Name: automated contraction analysis of human engineered heart tissue for cardiac drug safety screening
Uploaded: 2017-04-15T13:00:00.000+00:00
Duration: 10 min 39 s
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 / 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>

<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><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&amp;nbsp;</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&amp;nbsp;</td><td>Thermo scientific</td><td>70101</td><td>Adjust rotor speed to 40 rpm</td></tr><tr><td>T175 cell culture flask&amp;nbsp;</td><td>Sarstedt&amp;nbsp;</td><td>831,812,002</td><td></td></tr><tr><td>V-shaped sedimentation rack&amp;nbsp;</td><td>Custom made at UKE Hamburg</td><td>na</td><td></td></tr><tr><td>10&amp;times; DMEM</td><td>Gibco</td><td>52100</td><td></td></tr><tr><td>1-Thioglycerol&amp;nbsp;</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&amp;nbsp;</td><td>R&amp;amp;D systems</td><td>338-AC</td><td></td></tr><tr><td>Agarose&amp;nbsp;</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&amp;nbsp;</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>BMP-4</td><td>R&amp;amp;D systems</td><td>314-BP</td><td></td></tr><tr><td>Collagenase II&amp;nbsp;</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&amp;nbsp;</td><td>Sigma Aldrich</td><td>D4540</td><td></td></tr><tr><td>DNase II, type V (from bovine spleen)</td><td>Sigma&amp;nbsp;</td><td>D8764</td><td></td></tr><tr><td>Dorsomorphin&amp;nbsp;</td><td>abcam</td><td>ab120843</td><td></td></tr><tr><td>EDTA&amp;nbsp;</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&amp;nbsp;</td><td>Gibco</td><td>A1413302</td><td>For coating: 1:200 dilution</td></tr><tr><td>HBSS w/o Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;&amp;nbsp;</td><td>Gibco</td><td>14175-053</td><td></td></tr><tr><td>HEPES&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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 MgCl&lt;sub&gt;2&lt;/sub&gt;/CaCl&lt;sub&gt;2&lt;/sub&gt;</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&amp;nbsp;</td><td>Sigma Aldrich</td><td>P2443</td><td></td></tr><tr><td>Polyvinyl alcohol&amp;nbsp;</td><td>Sigma Aldrich</td><td>P8136</td><td></td></tr><tr><td>RPMI 1640&amp;nbsp;</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&amp;szlig;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&amp;nbsp;</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

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Pharmacology

Bioengineering

Unit 1

Cardiovascular Drugs: Antiarrhythmic and Heart Failure Drugs

SCIENTISTS IN ACTION

12.6K 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

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 ...

科研

生物工程

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

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 ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

在微流体平台内开发 3D 组织人类心脏组织

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

Hybrid Cell Analysis System to Assess Structural and Contractile Changes of Human iPSC-Derived Cardiomyocytes for Preclinical Cardiac Risk Evaluation

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold promise to serve as a human-relevant model in preclinical phases of drug discovery and safety pharmacology, their maturity is still controversial in the scientific community and under constant development. We present a hybrid contractility and impedance/extracellular field potential (EFP) technology, adding significant pro-maturation features to an industry-standard 96-well platform.
The impedance/EFP system monitors cellular functionality in real-time. Besides the beat rate of contractile cells, the electrical impedance spectroscopy readouts detect compound-induced morphological changes like cell density and integrity of the cellular monolayer. In the other component of the hybrid cell analysis system, the cells are cultured on bio-compliant membranes that mimic the mechanical environment of real heart tissue. This physiological environment supports the maturation of hiPSC-CMs in vitro, leading to more adult-like contractile responses including positive inotropic effects after treatment with isoproterenol, S-Bay K8644, or omecamtiv mecarbil. Parameters such as the amplitude of contraction force (mN/mm2) and beat duration also reveal downstream effects of compounds with influence on electrophysiological properties and calcium handling.
The hybrid system provides the ideal tool for holistic cell analysis, allowing preclinical cardiac risk assessment beyond the current perspectives of human-relevant cell-based assays.

The analysis of changes in contractile function and cellular integrity of human iPSC-derived cardiomyocytes is of immense importance for nonclinical drug development. A hybrid 96-well cell analysis system addresses both parameters in a real-time and physiological manner for reliable, human-relevant results, necessary for a safe transition into clinical stages.

用于评估人iPSC来源心肌细胞结构和收缩变化的杂交细胞分析系统，用于临床前心脏风险评估

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

2D人干细胞来源心肌细胞中心脏收缩力调节疗法的评估

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...

Preclinical Drug Testing in Scalable 3D Engineered Muscle Tissues

Accurately modeling healthy and disease conditions in vitro is vital for the development of new treatment strategies and therapeutics. For cardiac and skeletal muscle diseases, contractile force and kinetics constitute key metrics for assessing muscle function. New and improved methods for generating engineered muscle tissues (EMTs) from induced pluripotent stem cells have made in vitro disease modeling more reliable for contractile tissues; however, reproducibly fabricating tissues from suspended cell cultures and measuring their contractility is challenging. Such techniques are often plagued with high failure rates and require complex instrumentation and customized data analysis routines. A new platform and device that utilizes 3D EMTs in conjunction with a label-free, highly-parallel, and automation-friendly contractility assay circumvent many of these obstacles. The platform enables facile and reproducible fabrication of 3D EMTs using virtually any cell source. Tissue contractility is then measured via an instrument that simultaneously measures 24 tissues without the need for complex software analysis routines. The instrument can reliably measure micronewton changes in force, allowing for dose-dependent compound screening to measure the effect of a drug or therapeutic on contractile output. Engineered tissues made with this device are fully functional, generating twitch and tetanic contractions upon electrical stimulation, and can be analyzed longitudinally in culture over weeks or months. Here, we show data from cardiac muscle EMTs under acute and chronic dosing with known toxicants, including a drug (BMS-986094) that was pulled from clinical trials after patient fatalities due to unanticipated cardiotoxicity. Altered skeletal muscle function in engineered tissues in response to treatment with a myosin inhibitor is also presented. This platform enables the researcher to integrate complex, information-rich bioengineered model systems into their drug discovery workflow with minimal additional training or skills required.

This protocol provides methods for generating 3D engineered cardiac and skeletal muscle tissues and describes their use in preclinical drug screening modalities. The described methods utilize a magnetic sensing system to facilitate the simultaneous assessment of 24 tissues in parallel.

可扩展的3D工程肌肉组织中的临床前药物测试

Accurately modeling healthy and disease conditions in vitro is vital for the development of new treatment strategies and therapeutics. For cardiac and ...

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering is crucial for basic science cardiac research; in vitro human cell culture eliminates the interspecies differences of animal models, while a more tissue-like 3D environment (e.g., with extracellular matrix and heterocellular coupling) simulates in vivo conditions to a greater extent than traditional two-dimensional culture on plastic Petri dishes. However, each model system requires specialized equipment, for example, custom-designed bioreactors and functional assessment devices. Additionally, these protocols are often complicated, labor-intensive, and plagued by the failure of the small, delicate tissues.
This paper describes a process for generating a robust human engineered cardiac tissue (hECT) model system using induced pluripotent stem-cell-derived cardiomyocytes for the longitudinal measurement of tissue function. Six hECTs with linear strip geometry are cultured in parallel, with each hECT&#160;suspended from a pair of force-sensing polydimethylsiloxane (PDMS) posts attached to PDMS racks. Each post is capped with a black PDMS stable post tracker (SPoT), a new feature that improves the ease of use, throughput, tissue retention, and data quality. The shape allows for the reliable optical tracking of post deflections, yielding improved twitch force tracings with absolute active and passive tension. The cap geometry eliminates tissue failure due to hECTs slipping off the posts, and as they involve a second step after PDMS rack fabrication, the SPoTs can be added to existing PDMS post-based designs without major changes to the bioreactor fabrication process.
The system is used to demonstrate the importance of measuring hECT function at physiological temperatures and shows stable tissue function during data acquisition. In summary, we describe a state-of-the-art model system that reproduces key physiological conditions to advance the biofidelity, efficiency, and rigor of engineered cardiac tissues for in vitro applications.

Three-dimensional cardiac tissues bioengineered using stem-cell-derived cardiomyocytes have emerged as promising models for studying healthy and diseased human myocardium in vitro while recapitulating key aspects of the native cardiac niche. This manuscript describes a protocol for fabricating and analyzing high-content engineered cardiac tissues generated from human induced pluripotent stem-cell-derived cardiomyocytes.

设计生物反应器以改善工程心脏组织的数据采集和模型通量

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering ...

Contractions of Human-iPSC-derived Cardiomyocyte Syncytia Measured with a Ca-sensitive Fluorescent Dye in Temperature-controlled 384-well Plates

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac physiology and pharmacology. Various methods have been proposed to record this spontaneous activity and to evaluate drug effects, but many of these methods suffer from limited throughput and/or physiological relevance. We developed a high-throughput screening system to quantify the effects of exogenous compounds on hiPSC-CM's beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader. We describe how to prepare the cell plates and the compound plates and how to run the automated assay to achieve high sensitivity and reproducibility. We also describe how to transform and analyze the fluorescence data to provide reliable measures of drug effects on spontaneous rhythm. This assay can be used in drug discovery programs to guide chemical optimization away from, or toward, compounds affecting human cardiac function.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells are useful models of human cardiac physiology and pharmacology. Here we present a high-throughput screening system to quantify the effects of exogenous compounds on beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader.

温度控制384孔板中用 Ca 敏感荧光染料测定人 iPSC 的心肌细胞较多的收缩

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac ...

医学

3D Human Myocardial Tissue Generation Using Melt Electrospinning Writing of Polycaprolactone Scaffolds and hiPSC-Derived Cardiac Cells

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and regenerative medicine. This protocol describes the stepwise fabrication of 3D myocardial tissues with advanced mimicry of native cardiac structure by combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds with fibrin hydrogels and human induced pluripotent stem cell (hiPSC)-derived cardiac cells. The process involves embedding a mixture of cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts (hiPSC-CFs) within a fibrin matrix to create mini-tissues, with structural support provided by MEW-generated scaffolds. These fibrillar scaffolds are fabricated at the micro- to nanoscale, allowing for precise control over fiber architecture, which plays a key role in organizing cell distribution and alignment. Meanwhile, the fibrin matrix promotes cell viability and mimics the extracellular environment. Characterization of the generated tissues reveals well-organized sarcomeres within hiPSC-CMs, along with stable contractile activity. The tissues demonstrate consistent spontaneous beating as early as two days post-seeding, with sustained functionality over time. The combination of hiPSC-CFs with hiPSC-CMs enhances the structural integrity of the tissues while supporting long-term cell viability. This approach offers a reproducible, adaptable, and scalable method for creating biomimetic cardiac tissue models, providing a versatile platform for preclinical drug testing, mechanistic studies of cardiac disease, and potential regenerative therapies.

A reproducible method is presented to generate 3D myocardial tissues combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds and fibrin hydrogels with hiPSC-derived cardiomyocytes and fibroblasts. This technique offers precise control over scaffold architecture and can be applied in preclinical drug testing and cardiac disease modeling.

使用聚己内酯支架和 hiPSC 衍生的心肌细胞的熔融静电纺丝写入生成 3D 人体心肌组织

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and ...

Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety assessment models is a critical step during drug development. Currently, cardiac safety assessment is still highly dependent on animal studies. However, animal models are plagued by poor translational specificity to humans due to species-specific differences, particularly in terms of cardiac electrophysiological characteristics. Thus, there is an urgent need to develop a reliable, efficient, and human-based model for preclinical cardiac safety assessment. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as an invaluable in vitro model for drug-induced cardiotoxicity screening and disease modeling. hiPSC-CMs can be obtained from individuals with diverse genetic backgrounds and various diseased conditions, making them an ideal surrogate to assess drug-induced cardiotoxicity individually. Therefore, methodologies to comprehensively investigate the functional characteristics of hiPSC-CMs need to be established. In this protocol, we detail various functional assays that can be assessed on hiPSC-CMs, including the measurement of contractility, field potential, action potential, and calcium handling. Overall, the incorporation of hiPSC-CMs into preclinical cardiac safety assessment has the potential to revolutionize drug development.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising in vitro model for drug-induced cardiotoxicity screening and disease modeling. Here, we detail a protocol for measuring the contractility and electrophysiology of hiPSC-CMs.

微电极阵列和膜片钳记录在人诱导多能干细胞来源心肌细胞上的技术应用

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety ...