Name: automated contraction analysis of human engineered heart tissue for cardiac drug safety screening
Uploaded: 2017-04-15T13:00:00
Description: Watch this Scientific Journal Video about Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening at JoVE.com

作者非常感谢亚历山德拉·莫雷蒂和丹尼斯·施德其材料的实物捐助。我们承认，在实验药理学和UKE的毒理学部的大力支持IP和EHT工作组。作者的作品是由来自DZHK（德国中心心血管研究）和德国教育与研究部（BMBF），德国研究基金会（DFG居88 / 12-1，HA 3423 / 5-1资金支持），英国国家中心更换细化和在研究动物的减少（NC3Rs CRACK-IT授予35911-259146），英国心脏基金会的RM /30157分之13，欧洲研究理事会（高级格兰特IndivuHeart），德国心脏基金会和柏林自由UND Hansestadt汉堡。

注意：下面的步骤描述了一种细胞培养协议。请在无菌条件下进行，并使用预热媒体。 1.的hiPSC的心脏分化<ol><li> 培养的hiPSC <ol><li>涂层6孔板（1毫升/孔）或T75烧瓶（7毫升/瓶）具有减少的生长因子基底膜基质（ 例如 geltrex，1：200;见的材料表）的Dulbecco改良的Eagle培养基中稀释（DMEM），用于在37℃下30分钟。通过混合制备FTDA介质12基本DMEM ...

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiologic+force-frequency+response+in+engineered+heart+muscle+by+electromechanical+stimulation.">Physiologic force-frequency response in engineered heart muscle by electromechanical stimulation.</a> Biomaterials. 60, 82-91 (2015).</li><li>Eng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomous+beating+rate+adaptation+in+human+stem+cell-derived+cardiomyocytes.">Autonomous beating rate adaptation in human stem cell-derived cardiomyocytes.</a> Nat Commun. 7, 10312 (2016).</li><li>Huebsch, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniaturized+iPS-Cell-Derived+Cardiac+Muscles+for+Physiologically+Relevant+Drug+Response+Analyses.">Miniaturized iPS-Cell-Derived Cardiac Muscles for Physiologically Relevant Drug Response Analyses.</a> Sci Rep. 6 (24726), 1-12 (2016).</li><li>Masumoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myocardial+regenerative+potential+of+three-dimensional+engineered+cardiac+tissues+composed+of+multiple+human+iPS+cell-derived+cardiovascular+cell+lineages.">The myocardial regenerative potential of three-dimensional engineered cardiac tissues composed of multiple human iPS cell-derived cardiovascular cell lineages.</a> Sci Rep. 6 (29933), 1-10 (2016).</li><li>Ravenscroft, S. M., Pointon, A., Williams, A. W., Cross, M. J., Sidaway, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+Non-myocyte+Cells+Show+Enhanced+Pharmacological+Function+Suggestive+of+Contractile+Maturity+in+Stem+Cell+Derived+Cardiomyocyte+Microtissues.">Cardiac Non-myocyte Cells Show Enhanced Pharmacological Function Suggestive of Contractile Maturity in Stem Cell Derived Cardiomyocyte Microtissues.</a> Toxicol Sci. 152 (1), 99-112 (2016).</li><li>Zhang, J. H., Chung, T. D. Y., Oldenbutg, K. 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工程心脏组织提供了一个有价值的选择，以心血管疾病研究的工具盒。在24井EHTs已经为疾病建模8，14，药品安全筛选7，8，10，11，15，或基本心血管研究16，17证明是有价值的。 <p class...

心脏的副作用，如药物性长QT综合征导致市场撤回在过去几年里。统计数据表明，所有提款的约45％是由于心血管系统1的不良影响。这种昂贵的发展过程和批准后药物失效是制药公司最糟糕的情况。因此，研究和开发部门重点检测的这种不良心血管作用早期。出于经济和伦理问题，努力减少动物实验，并与新的体外筛选测定代替他们正在进行中。 一组建立了试验的包括在美国食品和药物管理局（FDA）和欧洲药品管理局（EMA）为致心律失常药物作用2临床前评价的准则。体细胞重编程，随后分化的技术人类诱导多能干细胞（hiPSC细胞）推动这一研究领域3。它现在提供了屏幕对人心肌细胞的新候选药物的可能性，并避免与物种间的差异问题。最近的心脏分化协议4，5不提供伦理关怀心肌无限量供应。然而，收缩力，最重要的，最特征的心肌细胞的活体参数测量，没有很好地建立起来。这与人类诱导多能干细胞衍生的心肌细胞（的hiPSC-CM）相比，成人心肌细胞作为相对不成熟6。一种可能的改进是从单电池7（工程化的心脏组织，EHT）工程师3维心脏组织。的EHT协议基于嵌入单个鼠或人心肌8 </SUP&gt;，9，10中的两个柔性聚二甲基硅氧烷（PDMS）柱11在24孔格式之间的纤维蛋白水凝胶。在几天之内的心肌细胞开始为单个细胞自发性收缩，并开始形成蜂窝网络。后7-10天，整个组织的宏观收缩可见。在此过程中的细胞外基质被重塑，从而导致直径和长度的减少。的EHT结果在PDMS的弯曲的缩短剩余时间内，即使后，在显影EHT到连续负载使心肌细胞。 EHTs继续在几个星期内进行auxotonic肌肉收缩。人类EHTs显示对生理和药理刺激指示它们用于药物筛选和疾病建模7适应性应答。 在这个手稿中，我们提出了一个generati强大和易于协议对人类EHT，和在hERG通道抑制剂存在收缩图案的浓度依赖性变化自动收缩的分析。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>EHT analysis intrument</td>
			<td>EHT Technologies GmbH</td>
			<td>A0001</td>
			<td>Software is included</td>
		</tr>
		<tr>
			<td>EHT PDMS rack</td>
			<td>EHT Technologies GmbH</td>
			<td>C0001</td>
			<td></td>
		</tr>
		<tr>
			<td>EHT PTFE spacer</td>
			<td>EHT Technologies GmbH</td>
			<td>C0002</td>
			<td></td>
		</tr>
		<tr>
			<td>EHT electrode</td>
			<td>EHT Technologies GmbH</td>
			<td>P0001</td>
			<td></td>
		</tr>
		<tr>
			<td>EHT pacing adapter/cable</td>
			<td>EHT Technologies GmbH</td>
			<td>P0002</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well-plate</td>
			<td>Nunc</td>
			<td>144530</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well-cell culture plate</td>
			<td>Nunc</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml falcon tube, graduated&#160;</td>
			<td>Sarstedt</td>
			<td>62,554,502</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Sarstedt</td>
			<td>831,830</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinner flask</td>
			<td>Integra</td>
			<td>182 101</td>
			<td></td>
		</tr>
		<tr>
			<td>Stirrer Variomag/ Cimarec Biosystem Direct&#160;</td>
			<td>Thermo scientific</td>
			<td>70101</td>
			<td>Adjust rotor speed to 40 rpm</td>
		</tr>
		<tr>
			<td>T175 cell culture flask&#160;</td>
			<td>Sarstedt&#160;</td>
			<td>831,812,002</td>
			<td></td>
		</tr>
		<tr>
			<td>V-shaped sedimentation rack&#160;</td>
			<td>Custom made at UKE Hamburg</td>
			<td>na</td>
			<td></td>
		</tr>
		<tr>
			<td>10&#215; DMEM</td>
			<td>Gibco</td>
			<td>52100</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Thioglycerol&#160;</td>
			<td>Sigma Aldrich</td>
			<td>M6145</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Phospho-L-ascorbic acid trisodium salt</td>
			<td>Sigma Aldrich</td>
			<td>49752</td>
			<td></td>
		</tr>
		<tr>
			<td>Activin-A&#160;</td>
			<td>R&#38;D systems</td>
			<td>338-AC</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose&#160;</td>
			<td>Invitrogen</td>
			<td>15510-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma Aldrich</td>
			<td>A1153</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqua ad injectabilia</td>
			<td>Baxter GmbH</td>
			<td>1428</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 PLUS insulin&#160;</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>BMP-4</td>
			<td>R&#38;D systems</td>
			<td>314-BP</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase II&#160;</td>
			<td>Worthington</td>
			<td>LS004176</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Biochrom</td>
			<td>F0415</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO&#160;</td>
			<td>Sigma Aldrich</td>
			<td>D4540</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase II, type V (from bovine spleen)</td>
			<td>Sigma&#160;</td>
			<td>D8764</td>
			<td></td>
		</tr>
		<tr>
			<td>Dorsomorphin&#160;</td>
			<td>abcam</td>
			<td>ab120843</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA&#160;</td>
			<td>Roth</td>
			<td>8043.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Gibco</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>Miltenyi Biotec</td>
			<td>130-104-921</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen (bovine)</td>
			<td>Sigma Aldrich</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr>
			<td>Geltrex&#160;</td>
			<td>Gibco</td>
			<td>A1413302</td>
			<td>For coating: 1:200 dilution</td>
		</tr>
		<tr>
			<td>HBSS w/o Ca2+/Mg2+&#160;</td>
			<td>Gibco</td>
			<td>14175-053</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES&#160;</td>
			<td>Roth</td>
			<td>9105.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Life technologies</td>
			<td>26050088</td>
			<td></td>
		</tr>
		<tr>
			<td>Human serum albumin&#160;</td>
			<td>Biological Industries</td>
			<td>05-720-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin, human</td>
			<td>Sigma Aldrich</td>
			<td>I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamin</td>
			<td>Gibco</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipidmix&#160;</td>
			<td>Sigma Aldrich</td>
			<td>L5146</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td>For EHT reconsitutionmix.</td>
		</tr>
		<tr>
			<td>N-Benzyl-p-Toluenesulfonamide</td>
			<td>TCI</td>
			<td>B3082-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS w/o MgCl2/CaCl2</td>
			<td>Biochrom</td>
			<td>14190</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127&#160;</td>
			<td>Sigma Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinyl alcohol&#160;</td>
			<td>Sigma Aldrich</td>
			<td>P8136</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640&#160;</td>
			<td>Gibco</td>
			<td>21875</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma Aldrich</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF&#223;1</td>
			<td>Peprotech</td>
			<td>100-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma Aldrich</td>
			<td>T7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin&#160;</td>
			<td>Sigma Aldrich</td>
			<td>T8158</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Biorbyt</td>
			<td>orb6014</td>
			<td></td>
		</tr>
		<tr>
			<td>hiPSC</td>
			<td>Custom made at UKE hamburg</td>
			<td>na</td>
			<td></td>
		</tr>
		<tr>
			<td>iCell cardiomyocytes kit</td>
			<td>Cellular Dynamics International</td>
			<td>CMC-100-010-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluricyte cardiomyocyte kit</td>
			<td>Pluriomics</td>
			<td>PCK-1.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Cor.4U - HiPSC cardiomyocytes kit</td>
			<td>Axiogenesis AG</td>
			<td>Ax-C-HC02-FR3</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellartis cardiomyocytes</td>
			<td>Takara Bio USA, Inc.</td>
			<td>Y10075</td>
			<td></td>
		</tr>
	</tbody>
</table>

automated contraction analysis of human engineered heart tissue for cardiac drug safety screening

心脏组织工程技术描述以构成三维力产生工程化组织。对于在基础研究和临床前药物开发这些程序的执行，必须制定规范条件下的自动生成和分析的协议是非常重要的。在这里，我们提出了一种技术来生成来自不同物种（大鼠，小鼠，人）的心肌细胞工程化的心脏组织（EHT）。该技术依赖于含有聚二甲基硅氧烷弹性（PDMS）柱之间解离的心肌细胞在24孔格式的血纤维蛋白凝胶的组装。三维的，力生成EHTs铸造后构成两个星期内。此过程允许每周几百EHTs的产生和在技术上由心肌细胞的可用性只有有限的（0.4-1.0×10 6 / EHT）。 auxotonic肌肉收缩的评价是在修改的孵育室与mechan执行对24孔板的iCal联锁，并放置在该腔室的顶部上的照相机。软件控制了摄像机的XYZ轴系统到每个EHT上移动。 EHT收缩是由一个自动数字识别算法检测到，并且力是基于EHT的缩短和弹性倾向和几何形状的PDMS帖子的计算。该方法允许标准化并在无菌条件下高数EHT的自动分析。对心肌收缩药物效果的可靠检测心脏药物开发和安全药理学的关键。我们证明，与hERG通道抑制剂E-4031的例子，该人的EHT系统上复制人的心脏收缩动力学药物反应，这表明它是心脏药物安全性筛选一个行之有效的手段。

心脏分化和EHT的制备的hiPSC分别在低生长因子基底膜基质膨胀，用EDTA和形成在旋转烧瓶过夜胚体（EB）解离。中胚层诱导3天后，心脏分化与Wnt抑制剂的启动。后分化方案的〜17天，跳动将EB解离为单细胞的II型胶原酶（ 图1）。 EHTs从新鲜分离的心肌细胞制备每100μL的组织1.0×10 6个细胞的EB解离（ 图2）</st...

Watch this Scientific Journal Video about Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening at JoVE.com

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

在这里，我们将展示人体工程心脏组织的产生从诱导多能干细胞（hiPSC细胞）衍生的心肌细胞。我们呈现由hERG通道抑制剂E-4031来分析收缩力和收缩图案的示例性改变的方法。这种方法显示的鲁棒性和适用性强心药物筛选的高水平。

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 ...

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Bioengineering

Unit 1

Cardiovascular Drugs: Antiarrhythmic and Heart Failure Drugs

SCIENTISTS IN ACTION

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

生物发光成像评估的免疫反应，植入心脏组织工程（东隧）

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

科研

生物工程

Self-reporting Scaffolds for 3-Dimensional Cell Culture

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

管式膜网络在心肌细胞的心房和心室分析

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

中空纤维生物反应器的<em&gt;在体内</em&gt;般的哺乳动物组织培养

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

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 ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

一种混合的3D组织工程<em&gt;体外</em&gt; /<em&gt;在硅片</em在具体的突变背景预测药物的有效性&gt;肺肿瘤模型

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

新生儿心脏支架：再生研究新矩阵

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

高通量自动 Microbioreactor 系统在 CHO 细胞模型 IgG1 生产中的应用

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

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

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 ...

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 ...