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 / F-12培养基含有2mM L-谷氨酰胺，0.5％青霉素/链霉素，5毫克/升运铁蛋白，5微克/升硒，0.1％人血清白蛋白，1×脂质混合，5 mg / L的胰岛素，50nM的dorsomorphin，2.5纳克/ ml激活素A，0.5毫微克/毫升人类TGFß1和30毫微克/毫升FGF2。 </li><li>种子的hiPSC（〜3×10 5/10平方厘米）在FTDA培养基和培养在37℃，5％CO 2，21％O 2，每天更换培养基（图1A）。在80％汇合时，通道（1：2-1：6）与乙二胺四乙酸（EDTA; 0.5毫米;培养时间4〜分钟）。 </li></ol></li><li> 胚胎体的形成（EB） <ol><li>在T75烧瓶中，以高密度培养的hiPSC。 <ol><li>用磷酸盐缓冲盐水洗与汇合的细胞培养瓶两次不含钙和镁（PBS）和0.5毫摩尔EDTA孵育〜10分钟。确保细胞不从烧瓶底部脱离。 </li><li>删除与玻璃吸管EDTA，冲洗掉用10mL PBS细胞，抽头烧瓶在工作台以分离细胞。 注意：使用细胞刮器，如果必要的。 </li><li>收集细胞在50mL锥形管中，加入体积的四分之一（ 例如，10毫升RMPI至40mL细胞悬浮液）的RPMI 1640培养基（含或1.8mM的钙的任何其他标准培养基）中，在250×g离心5分钟，并离心。 </li></ol></li><li>在FTDA介质悬浮细胞沉淀中添加4毫克/毫升的聚乙烯醇（PVA）和10μMY27632和计数在Neubauer室中的细胞。 </li><li>填充与细胞悬浮液在旋转瓶（补充30×10个6细胞/ 100mL的FTDA介质与PVA和Y-27632;参见步骤1.2.2）和引发EB形成在旋转烧瓶（调整磁力搅拌器的转子速度至40rpm ）在细胞培养孵育箱（37℃，5％CO 2，5％O 2）过夜。 </li></ol></li><li> 中胚层分化 <ol><li>在EB形成，外套细胞培养瓶适于与非离子表面活性剂（14毫升/ T175）过夜，在37℃的悬浮培养。 </li><li>第二天早晨，洗净表面活性剂包被的烧瓶两次，用PBS（30毫升/ T175）。再次加入PBS，并保持在孵化器，直到需要。 </li><li>除去从培养箱旋转器烧瓶中。 注：细胞悬浮液应是具有小宏观可见的EB（ 图1B）清楚。 </li><li>转移50毫升悬浮液至50mL锥形桶e和让EB的解决5-20分钟。 </li><li>去除上清，用7mL的RPMI 1640补充有2％PVA和10μMY-27632洗涤沉淀。转移悬浮液到刻度15mL锥形管中，估计EB体积（〜50-100微升）。 </li><li>传递旋转烧瓶的内容到未涂覆的T175烧瓶并离开此对沉降V形机架长达20分钟。除去上清液，并如上文所述洗涤胚状体。不补的超过200毫升的T175烧瓶沉淀的时间太长。如果初始旋转瓶体积超过200毫升，分离EB悬浮到几个T175烧瓶并在洗涤后再次结合的EB。 </li><li>在介质上去除洗涤介质和重悬为中胚层分化，这是补充有4毫克/毫升PVA，0.5％青霉素/链霉素，10mM的4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES），0.05％人血清白蛋白，5微克/毫升转铁蛋白，5纳克/ mL亚硒酸钠，0.1％的脂质混合物，250μM磷酸抗坏血酸，10μMY-27632，10ng / mL的骨形态发生蛋白-4（BMP-4），和3纳克/ ml激活素A，5纳克/毫升稳定FGF2。 </li><li>从表面活性剂涂布的T175烧瓶中取出PBS，用细胞悬液（200μL的EB在40毫升）填充。使用较小的瓶中小EB量。 </li><li>在悬浮液中三天（37℃，5％CO 2，5％O 2）培育的EB。交换介质的50％（相同的组成，在步骤1.3.7）每天（使用V形沉降机架）。准备喂食之前每天培养基的新鲜。 </li></ol></li><li> 心肌分化 <ol><li>之前准备新的表面活性剂涂覆容器的日期和处理上述（1.3.2）如所述。 </li><li>后中胚层诱导的三天，让定居的EB对沉降机架最多5分钟。除去大部分的培养基中，并用RPMI 1640补充有0.5％青霉素/链霉素和10mM HEPES洗。 </li><li>转移10 EB悬浮液mL至刻度15毫升管中并估计沉降后EB体积。 </li><li>重悬的EB仔细心脏分化培养基由RPMI 1640补充有0.5％青霉素/链霉素，10mM的HEPES，0.05％人血清白蛋白，5微克/毫升转铁蛋白，5纳克/ mL亚硒酸钠，0.1％的脂质混合物，250μM的磷酸-ascorbate，1μMY-27632，100nM的的Wnt抑制剂DS-I-7 13。 </li><li>转移的EB到新的表面活性剂包被的细胞培养瓶中（〜200μL的EB在46毫升/ T175）。 </li><li>孵育三天（37℃，5％CO 2，21％O 2），用50％介质变化（培养基组成见步骤1.4.4）在所述第二和第三天。不要在第一个48小时内改变介质。 </li><li>孵育在介质由RPMI 1640补充有500μM的1-硫代甘油，10mM的HEPES，0.5％青霉素/链霉素，1μMY-27632，2％无血清的神经细胞培养物补充物（ 例如 B27）胰岛素，100nM的Wnt信号的 - 抑制剂DS-I-7与接下来的四天50％的媒体的日常交流。 EB的开始应该在这段时间内跳动。 </li><li>心脏分化的一周后，切换到无的Wnt抑制，并用无血清的神经细胞培养物补充物的相同培养基中。每天改变介质的50％，除了第一天。 </li></ol></li><li> 人类心肌细胞的解离 注：2周分化方案后，EB中应显示自发收缩（ 图1C）。如果是这样，开始分解并准备胶原酶溶液。 <ol><li>通过称重和在无钙的Hanks&#39;平衡盐溶液解决200U / mL的无钙/镁（HBSS）制备胶原酶溶液，并添加1mM的HEPES，10μMY-27632，30μMN-苄基 - 对 - 甲苯磺酰胺（ BTS）。通过过滤（0.2微米的过滤器），并存储等分在-20℃下灭菌。在4℃下过夜解冻等分试样。 </li><li>在沉降机架解决EBS，吸出培养基并替换为预热20-25毫升HBSS。重复此洗涤步骤一次。 </li><li>吸HBSS，并用预热的胶原酶溶液（15mL / T175）替换。孵育在细胞培养箱4小时（37℃，5％CO 2，21％O 2）。 </li><li>制备封闭缓冲液中用DMEM补充有1％青霉素/链霉素和DNA酶（12微克/毫升）。 </li><li>点击板凳上的细胞培养瓶，解离的EB应瓦解和崩溃（如果没有，增加孵育时间）。轻轻磨碎的细胞悬浮液，并转移到锥形50mL试管。洗用封闭缓冲液（15毫升/ T175）瓶中，并转移至管中。 </li><li>离心机在100×g下10分钟。重悬在温暖的DMEM，磨碎细胞和细胞计数。 </li></ol></li></ol> 2.代工程心脏组织的（EHT） <ol><li>高压灭菌市售PDMS机架和聚四氟乙烯（PTFE）垫片（见材料和设备的电子表格; 图2），并在无菌条件下制备以下溶液，EHT代前一天。 <ol><li>通过称重并在PBS中，高压釜的适当体积中溶解制备2％的琼脂糖，立即储存在60℃。 </li><li>制备抑肽酶（33毫克/毫升）通过称重和在AQUA广告iniectabilia的适当体积的溶解，在-20℃下进行过滤灭菌（0.22微米过滤器），等分试样并储存。 </li><li>通过在-20℃的无菌纤维蛋白原溶解在无菌的0.9％NaCl（温），等分试样并储存的适当体积制备纤维蛋白原（200毫克/毫升）。 </li><li>通过在三个部分无菌凝血酶溶解制备凝血酶（100U / mL）的无菌PBS和2份AQUA广告iniectabilia，在小管子3微升的等分部分并储存在-20℃。 </li><li>通过在5mL AQUA广告iniectabilia溶解670毫克10×DMEM粉末制备10X DMEM，无菌过滤并储存于4℃。 </li><li>准备的2×DMEM通过混合2毫升10×DMEM用2mL热灭活的马血清，0.2毫升青霉素/链霉素和5.8毫升水上广告iniectabilia，过滤，在4℃的无菌和存储。 </li><li>制备EHT浇铸通过在4℃下用10％热灭活的胎牛血清，1％青霉素/链霉素和1％L-谷氨酰胺（200毫摩尔），商店混合DMEM培养基（ECM）。 </li></ol></li><li>通过吸取1.6mL的温暖琼脂糖（2％，PBS）以及将所述PTFE隔离物（ 图2A）至孔准备在24孔板铸模。 注：将立即隔离吸取到最多8个井后 - 琼脂凝固速度非常快。不要让在室温下琼脂糖超过5分钟。 </li><li>琼脂糖固化（〜10分钟，琼脂糖会变成不透明）后，取出PTFE隔离物（ 图2C），并放置在PDMS机架（ 图2B）进入铸模以便对PDMS帖子的伸入每个模具（ 图2D）。 </li><li&gt;重悬ECM的心肌细胞与15×10 6细胞/ mL的最小浓度。 </li><li>在上ice.For 24 EHTs圆底15mL的管中准备重建混合物的估计的体积的110％，吸管在表1中用于人，大鼠或小鼠的心肌细胞中列出的重构组合，分别为（10％的额外被包括在内）。 注：重构混合物的细胞浓度是人类（10×10 6个细胞/ mL），大鼠（4.1×10 6个细胞/ mL）和小鼠（6.8×10 6个细胞/ mL）EHTs不同。取决于细胞悬浮液的浓度，添加额外的ECM到2640微升的最终体积。纤维蛋白原必须是不冷不热的移液。慢慢地填补了吸管的尖端和纤维蛋白原增加时，重建组合不与含纤维蛋白原吸管触及冷重建混合物表面 - 在枪头纤维蛋白原的粘度会立即增加。 </li><li>磨碎重建组合10 - 一个血清移液管，直到纤维蛋白原凝块15次溶解。检查吸管同质重建组合。 </li><li>用于在一个凝血酶等分试样（3μL在200μL管）各EHT，移液管将100μL复溶混，混合并且将混合物尽可能快吸管进入琼脂糖时隙尽可能（ 图2E）。使用每个EHT一个新的过滤嘴。重悬每8个EHTs后的重建混合（研磨与血清吸管5-10倍）。 </li><li>一旦所有槽都充满了重建混合物，关闭细胞培养皿的盖子，并在37℃，7％CO 2温育2小时。 </li><li>在无菌罩下除去从培养箱和地点板。覆盖每个孔用少量的培养基（ 例如 DMEM，大约200 - 500μL），轻轻摇动平板上并在37℃再次孵育至少10分钟。加入DMEM的将缓解由纤维蛋白凝胶从琼脂糖铸造模具去除。 </l我&gt; <li>准备一个新的24孔板中以每孔由DMEM补充有10％热灭活的马血清，1％青霉素/链霉素，10μg/ mL的胰岛素，33微克/ mL抑肽酶在1.5毫升EHT-平台。 </li><li> 小心地从琼脂糖铸模除去PDMS机架和它们传送到填充介质-24孔板（图2F）。放置培养皿放回培养箱（37℃，7％CO 2，40％O 2对人类和大鼠EHTs，21％O 2对小鼠EHTs）和饲料星期一，星期三和星期五用新鲜制备的EHT-平台。 EHTs应铸造（图1D，2G）后7-10天呈现自发性收缩偏转PDMS架的帖子。 <ol><li>对于小鼠EHTs加入25微克/毫升5-胞嘧啶βDarabinofuranoside到培养基48小时，以限制成纤维细胞增殖。 </li></ol></li></ol> 3.收缩分析注：收缩分析是基于视频-光记录在市售的EHT分析仪器（见的材料数据表 ）。该仪器的中央部是一个孵育室（ 图3A）。随仪器提供该软件基于与已知的弹性模量和几何形状11（ 补充图1）PDMS柱的偏转计算收缩力。 <ol><li>转之前的测量机器2小时的加热装置上。的收缩分析开启气体供给和轴系统的电子供给之前立即。 </li><li> 为基线记录（在分析中的参考点），将24孔板在EHT-介质EHTs成EHT分析仪，并根据供应商手册开始收缩分析软件。 <ol><li>单击右侧面板上的“协议”选项卡，并输入有关研究的名称，用户，品种，CE相关信息LL类型，年龄，记录长度和补充说明。 </li><li>点击左侧面板上的“摄像机视图”选项卡，并开始自动数字检测模式摄像机的实时模式。单击右侧面板上的“设置”选项卡andadjust与XYZ相机位置在24孔盘的每个坐标很好。确保EHT在窗口的中心和相机的焦点。 <ol><li>确保细胞培养皿的盖子是不是有雾，因为这将损害数字识别和收缩分析。 注：图中识别算法是基于检测高对比度区域，并监测其位置的变化。在软件数字识别这些区域将标有蓝色十字架，与EHT的收缩运动。 </li><li>确保蓝色杂交的位置是在EHTs两者的顶端和底端与仅移动垂直匹配EHTs（ 图3B）的收缩。小号的每个孔AVE设置位置通过按下按钮“位置确定”。 </li></ol></li><li>接下来，单击左侧面板上的“参数”选项卡。这里显示的参数定义，通过该软件识别收缩值，并用绿化广场，标志着它的标准。这些值是用于正确识别峰收缩（ 图3C），无假的数据点（ 图3E-F）的非常重要的。 注：的物种相关的参数和示例的列表在图3H中示出。欲知详情，请参考软件手册。 </li><li>单击右侧面板上的“自动”选项卡，并通过激活“Start”按钮开始分析。该软件将从一个很好依次移动到下一个，记录EHT收缩和记录（左侧面板上显示的“实时”选项卡）中计算力。在分析中，PDF报告的结尾summari懋结果将自动生成。 </li></ol></li><li>保存并分析了PDF格式的报告生成汇总结果的系统。 </li><li>监控持续数天的反复测量收缩参数。该EHT会收缩力增加，直到它达到了一个平台期（通常在一天的文化15）。 </li><li> 药物筛选 <ol><li>测量的前一天制备无蛋白质的Tyrode溶液和在24孔板平衡（2毫升/孔）在细胞培养孵育箱（37℃，7％CO 2，40％O 2）过夜：120mM氯化钠， 5.4毫米氯化钾，1mM的MgCl 2的，0.1-5毫米氯化钙2，0.4mM的的NaH 2 PO 4，22.6毫碳酸氢钠 ，5mM葡萄糖，0.05mM的的Na 2 EDTA，25mM的HEPES。添加钙高达1.8 mM或先前确定的[EC 50]调查正性肌力作用。 </li><li>称重和溶解药物的DMSO和如果必要的话，过滤器无菌的。准备工作浓度的6种不同的1000倍子储备溶液（在DMSO中）（ 例如 0.3，1，3，10，30，100μM用于相应纳摩尔范围）。 </li><li>稀释在蒂罗德板的各股票（2μL在各2mL的孔），并充分混合。直到需要将平板放回培养箱中。 </li><li>通过在孔中的无菌罩和位置的碳电极下以第一蒂罗德填充24孔板启动基线测量。转移PDMS机架与放置在碳电极的顶部上的EHTs，盖上盖子，把该板放回培养箱中，等待的〜30分钟的平衡期。 </li><li>从孵化器转移EHTs在EHT分析仪联锁。关闭仪器，记录自发收缩的基线（20秒/ EHT）。 </li><li>碳电极连接到起搏电缆并启动EHTs（2.5 V，4毫秒脉冲持续时间，人力的电刺激：〜1赫兹，大鼠：〜2赫兹，小鼠：〜4赫兹）。记录了 “节奏” 基线（10秒/ EHT）。确保所有EHTs正确节奏。刺激频率和电压可能会有所不同，从细胞系到细胞系。 </li><li>基线记录后，从仪器中EHTs并与PDMS机架上顶部与第一药物浓度转移电极，向24孔板。立即传送EHTs放回EHT分析仪器的联锁和孵化那里（标准药物温育时间例如 20分钟）。 </li><li>重新连接起搏电缆，调整图中识别并记录收缩EHT响应第一药物浓度。 </li><li>对于以下的药物浓度，重复步骤3.5.7和3.5.8用含有较高药物浓度的24孔板。 </li><li>保存所有PDF格式的录音。检查每个记录为数字识别，绿色方块的正确定位识别收缩峰和工件（参见3.2.1和图3 </stron克&gt;）。 </li><li> 如果需要进行额外的离线分析。 <ol><li>通过点击“选择运行”选择从左侧面板上的“流程”数据库中的相应的运行。在左侧的“参数”选项卡上，收缩分析更改参数。现在，在“离线”选项卡中选择合适的面板上的“离线分析”来重新分析视频。 </li><li>要调整图形识别，因此录制新的视频文件，选择调整中的“实时”选项卡中的数字识别后“抽样审查”。注：抽样审查只能在一个时间再分析一个好，而离线分析可以通过点击按钮“上所有的孔，使用参数”，在左侧的“参数”面板应用新参数的所有井。这两种类型的离线分析会自动创建新的数据文件和PDF格式显示结果。 </li></ol></li><li>通过将B的结果分析实验iological复制和总结在浓度响应曲线的数据频率，收缩力，收缩时间和弛豫时间（ 图5D）和平均收缩峰值（ 图5C）/或图形显示。 注：PDMS机架和PTFE垫片可以反复使用（PDMS机架最多5次，和PTFE垫片无休止）。使用后，人工清洗与水，软化水和高压煮两次。注意潜在的遗留效应，当再次使用机架，因为药物可能被PDMS所吸收。 </li></ol></li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> J Biomol Screen. 4 (2), 67-73 (1999).</li><li>Vandenburgh, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Durg-screening+platform+based+on+the+contractility+of+tissue-engineered+muscle.">Durg-screening platform based on the contractility of tissue-engineered muscle.</a> Muscle Nerve. 37 (4), 438-447 (2008).</li></ol>

IM，TE和AH是EHT技术公司，德国的联合创始人。

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 ...

automated-contraction-analysis-human-engineered-heart-tissue-for

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Bioengineering

Unit 1

Cardiovascular Drugs: Antiarrhythmic and Heart Failure Drugs

SCIENTISTS IN ACTION

12.5K 次观看.  University Medical Center Hamburg-Eppendorf and DZHK (German Center for Cardiovascular Research). 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。 

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

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

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

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

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

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

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

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

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.

In cardiac myocytes, tubular membrane structures form intracellular networks. We describe optimized protocols for i) isolation of myocytes from mouse heart including quality control, ii) live cell staining for state-of-the-art fluorescence microscopy, and iii) direct image analysis to quantify the component complexity and the plasticity of intracellular membrane networks.

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. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

三维组织工程人食管黏膜型号生产，表征和潜在用途

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

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 dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system 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

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

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

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 mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

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

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

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

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in 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 ...

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

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

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

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular network comprising several levels of complexity to provide oxygen and nutrients within bioengineered heart tissues. Our laboratory has developed a three-dimensional in vitro model of the human heart, known as the "cardiac spheroid" or "CS". This presents biochemical, physiological, and pharmacological features typical of the human heart and is generated by co-culturing its three major cell types, such as cardiac myocytes, endothelial cells, and fibroblasts. Human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs or iCMs) are co-cultured at ratios approximating the ones found in vivo with human cardiac fibroblasts (HCFs) and human coronary artery endothelial cells (HCAECs) in hanging drop culture plates for three to four days. The confocal analysis of CSs stained with antibodies against cardiac Troponin T, CD31 and vimentin (markers for cardiac myocytes, endothelial cells and fibroblasts, respectively) shows that CSs present a complex endothelial cell network, resembling the native one found in the human heart. This is confirmed by the 3D rendering analysis of these confocal images. CSs also present extracellular matrix (ECM) proteins typical of the human heart, such as collagen type IV, laminin and fibronectin. Finally, CSs present a contractile activity measured as syncytial contractility closer to the one typical of the human heart compared to CSs that contain iCMs only. When treated with a cardiotoxic anti-cancer agent, such as doxorubicin (DOX, used to treat leukemia, lymphoma and breast cancer), the viability of DOX-treated CSs is significantly reduced at 10 &#181;M genetic and chemical inhibition of endothelial nitric oxide synthase, a downstream target of DOX in HCFs and HCAECs, reduced its toxicity in CSs. Given these unique features, CSs are currently used as in vitro models to study heart biochemistry, pathophysiology, and pharmacology.

This protocol aims to fabricate 3D cardiac spheroids (CSs) by co-culturing cells in hanging drops.&#160;Collagen-embedded CSs are treated with doxorubicin (DOX, a cardiotoxic agent) at physiological concentrations to model heart failure. In vitro testing using DOX-treated CSs may be used to identify novel therapies for heart failure patients.

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 ...