人工程心脏组织的收缩自动分析心脏药物安全筛选
Summary
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。
Abstract
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。
Introduction
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>，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。
Protocol
Representative Results
Discussion
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…Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| EHT analysis intrument | EHT Technologies GmbH | A0001 | Software is included |
| EHT PDMS rack | EHT Technologies GmbH | C0001 | |
| EHT PTFE spacer | EHT Technologies GmbH | C0002 | |
| EHT electrode | EHT Technologies GmbH | P0001 | |
| EHT pacing adapter/cable | EHT Technologies GmbH | P0002 | |
| 24-well-plate | Nunc | 144530 | |
| 6 well-cell culture plate | Nunc | 140675 | |
| 15 ml falcon tube, graduated | Sarstedt | 62,554,502 | |
| Cell scraper | Sarstedt | 831,830 | |
| Spinner flask | Integra | 182 101 | |
| Stirrer Variomag/ Cimarec Biosystem Direct | Thermo scientific | 70101 | Adjust rotor speed to 40 rpm |
| T175 cell culture flask | Sarstedt | 831,812,002 | |
| V-shaped sedimentation rack | Custom made at UKE Hamburg | na | |
| 10× DMEM | Gibco | 52100 | |
| 1-Thioglycerol | Sigma Aldrich | M6145 | |
| 2-Phospho-L-ascorbic acid trisodium salt | Sigma Aldrich | 49752 | |
| Activin-A | R&D systems | 338-AC | |
| Agarose | Invitrogen | 15510-019 | |
| Aprotinin | Sigma Aldrich | A1153 | |
| Aqua ad injectabilia | Baxter GmbH | 1428 | |
| B27 PLUS insulin | Gibco | 17504-044 | |
| BMP-4 | R&D systems | 314-BP | |
| Collagenase II | Worthington | LS004176 | |
| DMEM | Biochrom | F0415 | |
| DMSO | Sigma Aldrich | D4540 | |
| DNase II, type V (from bovine spleen) | Sigma | D8764 | |
| Dorsomorphin | abcam | ab120843 | |
| EDTA | Roth | 8043.2 | |
| Fetal calf serum | Gibco | 10437028 | |
| FGF2 | Miltenyi Biotec | 130-104-921 | |
| Fibrinogen (bovine) | Sigma Aldrich | F8630 | |
| Geltrex | Gibco | A1413302 | For coating: 1:200 dilution |
| HBSS w/o Ca2+/Mg2+ | Gibco | 14175-053 | |
| HEPES | Roth | 9105.4 | |
| Horse serum | Life technologies | 26050088 | |
| Human serum albumin | Biological Industries | 05-720-1B | |
| Insulin, human | Sigma Aldrich | I9278 | |
| L-Glutamin | Gibco | 25030-024 | |
| Lipidmix | Sigma Aldrich | L5146 | |
| Matrigel | BD Biosciences | 354234 | For EHT reconsitutionmix. |
| N-Benzyl-p-Toluenesulfonamide | TCI | B3082-25G | |
| PBS w/o MgCl2/CaCl2 | Biochrom | 14190 | |
| Penicillin/Streptomycin | Gibco | 15140 | |
| Pluronic F-127 | Sigma Aldrich | P2443 | |
| Polyvinyl alcohol | Sigma Aldrich | P8136 | |
| RPMI 1640 | Gibco | 21875 | |
| Sodium selenite | Sigma Aldrich | S5261 | |
| TGFß1 | Peprotech | 100-21 | |
| Thrombin | Sigma Aldrich | T7513 | |
| Transferrin | Sigma Aldrich | T8158 | |
| Y-27632 | Biorbyt | orb6014 | |
| hiPSC | Custom made at UKE hamburg | na | |
| iCell cardiomyocytes kit | Cellular Dynamics International | CMC-100-010-001 | |
| Pluricyte cardiomyocyte kit | Pluriomics | PCK-1.5 | |
| Cor.4U – HiPSC cardiomyocytes kit | Axiogenesis AG | Ax-C-HC02-FR3 | |
| Cellartis cardiomyocytes | Takara Bio USA, Inc. | Y10075 |
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