模具放在预冷的冰块上，以加速琼脂糖的持续时间。一旦琼脂糖变为半透明，小心地弯曲微模的边缘以失去琼脂糖复制品，并从各个侧面轻轻剥离复制品，将其从硅胶微模中分离出来。将含有81个圆形凹槽的琼脂糖微量组织托盘转移到无菌的12孔板中，然后将两毫升PBS加入琼脂糖微量组织托盘中，并在显微镜下检查是否有任何捕获的气泡或不规则形状的孔。将琼脂糖托盘浸没在两毫升70%乙醇中过夜，然后在生物安全柜中进行紫外线处理一小时。除去70%乙醇，用蒸馏水洗涤两次，一次用两毫升PBS洗涤。一旦在胰蛋白酶消化和中和后计数诱导多能干细胞衍生的心肌细胞，心脏成纤维细胞和内皮细胞，将细胞悬浮液放在冰上。在新管中，混合诱导多能干细胞衍生的心肌细胞，心脏成纤维细胞和内皮细胞。然后，从孔和细胞接种室中取出PBS而不接触凹槽，并滴加200微升细胞悬浮液。让细胞在37摄氏度下在二氧化碳培养箱中沉降两个小时。在琼脂糖模具周围加入微组织制造介质以覆盖内腔表面。24小时后，观察圆形凹槽中的自组装和紧凑型球体。每两天更换培养基以保持球体。通常，48小时后，可以观察到球体的自发跳动。在基底膜基质培养基或明胶包被的腔室载玻片上分别培养各个细胞类型。轻轻地将心脏微量组织从圆形凹槽中冲洗出来，并将它们收集在15毫升的编年史管中。吸出培养基并用一毫升PBS冲洗细胞或微组织。然后，用含有4.2%PFA的固定缓冲液固定细胞或微组织，并在室温下孵育。吸出PFA并用一毫升渗透溶液孵育细胞或微组织。然后，吸出溶液并用两到三毫升PBS冲洗。向细胞中加入500至1000微升的封闭溶液，并孵育至少一小时用于腔室载玻片，三到四个小时用于微组织。将含有偶联抗体的样品在封闭溶液中孵育一小时用于腔室载玻片，并在4摄氏度下过夜用于心脏微组织。用500微升0.1%吐温20洗涤腔室载玻片三次，每次洗涤间隔五分钟，然后用PBS进行最终洗涤。用两毫升0.1%吐温20洗涤心脏微组织五次，每次洗涤间隔20分钟。然后，再进行20分钟的最终洗涤。在共聚焦显微镜检查之前，用DAPI孵育细胞或微组织，然后小心地将心脏微组织转移到35毫米玻璃底培养皿中，并加入PBS以浸没微组织。使用宽或一毫升移液器吸头将微组织从圆形凹槽中冲洗出来，使其沉淀15毫升，并使其沉降。然后，吸出培养基并用一毫升PBS冲洗。加入200至300微升酶消化缓冲液，并在37摄氏度下孵育10分钟。然后轻轻混合微量组织一分钟，并重复孵育。孵育后，用常规的一毫升移液器吸头大力混合微量组织，将其消化成单细胞。获得浑浊的细胞悬浮液，并立即用含有5%FBS的5毫升培养基中和细胞悬浮液。通过40微米细胞过滤器过滤细胞悬浮液，并计数细胞总数。将单细胞悬浮液以300倍G在4摄氏度下离心五分钟。吸出上清液并将细胞与FITC膜联蛋白V和碘化丙啶或TO-PRO-3死细胞排除染料重悬于膜联蛋白结合缓冲液中，并在冰上孵育10分钟。孵育后，向细胞悬浮液中加入300微升膜联蛋白结合缓冲液，并将其转移到圆底FACS管中进行流式细胞术分析。使用相差显微镜观察纯化的诱导多能干细胞衍生的心肌细胞，内皮细胞和心脏成纤维细胞。使用免疫染色和流式细胞术测定不同细胞类型的纯度。肌钙蛋白T作为心肌细胞的标志物，纯度超过90%。将血小板内皮细胞粘附分子CD31和vimentin作为内皮细胞和心脏成纤维细胞的标志物，纯度分别超过98%和96%。免疫荧光染色显示，心肌细胞最重，占据微组织中心，而内皮细胞散布在整个微组织中，心脏成纤维细胞主要占据外围。使用这种速度一和Liberase TL对微组织进行消化，在培养两周后产生了高度活的细胞比例和更少的凋亡细胞。心脏微量组织暴露于高浓度阿霉素诱导剂量依赖性心脏毒性的一小时。微组织收缩力和载体运动产生了伪热图，说明了整个微组织的平均收缩曲线。心脏微组织的收缩运动产生正峰，其测量为收缩速度，松弛速度和节拍率，其计算为两个收缩周期之间的时间。在培养物中，心脏微组织的收缩性在四周内保持一致。细胞加热是该特定方案中最重要的步骤之一，其中必须注意小心地将细胞悬浮液滴入琼脂糖阵列中，以便在微孔内均匀分布，并防止溢出。使用这些微量组织的优化辐射方案，人们可以将其置于高通量测序技术中，例如单细胞RNA-seq或单细胞ATAC-seq，以了解由于不同的处理条件而产生的细胞特异性基因表达模式。

Research

JoVE Journal

Bioengineering

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

心肌在心脏组织工程中的纤维蛋白凝胶的封装

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

科研

生物工程

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

人类心脏静脉系统使用对比计算机断层扫描灌注固定的标本解剖重建

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

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 ...

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

使用3D生物印刷创建心脏组织展示球体的机械整合

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

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 ...

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. ...

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

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

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

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

用于循环作用电位记录的多井微电极阵列上的人类 iPSC 衍生心肌细胞网络

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

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

人体器官性心脏切片双电压和钙光学映射的临床前心脏电生理评估

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