这项工作是由美国国立卫生研究院（1F30HL118923-01A1）到TJC，NIH / NHLBI PEN合同HHSN268201000045C到KDC，香港TRS T13-706 / 11（KDC），美国国立卫生研究院（R01 HL113499），以BDG，美国的研究资助局的支持心脏协会（12PRE12060254）到RJ和香港研究资助局（TBRS，T13-706 / 11）RL额外的资金由美国国立卫生研究院DRB 5T32GM008553-18并在系统和发展NIDCR，跨学科培训实习提供给TJC生物学与出生缺陷T32HD075735。作者还希望衷心感谢亚瑟Autz在纽约城市学院的赞恩中心协助加工技术援助生物反应器马姆杜Eldaly。我们也感谢肯尼斯Boheler对心脏分化咨询医生，和Joshua野兔博士慷慨地提供人类骨髓间充质干细胞。

注:执行使用HEPA过滤II级生物安全柜在无菌条件下，所有的细胞操纵和由通过0.2μm过滤器过滤他们消毒所有的解决方案。无论是在相同的无菌条件或层流罩进行组织结构和功能测试。 1.在心脏分化准备H7人类胚胎干细胞的播种<ol><li> （1天）准备基底膜基质<ol><li>解冻的hESC合格基底膜基质的150微升等分试样在冰上过夜，在4℃。 </li></ol></li><li>在涂层板胚胎干细胞（0-4天），电镀<ol><li>稀释解冻矩阵成12毫升的冰冷DMEM / F12拌匀。 </li><li>传输1毫升的DMEM / F12-基​​质溶液到6孔组织培养处理过的培养皿的每个孔中的。矩阵罐外套两个6孔平板的每个等分试样。 </li><li>在室温下孵育的包被的板至少1小时。 注意:在我们的经验，用石蜡密封包被的培养皿可以存储在基质溶液在4℃下使用前长达10天。 </li><li>在大约75％汇合时，从使用6-毫升非酶解试剂的10cm皿解离H7人类胚胎干细胞。 5分钟后，使用一次性细胞刮刀轻轻刮去从培养表面的细胞和细胞悬浮液转移到一个无菌的15毫升离心管中。 </li><li>除去0.5毫升从15ml试管和转移的细胞解离溶液的一个新的无菌的15毫升管（离开5.5毫升解离试剂，以进一步分离所述人类胚胎干细胞），用于干细胞系的传播。 </li><li>沉淀0.5萃取池在300×g离心20℃5分钟。除去上清液和8毫升含有1％青霉素-链霉素的多能干细胞培养基轻轻悬浮然后转移到一个新的，包衣的10cm组织培养皿，保持37℃和5％的CO 2以维持干细胞系。 注意:在媒体的变化保持多能性干细胞介质上冰。 </li><li>同时，添加5.5微升的10mM ROCK抑制剂Y-27632的剩余5.5毫升离解的溶液，并继续在室温下孵育5〜10分钟。轻轻地用5毫升血清吸管混合细胞。继续孵育直到单细胞悬浮液实现，然后沉淀细胞，在300×g离心，在室温下5分钟。 </li><li>悬浮细胞在5ml多能干细胞介质，并使用血球计数器进行细胞计数。 </li><li>种子以每孔14万个细胞的密度的6孔培养皿的每个孔中。种子两个6孔板创建一共有六个定义的组织。电镀后剩余的细胞进行处理。 </li><li>填充井到1ml与多能干细胞培养基，并在37℃，5％的CO 2下孵育。二十四小时后，去除介质和加入2毫升的新鲜多能干细胞培养基到每个孔中（ 图1A </str翁&gt;）。 </li><li>检查板的汇合处的每一天，开始分化一旦细胞达到约75％汇合（ 图1B - D）。 </li></ol></li></ol>人类胚胎干细胞2（4-24天）分化为心肌30,31 <ol><li> （4-7天，分化0-3天），中胚层诱导<ol><li>通过组合500毫升RPMI 1640中，用10ml B27添加物（无胰岛素）和5的青霉素-链霉素母液（10,000微克/毫升链霉素万国际单位/毫升青霉素）毫升制备的RPMI分化培养基I（ 表1）。等分试样，在冰上，入50ml试管并储存在4℃。 </li><li> （第4天，分化0天）加入2.4微升的小分子GSK3抑制剂CHIR99021（10毫米的股票，6μM终浓度）准备中胚层诱导媒体12 RPMI分化培养基毫升一，从每个孔中取出多能干细胞介质一个次用2ml中胚层诱导培养基的更换，每孔。返回板的孵化器。 注:显着的细胞死亡通常与另外CHIR99021（ 图1E）的发生。单层将恢复，但冲洗死细胞远用DMEM / F12冲洗是很重要的。 </li><li>如上所述（第5天，分化第1天）准备新鲜的中胚层诱导培养基。除去从各旧媒体以及并用1ml的DMEM / F12的冲洗一次，每孔。 2毫升新鲜中胚层诱导培养基添加到每个孔中。 注:冲洗0-10天，每天大大增加了心肌细胞的产量和纯度。 </li><li> （第6天，分化2天）取出心脏诱导媒体和每孔的10ml DMEM / F12冲洗一次。替换用2ml我（没​​有小分子添加，但仍与B27（没有胰岛素）补充）的RPMI分化培养基冲洗，并返回到培养箱中。 </li></ol></li><li> （7-13天，分化大Ÿ3-10）心脏中胚层诱导<ol><li> （7天，分化3日）加入6微升的小分子抑制剂Wnt信号IWR-1（10毫米的股票，5微米决赛）的准备心脏中胚层诱导媒体12毫升RPMI分化培养基的一</li><li>除去从各介质孔，每孔1的10ml DMEM / F12冲洗一次，并用2ml心脏中胚层诱导培养基的更换，每孔。 </li><li>如上文所述（第8天，分化第4天）准备额外12毫升心脏中胚层诱导媒体。移除介质之前加入的那一天，以每孔1的10ml DMEM / F12冲洗一次，用每阱2毫升的新鲜心脏中胚层分化培养基的更换，并返回到培养箱中。 </li><li> （9-10天，分化每天5-6）在这两天，取出心脏中胚层诱导媒体和冲洗每孔用1毫升DMEM / F12的。 </li><li>加入2毫升的新鲜RPMI分化培养基的我（不小分子添加，但仍与B27（无胰岛素）补充）到每个孔中并将板返回孵化器。 </li></ol></li><li> （11-24天，分化日7-20）胚胎源性心肌细胞组织/成熟<ol><li>通过组合500毫升RPMI 1640中，用10ml B27添加物（胰岛素）和5的青霉素-链霉素原液毫升制备的RPMI分化培养基II（ 表1）。等分试样，在冰上，入50ml试管并储存在4℃。 </li><li> （第11天，分化第7天）从各取出的RPMI分化培养基（不含胰岛素）以及并用1ml的DMEM / F12冲洗。用2ml的新的RPMI分化培养基II（胰岛素）的每个取代孔，并返回到培养箱中。 注意:自发跳动应先天7和10之间观察到如果在此期间没有观察到打浆，则通常表明分化效率差。该协议可以持续15日为止的一天为了观察跳动，但如无跳动是第15天观察到的最好是圣一场新的艺术分化。 </li><li> （12-24天，分化日8-20）每天，删除旧的分化培养基并用2ml新鲜的RPMI分化培养基Ⅱ的取代每孔以允许打浆单层细胞成熟和组织（ 图1F， 视频图1 ）。 注意:根据所述残留的细胞死亡，可能有必要通过10分化一天/ 1的10ml DMEM F12冲洗。 </li></ol></li></ol> 3.（第24天，分化第20天）心肌细胞和成纤维细胞样细胞的分离<ol><li>从单层细胞分离<ol><li>除去分化培养基并用1ml的PBS冲洗一次。 </li><li>用1毫升的酶解溶液（0.04％胰蛋白酶/ 0.03％EDTA）至各孔离解的单层。 </li><li>移动板进入培养箱10分钟。同时，添加12微升ROCK抑制剂的至6ml的胰蛋白酶中和溶液。 </li><li> GentlY添加1ml含有ROCK抑制剂到板的各孔中，以中和胰蛋白酶溶液的胰蛋白酶中和溶液。 </li><li>使用无菌移液管，轻轻混匀每孔掰开的细胞团。 </li><li>传送从6孔板全部12毫升至15毫升离心管中。 注意:由于两个6孔平板在这个协议中使用的，两个15毫升管将需要。 </li><li>传输3毫升的PBS至板的一个孔中，然后在相同3毫升依次传送到每个随后的井以收集任何残留的细胞上的菜。传送从最终剩余3毫升井到含有细胞相同的15ml离心管中。 </li><li>粒料在300×g离心在4℃下5分钟。 </li></ol></li><li>准备细胞通过FACS活细胞分选<ol><li>通过加入5ml胎牛血清至45毫升的PBS在冰上用50μlROCK抑制剂的制备染色缓冲液。 </li><li>从细胞象素除去上清让（在3.1.8）和1.2毫升染色缓冲液悬浮。 </li><li>转移200微升细胞悬浮液到一个新的，预冷的50毫升离心管置于冰上。这将是阴性染色对照。 </li><li>传送剩下的1 ml的细胞悬浮液到一个新的，预冷的50毫升离心管在冰上，加入2微升SIRPα-PE / Cy7的（1:500稀释度）和4微升CD90-FITC（1:250稀释度） 。轻轻地用移液管混合细胞悬浮液，并返回到冰上。 </li><li> 1小时孵育两个阴性对照和样品，在一个摇杆振动筛，在冰上，在4℃下。 </li><li>通过加入3ml RPMI培养基（胰岛素）的两个15ml离心管中制备样品收集管中。加入3微升ROCK抑制剂的每管并储存在冰上。 </li><li>沉淀经染色的细胞在300×g离心5分钟，在4℃下，用每漂洗至少10毫升冰冷的PBS中漂洗两次。 </li><li> 1微升的DAPI（1微克/毫升）添加到5ml染色缓冲液中。平缓重悬以1-3毫升用移液管将含DAPI染色缓冲的样品沉淀。 </li><li>添加500μl的染色缓冲液（无DAPI添加）的阴性对照。 </li><li>通过40微米的细胞滤网过滤轻轻两个阴性对照和样品以除去细胞团块并转移至聚苯乙烯在冰上FACS管。立即将样品细胞分选。 </li><li>要建立活细胞类似的排序方法18，使用阴性对照，设置闸门，选择活细胞（DAPI负），并收集两个FITC +（ 即 ，CD90 +成纤维细胞）和PE / Cy7的+（ 即 ，SIRPα+心肌细胞）在20psi（ 图2）独立地人口。 注:在设置栅极之后，阴性对照可固定在4％PFA通过染色心脏肌钙蛋白-T来确定分化效率。 注意:有些学者喜欢使用同种型对照，而不是未染色的对照来设置门电路以补偿用于非特异性抗体结合。所谓的荧光减一（FMO）控制是另一种可能性。由于FITC，DAPI和PE-Cy7的信号的清晰的双峰分布，我们从阳性群体门控，保守瞄准远到正栅极，这可能排除某些真阳性而有助于减少任何误报。 </li></ol></li><li>在组织工程准备细胞再聚集<ol><li>小区的排序后，在1ml DMEM中含有10％新生牛血清，1％青霉素 - 链霉素和0.2％两性霉素B（"NBS介质"）沉淀都收集管和重悬。 </li><li>重组的SIRPα+和CD90 +细胞在3:1的比例和板组合的细胞在非组织培养在为60 平方厘米（10厘米培养皿）2百万个细胞的密度处理培养皿。加入10 mL NBS媒体和101，ROCK抑制剂Y-27632 l的。 </li><li>放置细胞悬浮液中的组织培养孵化48小时，以使细胞再聚集成小簇。 </li></ol></li></ol> 4.人类心脏组织工程<ol><li>制造多组织生物反应器 注意:要补充图3A插图，有详细的生物反应器的设计原理图CAD文件可向作者请求。 <ol><li>机通过钻0.5mm直径六个均匀间隔的孔成聚四氟乙烯9×33×3.25毫米长方体的PDMS母模。 </li><li>使用立铣刀，机从聚砜测量25×35×11 立方毫米的框架。该帧的目的是为了保持与PDMS讯息对准在底板的孔中（从上方取得的铸造构造的）。 </li><li>使用1毫米的端铣刀，机器6个孔（6×1×1毫米3），4毫米分割成一个20×40×5毫米3一块黑色的聚四氟乙烯，以形成基板。 </li><li>混合在一个10的弹性体基础和用于聚二甲基硅氧烷（PDMS）固化剂1 w / w的比率，并添加到自定义聚四氟乙烯模具（步骤4.1.1）来创建六个力传感讯息两排，并孵育过夜，并在真空在80℃。固化后，轻轻地从母模取出PDMS，仔细标记每个岗位为增强对比度和自动实时后偏转跟踪黑色永久性标记的顶部。 注意:聚四氟乙烯是相当软，并很容易被损坏。清洗，PDMS铸造，以确保系统和一致的PDMS后几何长寿母模前时要小心。为0.5mm导线可以用来清洁每次使用后支柱上的孔，但照顾到不刮孔的内部。 注意:另一种方法是脱气真空下的PDMS在室温下几个小时，然后让在环境压力下将混合物固化。这可能导致在固化过程中与PDMS形成较少的残留气泡。 </li><li>消毒所有组件蒸汽高压灭菌器。 注:PDMS母模（步骤4.1.1）和聚砜帧（步骤4.1.2）都是可重复使用。自投（步骤4.1.4）中创建的PDMS职位也可重复使用，但只有约10用途。然而，由于使用模具师傅可以根据需要创建更多的PDMS的职位。 </li></ol></li><li>收集Reaggregated心肌细胞<ol><li>除去从培养箱的reaggregated细胞和所有10个毫升再聚集媒体转移到50毫升离心管中。 </li><li>冲洗用3毫升的PBS在板和漂洗转移到含有再聚集媒体相同的50ml离心管中。 </li><li>添加3毫升0.04％胰蛋白酶/ 0.03％EDTA至10 cm培养皿，并返回到培养箱中5分钟。 </li><li> 5分钟后，检查用倒置化合物显微镜板在10倍的放大倍率，以确保完全细胞dissocia化从盘。如果一些残留的集群仍然附着，轻轻搅动板分离的团块。如果簇保持连接，返回到培养箱中另一2-3分钟。不要孵化时间超过十分钟显著细胞死亡，可能会出现。 </li><li>一旦所有的细胞分离，加入3ml胰蛋白酶中和溶液。轻轻混匀与胰蛋白酶的细胞溶液，并转移到含有再聚集媒体的50ml离心管中的中和溶液，并用PBS冲洗一次。 </li><li>冲洗用5ml PBS的整个盘并转移到含有细胞同样的50毫升管。 </li><li>沉淀细胞，在300×g离心，在室温下5分钟。 </li><li>去除上清，悬浮在1毫升NBS媒体的颗粒，然后转移到一个1.5 ml离心管。 </li><li>沉淀，在300×g离心，在室温下5分钟，去除上清。心脏细胞（CD90 +基质细胞和SIRPα+ MYOCytes）现在已经准备好为组织构建。 </li></ol></li><li> （可选）收取利息的补充细胞 注意:除了仅含SIRPα+心肌细胞和CD90 +成纤维细胞样细胞中的定义组织中，也可以添加感兴趣的额外的细胞以询问其对组织的功能的效果。例如大鼠MSC已经显示增强大鼠的工程心脏组织1的功能。以下任选的步骤描述了补充细胞来定义的系统的集合。 <ol><li>收集辅助细胞类型的利益使用0.25％胰蛋白酶/ 0.1％的EDTA（ 例如 ，间充质干细胞）。 </li><li>沉淀细胞，在300×g离心在室温下5分钟，然后在5毫升适当的细胞培养介质的悬浮为感兴趣的细胞类型。例如，对于干细胞，可以使用补充有20％胎牛血清，1％青霉素 - 链霉素和0.2％两性霉素B至培养的C厄尔。 </li><li>使用血球进行细胞计数，然后在300 xg离心再次沉淀细胞，在室温下5分钟。 </li><li>除去在1ml细胞培养基和转移的上清，重悬到1.5ml微量离心管中。 </li><li>沉淀细胞，在300×g离心，在室温下5分钟。 </li><li>去除上清。补充细胞现在已经准备好组织建设。 注意:如果所述补充细胞以在组织中的细胞总数的10％的浓度加入，则所定义的组织，这将需要每组织50000补充细胞作为每个组织含有50万心脏细胞（均CD90 +基质细胞和SIRPα+细胞）。 </li></ol></li><li>创建人工程心脏组织 注:储存在冰上，并在室温温度下，所有的解决方案的细胞。下面列出的所有卷每个组织。通常情况下，大约6种组织可以从两个构造6孔复心脏分化的阿泰。 <ol><li>稀释60.0微升5毫克/毫升的胶原库存的3.125毫克/毫升与1.5微升的1M NaOH中，10倍的PBS 9.6微升和24.9微升的灭菌超去离子水。 关键的一步:避免在制备过程中引入气泡的每个解决方案，气泡会扰乱适当的组织形成。 </li><li>同时添加10倍的MEM 12.0微升和0.2N的HEPES pH为9至稀胶原混合物以创建胶原组合。为了避免引入气泡进入胶原组合加入两种溶液走在15ml离心管的一侧。 </li><li>基底膜基质添加到0.9毫克/毫升至胶原混合的最终浓度，并储存在冰上，以创建最终组织组合。胶原的最终浓度应为2毫克/毫升。 </li><li>添加reaggregated细胞500,000到组织混合物（肌细胞+成纤维细胞浓度为20万个/ ml）和填充到25微升的每一个终体积与任一无细胞NBS介质或感兴趣的（ 例如 ，的hMSCs）补充细胞类型的50,000个细胞并充分混合组织，以形成均匀的细胞悬浮液。 注意:补充的细胞数将来自不同的应用而变化。这里使用的10％的补充。 </li><li>小心，吸管25微升细胞悬浮液到每个生物反应器中的底板的六个孔中的，而不引入气泡进入井中。 </li><li>推PDMS力传感器的两行上的聚砜框架的任一侧，形成6对相对帖子，然后反在底板顶部的框架，使一对柱进入每个孔含有细胞悬浮液。 注:聚砜框架包括标签在PDMS对齐提供帮助。 </li><li>小心地将生物反应器，底板下来，到60毫米的菜，然后将菜没有它的10cm皿内盖，放置在顶部的10厘米盖，并移动整个生物反应器组件于组织培养孵化器，等待两个小时的组织到凝胶。 </li><li> 2小时后，从孵化器中取出的生物反应器，并添加14毫升NBS媒体向整个组件，足以覆盖基板上。返回到孵化器，并每天换一半的媒体。 </li><li>四十八个小时后，小心地轻轻底板的每侧几毫米一次离开所述帧去除基板，改变介质，然后返回所述生物反应器，组织朝下，向媒体。 </li><li>每天都在不断变化，国家统计局培养基的一半。 注意:在组织的自发收缩可以组织结构后，早在3天观察到的，早5天至第7产生可测量的抽搐的力。 </li></ol></li></ol>

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biowire:+a+platform+for+maturation+of+human+pluripotent+stem+cell-derived+cardiomyocytes.">Biowire: a platform for maturation of human pluripotent stem cell-derived cardiomyocytes.</a> Nature Methods. 10 (8), 781-787 (2013).</li><li>Ma, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+filamentous+human+diseased+cardiac+tissue+model.">Three-dimensional filamentous human diseased cardiac tissue model.</a> Biomaterials. 35 (5), 1367-1377 (2014).</li><li>Baker, B. M., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deconstructing+the+third+dimension:+how+3D+culture+microenvironments+alter+cellular+cues.">Deconstructing the third dimension: how 3D culture microenvironments alter cellular cues.</a> J. Cell Sci. 125 (13), 3015-3024 (2012).</li><li>Pontes Soares, C., Midlej, V., de Oliveira, M. E. W., Benchimol, M., Costa, M. L., Mermelstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2D+and+3D-organized+cardiac+cells+shows+differences+in+cellular+morphology,+adhesion+junctions,+presence+of+myofibrils+and+protein+expression.">2D and 3D-organized cardiac cells shows differences in cellular morphology, adhesion junctions, presence of myofibrils and protein expression.</a> PloS One. 7 (5), e38147 (2012).</li><li>Burridge, P. W., Keller, G., Gold, J. D., Wu, J. 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Res. 111 (3), 344-358 (2012).</li><li>Kim, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-cardiomyocytes+influence+the+electrophysiological+maturation+of+human+embryonic+stem+cell-derived+cardiomyocytes+during+differentiation.">Non-cardiomyocytes influence the electrophysiological maturation of human embryonic stem cell-derived cardiomyocytes during differentiation.</a> Stem Cells Dev. 19 (6), 783-795 (2010).</li><li>Dubois, N. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracoronary+bone+marrow+cell+transfer+after+myocardial+infarction:+5-year+follow-up+from+the+randomized-controlled+BOOST+trial.">Intracoronary bone marrow cell transfer after myocardial infarction: 5-year follow-up from the randomized-controlled BOOST trial.</a> Eur. Heart J. 30 (24), 2978-2984 (2009).</li><li>Meyer, G. 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Heart J. 32 (14), 1736-1747 (2011).</li><li>Simari, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+mononuclear+cell+therapy+for+acute+myocardial+infarction:+a+perspective+from+the+cardiovascular+cell+therapy+research+network.">Bone marrow mononuclear cell therapy for acute myocardial infarction: a perspective from the cardiovascular cell therapy research network.</a> Circ. Res. 114 (10), 1564-1568 (2014).</li><li>Bhattacharya, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+differentiation+of+human+pluripotent+stem+cells+to+cardiomyocytes+and+characterization+by+flow+cytometry.">High efficiency differentiation of human pluripotent stem cells to cardiomyocytes and characterization by flow cytometry.</a> J. Vis. Exp. 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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+universal+system+for+highly+efficient+cardiac+differentiation+of+human+induced+pluripotent+stem+cells+that+eliminates+interline+variability.">A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability.</a> PloS One. 6 (4), e18293 (2011).</li><li>Kean, T. J., Lin, P., Caplan, A. I., Dennis, 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=MSCs:+Delivery+Routes+and+Engraftment,+Cell-Targeting+Strategies,+and+Immune+Modulation.">MSCs: Delivery Routes and Engraftment, Cell-Targeting Strategies, and Immune Modulation.</a> Stem Cells Int. , 732742 (2013).</li><li>Trachtenberg, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rationale+and+design+of+the+Transendocardial+Injection+of+Autologous+Human+Cells+(bone+marrow+or+mesenchymal)+in+Chronic+Ischemic+Left+Ventricular+Dysfunction+and+Heart+Failure+Secondary+to+Myocardial+Infarction+(TAC-HFT)+trial:+A+randomized,+double-blind,+placebo-controlled+study+of+safety+and+efficacy.">Rationale and design of the Transendocardial Injection of Autologous Human Cells (bone marrow or mesenchymal) in Chronic Ischemic Left Ventricular Dysfunction and Heart Failure Secondary to Myocardial Infarction (TAC-HFT) trial: A randomized, double-blind, placebo-controlled study of safety and efficacy.</a> Am. Heart J. 161 (3), 487-493 (2011).</li><li>Williams, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramyocardial+stem+cell+injection+in+patients+with+ischemic+cardiomyopathy:+functional+recovery+and+reverse+remodeling.">Intramyocardial stem cell injection in patients with ischemic cardiomyopathy: functional recovery and reverse remodeling.</a> Circ. Res. 108 (7), 792-796 (2011).</li><li>Razeghi, P., Young, M. E., Alcorn, J. L., Moravec, C. S., Frazier, O. 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The authors declare that they have no competing financial interests.

定义人工程心脏组织（HECT）的建设可以提供人体心肌细胞的功能更一致和可靠的模型。关键的是，系统中的所有细胞和细胞外成分是已知的并且根据需要，从而消除从分化过程产生的其他未知的细胞类型的混杂影响可以被操纵。以平衡快速的细胞生长和高收率，优选的分化开始在人类胚胎干细胞的75％汇合时，理想的电镀后四天。此外，这两个排序后的细胞解离和再凝集期间使用ROCK抑制剂Y-27632?...

心脏组织工程已在过去十年中极大地推进，以多组发布最近来自鼠心肌细胞1-6和，充分发挥功能，打浆组织，人干细胞衍生的心肌细胞7-12的结果。心脏组织工程领域是由两个主要和基本上独立目标驱动:1），以开发可移植到衰竭心脏改善功能4-6外源性移植物;和2） 的体外模型开发用于研究生理学和疾病，或作为筛选工具用于治疗发展2,7。 三维（3-D）的细胞培养物被认为是开发下一代筛选工具，作为3维矩阵反映更自然心脏微环境比传统的二维单层细胞培养至关重要;的确细胞生物学的一些方面是在2-D对的3-D培养物13,14根本不同</suP&gt;。细胞外基质，及细胞群:此外，工程化的心脏组织被从完全定义的组件构成。对于传统的设计的人心脏组织中，而细胞外基质组合物（通常为纤维蛋白9或胶原7,8,10）被严格控制，输入单元组合物不太明确，与细胞的整个混合物从一个定向心脏分化任胚胎干细胞（ESC 7,9）或诱导的多能干细胞（IPSC的10,12）被添加到组织中。取决于具体的细胞系和所使用的分化方案的效率，所得到的心肌细胞的百分比可以从低于25％的范围内，以90％以上，具体的心肌的表型（ 即 ，ventricular-，atrial-或起搏器状）也可以有所不同，甚至非心肌分数可以是高度异质15,16和改变的分化心肌米到期yocytes 17。 最近心脏组织工程工作一直试图控制单元的输入口，与任一心脏记者人胚胎干细胞系8或细胞表面标记18被用来分化的心肌细胞成分分离。虽然最初只心肌细胞组成的组织，似乎是理想的，这是事实上并非如此;完全的心肌细胞组成hECTs无法压缩成功能的组织，具有一定的群体找到一个3:心肌细胞比为1:成纤维细胞产生了最高的抽搐力8。通过使用各种小区选择方法，其中包括表面标记活细胞分选，可以创建具有限定的细胞群体hECTs。而非心脏间质细胞的标记物已经有一段时间，如推定的成纤维细胞标记物CD90 19,20，心肌细胞的表面标记已经比较困难鉴别。 SIRPα是确定用于人类心肌细胞18中的第一心脏表面标记之间并已被证明是心脏谱系高度选择性。最近，我们已发现，双分拣SIRPα+和CD90 -细胞产生几乎纯的心肌细胞，与CD90 +群体表现出成纤维细胞样表型（Josowitz，未发表的观察结果）。基于这些发现收集，在此，我们介绍了如何创建使用3 hECTs:SIRPα+ / CD90的结合1 -心肌细胞和CD90 +成纤维细胞。 工程师一个完全确定的人类心脏组织的能力，不仅是用于创建健壮的筛选工具，而且还为开发模型系统研究新兴细胞和基于基因的心脏治疗至关重要。在心脏衰竭特别是，众多的细胞疗法，利用细胞类型，包括间质干细胞（MSC）21 </SUP&gt;，心脏干细胞22和骨髓单核细胞23-25，已经在临床试验中进行测试。虽然许多初步结果已被看好21,23,25，最初的好处往往削弱随着时间的推移26-29。类似的趋势已经报道鼠工程心脏组织，这显示显著功能的好处，由于MSC的补充，但效益长期培养1期间不会持续。基础的亚最佳性能是我们有限理事细胞疗法的机制的知识。如何治疗细胞发挥其有益的影响，以及肌细胞nonmyocyte相互作用的潜在的负面影响更深的了解，将有助于提高治疗的临床收益和显著效益的持续发展，以最小的副作用，患者的心脏衰竭。 在这里，我们描述了使用定义hECTs来interrog吃的基于细胞的疗法的机制。受控组织构成是必要的识别影响心肌性能的具体因素。直接补充hECTs与感兴趣的治疗性细胞类型（ 例如 ，干细胞），可以揭示在心肌细胞性能的影响，如我们在大鼠的ECT 1已经证明。 以下多步协议开始指示心脏干细胞的分化，其次多的组织的生物反应器的制造，并与组织结构和功能分析的说明结束。我们的实验中使用的是美国国立卫生研究院批准H7人类胚胎干细胞（hESC细胞）线进行。但是，以下的协议也已使用额外的人类胚胎干细胞系，并与类似的结果3诱导多能干细胞（hiPSC）行测试。我们已发现，在心肌细胞的分化和成功HECT制造效率可以细胞系相关的，特别是FO从个别病人衍生řhiPSC线。按照此协议，两个6孔皿镀有共168万人类胚胎干细胞（每孔14万个细胞），它区分了20天，分选，足以花费六个定义组织后产生大约250万细胞。

<table border="0">
	<tbody>
		<tr>
			<td>Cell Culture</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma-Aldrich</td>
			<td>A2411</td>
			<td>Prepare a 2.5 mg/ml stock in DMSO and filter-sterilize</td>
		</tr>
		<tr>
			<td>B27 with Insulin</td>
			<td>Life Technologies</td>
			<td>17505055</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 without Insulin</td>
			<td>Life Technologies</td>
			<td>A1895601</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Stemgent</td>
			<td>04-0004</td>
			<td>Create 6 &#956;M stock, then aliquot and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Essential 8 Media</td>
			<td>Life Technologies</td>
			<td>A1517001</td>
			<td></td>
		</tr>
		<tr>
			<td>H7 Human Embryonic Stem Cells</td>
			<td>WiCell</td>
			<td>WA07</td>
			<td></td>
		</tr>
		<tr>
			<td>hESC Qualified Matrix, Corning Matrigel</td>
			<td>Corning</td>
			<td>354277</td>
			<td>Thaw on ice at 4 &#176;C overnight then aliquot 150 &#956;l into separate tubes and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>IWR-1</td>
			<td>Sigma-Aldrich</td>
			<td>I0161</td>
			<td>Create 10 mM stock and aliquot. Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Neonatal Calf Serum</td>
			<td>Life Technologies</td>
			<td>16010159</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-enzymatic Dissociation Reagent: Gentle Cell Dissociation Reagent</td>
			<td>Stem Cell Technologies</td>
			<td>7174</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td>Keep refrigerated</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK Inhibitor)</td>
			<td>Stemgent</td>
			<td>04-0012</td>
			<td>Resuspend to a 10 mM stock concentration, aliquot and store at -20 &#176;C.&#160; Avoid freeze thaw cycles.</td>
		</tr>
		<tr>
			<td>Cell Sorting</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>4&#8217;,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)</td>
			<td>Life Technologies</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>CD90-FITC</td>
			<td>BioLegend</td>
			<td>328107</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzymatic Dissociation Reagent: Cell Detach Kit I (0.04 % Trypsin/ 0.03% EDTA, Trypsin neutralization solution and Hanks Buffered Salt Solution)&#160;</td>
			<td>PromoCell</td>
			<td>C-41200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologics</td>
			<td>S11250</td>
			<td></td>
		</tr>
		<tr>
			<td>SIRP&#945;-PE/Cy7</td>
			<td>BioLegend</td>
			<td>323807</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Construction</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>0.25% Trypsin/0.1% EDTA</td>
			<td>Fisher Scientific</td>
			<td>25-053-CI</td>
			<td>Optional: For collection of supplemental cells of interest</td>
		</tr>
		<tr>
			<td>10x MEM</td>
			<td>Sigma-Aldrich</td>
			<td>M0275-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>10X PBS Packets</td>
			<td>Sigma-Aldrich</td>
			<td>P3813</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen, Bovine Type I</td>
			<td>Life Technologies</td>
			<td>A10644-01</td>
			<td>Keep on ice</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Life Technologies</td>
			<td>11330057</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagles Medium (DMEM), High Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D5648</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3784</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>1.5 ml microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>NC0536757</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml polyproylene centrifuge tube</td>
			<td>Corning</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml Polystyrene Round-Bottom Tube</td>
			<td>Corning</td>
			<td>352235</td>
			<td>With integrated 35 &#956;m cell strainer</td>
		</tr>
		<tr>
			<td>50 ml polyproylene centrifuge tube</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well flat bottom tissue-culture treated plate</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Scraper, Disposable</td>
			<td>Biologix</td>
			<td>70-2180</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysulfone</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polytetrafluoroethylene (Teflon)</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Olympus</td>
			<td>SZ-61</td>
			<td>Or similar, must have a mount for the high speed camera to attach</td>
		</tr>
		<tr>
			<td>Electrical Pacing System</td>
			<td>Astro-Med, Inc</td>
			<td>Grass S88X Stimulator</td>
			<td></td>
		</tr>
		<tr>
			<td>High Speed Camera</td>
			<td>Pixelink</td>
			<td>PL-B741U</td>
			<td>Or similar, but must be capable of 100 frames per second for accurate data acquisition</td>
		</tr>
		<tr>
			<td>Plate Temperature Control</td>
			<td></td>
			<td></td>
			<td>Used to maintain media temperature during data acqusition.</td>
		</tr>
		<tr>
			<td>Custom Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>LabView Post-tracking Program</td>
			<td></td>
			<td></td>
			<td>available upon request from the authors</td>
		</tr>
	</tbody>
</table>

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.

以获得心肌细胞，的Boheler和廉分化方法略加修改使用30,31。当务之急是在分化过程中的细胞生长的对数阶段开始，也即初始种群足够汇合排序（约75％是最优的）后，得到的细胞的可用数目。典型地，对于H7的hESCs，在保持在37℃的每一个的6孔培养皿的孔140000人类胚胎干细胞中必不可少8媒体和5％CO 2培养箱的密度电镀后4天产生充分融合培养开始分化，如所示在图1A中- <s...

Watch this Scientific Journal Video about Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy at JoVE.com

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

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 ...

construction-defined-human-engineered-cardiac-tissues-to-study

Research

JoVE Journal

Bioengineering

9.3K 次观看.  Icahn School of Medicine at Mount Sinai. 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.

视频: Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Shock Wave Application to Cell Cultures

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to target cells or tissue without any subsequent damage. Therefore, in vitro experiments are of increasing interest. Various methods of applying shock waves onto cell cultures have been described. In general, all existing models focus on how to best apply shock waves onto cells.
However, this question remains: What happens to the waves after passing the cell culture? The difference of the acoustic impedance of the cell culture medium and the ambient air is that high, that more than 99% of shock waves get reflected! We therefore developed a model that mainly consists of a Plexiglas built container that allows the waves to propagate in water after passing the cell culture. This avoids cavitation effects as well as reflection of the waves that would otherwise disturb upcoming ones. With this model we are able to mimic in vivo conditions and thereby gain more and more knowledge about how the physical stimulus of shock waves gets translated into a biological cell signal (&#8220;mechanotransduction&#34;).

Shock waves nowadays are well known for their regenerative effects. Therefore in vitro experiments are of increasing interest. We therefore developed a model for in vitro shock wave trials (IVSWT) that enables us to mimic in vivo conditions thereby avoiding distracting physical effects.

冲击波应用细胞培养

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to ...

科研

生物工程

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure. 
Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive. 
In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve.

Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

在小型组织培养体系培养老鼠心脏瓣膜

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

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

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

人工程心脏组织的收缩自动分析心脏药物安全筛选

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor analytes against the background of human serum are crucial.
Nanoimprinting lithography (NIL) was used to fabricate, at a low cost, sensing areas as large as 1.5 mm x 1.5 mm. The sensing surface was made of high-fidelity arrays of nanoholes, each with an area of about 140 nm2. The great reproducibility of NIL made it possible to employ a one-chip, one-measurement strategy on 12 individually manufactured surfaces, with minimal chip-to-chip variation. These nanoimprinted localized surface plasmon resonance (LSPR) chips were extensively tested on their ability to reliably measure a bioanalyte at concentrations varying from 2.5 to 75 ng/mL amidst the background of a complex biofluid-in this case, human serum. The high fidelity of NIL enables the generation of large sensing areas, which in turn eliminates the need for a microscope, as this biosensor can be easily interfaced with a commonly available laboratory light source. These biosensors can detect cardiac troponin in serum with a high sensitivity, at a limit of detection (LOD) of 0.55 ng/mL, which is clinically relevant. They also show low chip-to-chip variance (due to the high quality of the fabrication process). The results are commensurable with widely used enzyme-linked immunosorbent assay (ELISA)-based assays, but the technique retains the advantages of an LSPR-based sensing platform (i.e., amenability to miniaturization and multiplexing, making it more feasible for POC applications).

This work describes a nanoimprinting lithography method to fabricate high-quality sensing arrays that work on the principle of extraordinary optical transmission. The biosensor is low-cost, robust, easy to use, and can detect cardiac troponin I in serum at clinically relevant concentrations (99th percentile cutoff &#8764;10-400 pg/mL, depending on the assay).

用超常光透射法定量测定人血清中的心脏标志物

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

MicroRNA 移植成人骨髓造血干细胞促进细胞工程与磁性靶向结合的协议

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

基于网模的无支架三维心脏组织创建方法

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

人体器官性心脏切片双电压和钙光学映射的临床前心脏电生理评估

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

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 ...

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 ...

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 ...