Name: preparation of mesh-shaped engineered cardiac tissues derived from human ips cells for in vivo myocardial repair
Uploaded: 2020-06-09T13:00:00
Description: Watch this Scientific Journal Video about Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair at JoVE.com

这项工作得到了路易斯维尔大学Kosair慈善儿科心脏研究项目和RIKEN生物系统动力学研究中心的有机物项目的支持。我们发布的协议中使用的HIPSC由日本京都大学 iPS 细胞研究和应用中心提供。

1. 维护高保和心血管分化
<ol><li>在从小鼠胚胎成纤维细胞（MEF-CM）中提取的有条件介质中，在具有人类基本成纤维细胞生长因子（hbFGF）的中，在薄涂层基底膜基质（生长因子减少，1：60稀释）4上扩展和维护hiPSC。 注：我们使用了hiPSCs（4因子（10月3/4，Sox2，Klf4和c-Myc）行：201B6）。在每个细胞系的适当浓度下添加 hbFGF。拉米宁-511片段也可用于培养皿的涂层，而不是地下室膜?...

<ol>
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H., 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=Engineered+heart+tissue+grafts+improve+systolic+and+diastolic+function+in+infarcted+rat+hearts.">Engineered heart tissue grafts improve systolic and diastolic function in infarcted rat hearts.</a> Nature Medicine. 12, 452-458 (2006).</li><li>Fujimoto, K. L., 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=Engineered+fetal+cardiac+graft+preserves+its+cardiomyocyte+proliferation+within+postinfarcted+myocardium+and+sustains+cardiac+function.">Engineered fetal cardiac graft preserves its cardiomyocyte proliferation within postinfarcted myocardium and sustains cardiac function.</a> Tissue engineering. Part A. 17, 585-596 (2011).</li><li>Lancaster, J. J., 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=Surgical+treatment+for+heart+failure:+cell-based+therapy+with+engineered+tissue.">Surgical treatment for heart failure: cell-based therapy with engineered tissue.</a> Vessel Plus. 2019, (2019).</li><li>Bian, W., Liau, B., Badie, N., Bursac, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesoscopic+hydrogel+molding+to+control+the+3D+geometry+of+bioartificial+muscle+tissues.">Mesoscopic hydrogel molding to control the 3D geometry of bioartificial muscle tissues.</a> Nature protocols. 4, 1522-1534 (2009).</li><li>Zhang, D., 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=Tissue-engineered+cardiac+patch+for+advanced+functional+maturation+of+human+ESC-derived+cardiomyocytes.">Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes.</a> Biomaterials. 34, 5813-5820 (2013).</li><li>Christoforou, N., 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=Induced+pluripotent+stem+cell-derived+cardiac+progenitors+differentiate+to+cardiomyocytes+and+form+biosynthetic+tissues.">Induced pluripotent stem cell-derived cardiac progenitors differentiate to cardiomyocytes and form biosynthetic tissues.</a> PloS one. 8, 65963 (2013).</li><li>Nakane, T., 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=Impact+of+Cell+Composition+and+Geometry+on+Human+Induced+Pluripotent+Stem+Cells-Derived+Engineered+Cardiac+Tissue.">Impact of Cell Composition and Geometry on Human Induced Pluripotent Stem Cells-Derived Engineered Cardiac Tissue.</a> Scientific Reports. 7, 45641 (2017).</li><li>Kowalski, W. 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在完成对线性格式（hiPSC衍生的ECT5）的调查后，我们调整了协议，将hiPSC衍生的CMs、EC和MC混合在一起，以促进ETC内血管细胞的体外扩张，以及ECT和接受者心肌之间随后的体内血管耦合。
为了促进更大、可植入的网格 ECT 几何的生成，我们使用薄的 PDMS 表设计 3D 模具，加载柱排列在交错位置。在初步实验中，我们注意到ETS在凝胶压实过程中粘附在PDMS柱上，因?...

许多临床前研究和临床试验已经确认了细胞为基础的心脏再生疗法对心脏1，2，3,2失败的有效性。在各种细胞类型中，人类诱导多能干细胞（hiPSCs）凭借其增殖能力、产生各种心血管血统4、5,和全源性的潜力，是有希望的细胞来源。此外,，组织工程技术已经使得将数百万个细胞转移到受损的心脏5，6，7，8。6,758
此前，我们报告了从hiPSC衍生的心血管系生成三维（3D）线性工程心脏组织（ECTs），使用商用的培养系统进行3D生物人工组织5,5，7。我们发现，ECT内血管内皮细胞和壁画细胞与心肌细胞共存，促进了结构和电生理组织的成熟。此外，我们验证了在免疫耐受大鼠心肌梗塞模型中植入hiPSC-ECTs的治疗潜力，以改善心脏功能，再生心肌，增强血管生成5。然而，此方法构建的线性 EC 是 1 mm x 10 mm 圆柱体，因此不适合植入临床前研究与较大的动物或临床用途。
基于成功利用组织模具利用大鼠骨骼肌细胞和心肌细胞9、人类ESC衍生心肌细胞10和小鼠 iPSCs 11生成多孔工程组织形成，我们开发了一种协议，利用聚二11氧化硅氧烷 （PDMS） 霉菌生成可扩展的 hiPSC 衍生更大的可植入组织。我们评估了一系列模具几何形状，以确定最有效的模具特性。与缺少毛孔或结的薄板或线性格式相比，具有多个束和结的网状 EC 在细胞生存能力、组织功能和可伸缩性方面表现出出色的特性。我们把网状ECT植入大鼠心肌梗死模型，并确认其治疗效果类似于植入的圆柱形ECT12。在这里，我们描述了生成 hiPSC 派生网格形状的 ECT 的协议。

<table>
	<tbody>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dishes 100x20 mm style</td>
			<td>Falcon/ Thomas scientific</td>
			<td>9380C51</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiwell Plates For Cell Culture 6well 50/CS</td>
			<td>Falcon / Thomas scientific</td>
			<td>6902A01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning</td>
			<td>761036</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accumax</td>
			<td>Innovative Cell Technologies</td>
			<td>AM-105</td>
			<td></td>
		</tr>
		<tr>
			<td>BMP4, recombinant (10&#181;g)</td>
			<td>R&#38;D</td>
			<td>RSD-314-BP-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen, Type I solution from rat tail</td>
			<td>Sigma</td>
			<td>C3867</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth factor-reduced Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>Human VEGF (165) IS, premium grade</td>
			<td>Miltenyi</td>
			<td>130-109-385</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127, 0.2 &#181;m filtered (10% Solution in Water)</td>
			<td>Molecular Probes</td>
			<td>P-6866</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human bFGF</td>
			<td>WAKO</td>
			<td>060-04543</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human/Mouse/Rat ActivinA (50&#181;g)</td>
			<td>R&#38;D</td>
			<td>338-AC-050</td>
			<td></td>
		</tr>
		<tr>
			<td>rh Wnt-3a (10&#181;g)</td>
			<td>R&#38;D</td>
			<td>5036-WN</td>
			<td></td>
		</tr>
		<tr>
			<td>Versene solution</td>
			<td>Gibco</td>
			<td>15040066</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Culture medium and supplements</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x MEM</td>
			<td>Invitrogen</td>
			<td>11430</td>
			<td></td>
		</tr>
		<tr>
			<td>2 Mercaptro Ethanol</td>
			<td>SIGMA</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement minus insulin</td>
			<td>Gibco</td>
			<td>A1895601</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose</td>
			<td>Gibco</td>
			<td>11965084</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (500ml)</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (500ml)</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1x</td>
			<td>Gibco</td>
			<td>10010-031</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (5000 U/mL)</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640 medium</td>
			<td>Gibco</td>
			<td>21870092</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;MEM</td>
			<td>Invitrogen</td>
			<td>11900024</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flowcytometry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-TRA-1-60, FITC, Clone: TRA-1-60, BD Biosciences</td>
			<td>BD / Fisher</td>
			<td>560380</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Troponin T, Cardiac Isoform Ab-1, Clone: 13-11, Thermo Scientific Lab Vision</td>
			<td>Fisher</td>
			<td>MS-295-P0</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACS Clean Solution</td>
			<td>BD</td>
			<td>340345</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSFlow Sheath Fluid</td>
			<td>BD</td>
			<td>342003</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSRinse Solution</td>
			<td>BD</td>
			<td>340346</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Tube with Cell Strainer Cap (Case of 500)</td>
			<td>Corning</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (500ml)</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation</td>
			<td>Molecular Probes</td>
			<td>L34957</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGFRb; anti-CD140b, R-PE, Clone: 28D4, BD Biosciences</td>
			<td>BD / Fisher</td>
			<td>558821</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma-Aldrich</td>
			<td>47306-50G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>VEcad-FITC; anti-CD144, FITC, Clone: 55-7H1, BD Biosciences</td>
			<td>BD / Fisher</td>
			<td>560411</td>
			<td></td>
		</tr>
		<tr>
			<td>Zenon Alexa Fluor 488 Mouse IgG1 Labeling Kit</td>
			<td>Molecular Probes</td>
			<td>Z25002</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of mesh-shaped engineered cardiac tissues derived from human ips cells for in vivo myocardial repair

目前的协议描述了产生可扩展的网状工程心脏组织（ECTs）的方法，这些组织由来自人类诱导多能干细胞（hiPSCs）的心血管细胞组成，这些细胞朝着临床使用的目标发展。HiPSC衍生的心肌细胞、内皮细胞和血管壁画细胞与凝胶基质混合，然后倒入具有矩形内部交错柱的聚二甲基硅氧烷（PDMS）组织模具中。到培养日，14个ETS成熟成一个1.5厘米x1.5厘米的网状结构与0.5毫米直径的硫纤维束。心肌细胞与每个束的长轴对齐，并自发地同步跳动。这种方法可以放大到更大的（3.0 厘米 x 3.0 厘米）网格 ECT，同时保持构造成熟度和功能。因此，从hiPSC衍生心脏细胞产生的网状EEC对于心脏再生范式可能是可行的。

图1A，B 显示了CM+EC和MC协议的示意图。从CM+EC协议和MC从MC协议诱导CM和EC后，细胞混合调整最终MC浓度，以代表10-20%的总细胞。2 厘米宽的组织模具是根据 0.5 mm 厚 PDMS 板材（图2A，B） 的设计图纸制造的。600万个CM+EC+MC细胞与胶原蛋白一结合，并用基质和马球407预涂的组织模具（图2C）在初步实验中，我们制?...

Watch this Scientific Journal Video about Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair at JoVE.com

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

本协议产生网状工程心脏组织，其中包含来自人类诱导多能干细胞的心血管细胞，以便研究心脏病的细胞植入疗法。

从人体 iPS 细胞中提取的网状工程心脏组织用于活体心肌修复

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from ...

该协议描述了一种高贵且简单的方法，用于创建从人体诱导身体部位和STEM细胞衍生心脏细胞中提取的网状工程心脏组织。组织几何的灵活性和保存的组织功能的可伸缩性是该技术的主要优点。植入的网状ET可以在大鼠心肌梗死模型中恢复心脏结构和功能。确认其用于心脏再生治疗的可行性。这些技术需要可重复的人类STEM细胞处理和组织培养技能，以取得一致的结果。首先，将固化的 PDMS 板材切割到适当的尺寸，以便将纸张与硅胶粘合，以制造一个 21 由 20.5 毫米的矩形托盘，其中 7 毫米长 0.5 毫米宽，2.5 毫米高矩形柱位于交错位置。两行柱子之间的水平间距应为 2.5 毫米。在 120 摄氏度下将托盘在 120 摄氏度下自动安装 20 分钟，然后将 1%Poloxamer 407 涂在 PBS 中的模具上，使用 1 小时。然后用 PBS 彻底冲洗模具，将模具放入六井板的一个井中。对于血统分析，结合心肌细胞的细胞加上内皮和血管壁画细胞方案。使壁画细胞的最终浓度是10至20%，在细胞总数中，每体6个细胞的6倍。将133微升酸可溶鼠尾胶原蛋白一型溶液和17微升碱缓冲液混合到17微升，在冰上达到10倍最小基本介质。然后将67微升的基底膜基质添加到胶原蛋白溶液中。接下来，将细胞离心，在167微升高葡萄糖Dulbeco的最小基本介质中重新吸收颗粒，并辅以胎儿牛血清和抗生素。在冰上混合基质溶液上的细胞。然后小心地将 400 微升的细胞基质悬浮液倒入 Poloxamer 407 涂层 PDMS 组织模具上。在37摄氏度的标准细胞培养箱中孵育细胞基质混合物，在60分钟内孵育5%的二氧化碳。组织形成后，用四毫升ECT培养剂小心浸泡模具，将板回细胞培养箱14天。在 ECT 植入之前，您消毒细钳，以小心地将 ECT 从装载柱上拆下。组织模具可以制造各种图案，柱长度和后间距，以产生不同的最终ECT几何形状。对于此分析，使用了具有 7 毫米长柱的模具，其间隔为 2.5 毫米。Poloxamer 407 可防止细胞粘附在模具上，并在 14 天内快速压实凝胶后形成特征网状结构。即使在从模具中释放 ECT 后，结构也得到维护。制造组织结构大约1.5厘米宽，0.5毫米厚。网格中每个束的宽度平均约为 0.5 毫米。可以生成一个 3 厘米的最终宽度网格 ECT，其中包含 2400 万个细胞，该模组具有相同交错的后设计。模具的尺寸是 4 倍。这种较大的网格 ECT 也可以轻松地从模具中去除，并生成相当于较小网格 ECT 的局部力行为。重要的是将细胞基质混合物均匀地倒入组织模具中，而不产生气泡，以防止最终凝胶中出现缺陷。承包商的功能特征。第四频率关系可以使用内置系统执行。我们的最终目标是将ECT范式转化为临床疗法，利用大型动物临床前研究。

Research

JoVE Journal

Bioengineering

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 ...

科研

生物工程

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

在心肌结构，功能的体内定量评估，灌注与活力使用心脏微型计算机断层扫描

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

中的应用<em&gt;在体内</em&gt;评定组织的鼠胫骨前的功能测试工程化骨骼肌修复

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

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 ...

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 ...

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 ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

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 ...

Cardiac Spheroids as in vitro Bioengineered Heart Tissues to Study Human Heart Pathophysiology

心脏球形作为体外生物工程心脏组织研究人类心脏病理生理学

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

成纤维细胞衍生的人类工程结缔组织用于筛查应用

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Engineered Lung Tissues Prepared from Decellularized Lung Slices

由去细胞化肺切片制备的工程肺组织

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex ...