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Bioengineering

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

Published: March 14, 2021
doi:
10.3791/61879
, , ,
1Stanford Cardiovascular Institute,Stanford University School of Medicine, 2Department of Medicine, Division of Cardiovascular Medicine,Stanford University School of Medicine, 3Department of Radiology,Stanford University School of Medicine

Summary

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。

Abstract

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。

Introduction

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。

Protocol

1. 培养基、试剂、培养板制备 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。 心肌细胞分化培养基 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。 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?…

Representative Results

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?…

Discussion

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?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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）。

Materials

12-well plates Fisher Scientific 08-772-29
3D micro-molds Microtissues 12-81 format
6-well plates Fisher Scientific 08-772-1B
AutoMACS Rinsing Solution Thermo Fisher Scientific NC9104697
B27 Supplement minus Insulin Life Technologies A1895601
B27 Supplement plus Insulin Life Technologies 17504-044
BD Cytofix BD Biosciences 554655
BD Matrigel, hESC-qualified matrix BD Biosciences 354277
Cardiac Troponin T Antibody Miltenyi 130-120-403
CD144 (VE-Cadherin) MicroBeads Miltenyi 130-097-857
CD31 Antibody Miltenyi 130-110-670
CD31 Microbeads Miltenyi 130-091-935
CHIR-99021 Selleckchem S2924
DDR2 Santa Cruz Biotechnology sc-81707
Dead Cell Apoptosis Kit with Annexin V FITC and PI Thermo Fisher Scientific V13242
Dispase I Millipore Sigma 4942086001
DMEM, high glucose (4.5g/L) no glutamine medium 11960044
DMEM/F-12 basal medium Gibco 11320033
Dulbecco's phosphate buffered saline (DPBS), no calcium, no magnesium Life Technologies 14190-136
EGM2 BulletKit Lonza CC-3124
Fetal bovine serum Life Technologies 10437
FibroLife Serum-Free Fibroblast LifeFactors Kit LifeLIne Cell Technology LS-1010
Glucose free RPMI medium Life Technologies 11879-020
Goat serum Life Technologies 16210-064
Human FGF-basic Thermo Fisher Scientific 13256029
Human VEGF-165 PeproTech 100-20
IWR-1-endo Selleckchem S7086
Liberase TL Millipore Sigma 5401020001
LS Sorting Columns Miltenyi 130-042-401
MACS BSA Stock solution Miltenyi 130-091-376
MACS Rinsing Buffer Miltenyi 130-091-222
MidiMACS Separator Miltenyi 130-042-302
RPMI medium Life Technologies 11835055
SB431542 Selleckchem S1067
TO-PRO 3 Thermo Fisher Scientific R37170
Triton X-100 Millipore Sigma X100-100ML
TrypLE Select 10X Thermo Fisher Scientific red
Vimentin Alexa Fluor® 488-conjugated Antibody R&D Systems IC2105G
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Tags

Human IPSC-derived CardiomyocytesCardiac FibroblastsEndothelial CellsAgarose MicromoldCardiac SpheroidsDisease ModelingDrug TestingContractile FunctionImagingCell SuspensionCell SeedingMedium Change
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Thomas, D., Kim, H., Lopez, N., Wu, J. C. Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells. J. Vis. Exp. (169), e61879, doi:10.3791/61879 (2021).
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