Name: creation of cardiac tissue exhibiting mechanical integration of spheroids using 3d bioprinting
Uploaded: 2017-07-02T13:00:00.000+00:00
Duration: 4 min 40 s
Description: Watch this Scientific Journal Video about Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting at JoVE.com

作者承认以下资金来源:神经科学心血管研究基金和马里兰州干细胞研究基金（2016-MSCRFI-2735）。

1.心肌细胞的制备<ol><li>如上所述，在用基底膜基质包被的6孔板上产生和培养人诱导的多能干细胞（hiPSC）。 </li><li>使用先前描述的方法11,12将hiPSC分化为hiPSC来源的心肌细胞（hiPSC-CM）。 </li><li>分化后第19天，使用2mL胰蛋白酶/ EDTA的胰蛋白酶/ EDTA 0.05％的室温分离5分钟。 </li><li>在光学显微镜下监测心肌细胞以观察细胞解离。 </li><li>使用2mL胰蛋白酶抑制剂中和胰蛋白酶0.0125％。 </li><li>放置分离的心肌细胞，并使用电动吸管填充物将其转移到一个50 mL锥形管中。 </li><li>在室温下以250×g离心细胞悬液5分钟，得到沉淀物。 </li><li>将沉淀物重新悬浮在5mL Roswell Park中补充有B-27（RPMI / B-27细胞培养基）的纪念研究所（RPMI）细胞培养基。 </li><li>移取细胞培养液中的20μL细胞，并用等量的0.4％台盼蓝溶液进行染色。 </li><li>使用自动细胞计数器或手动血细胞计数器来计数和获得细胞悬浮液的浓度和细胞活力。 </li></ol> 2.成纤维细胞的制备<ol><li>启动人心脏成纤维细胞（HCF）（成人心室型）细胞系13 。 </li><li>通过并根据HCF培养方案分离它们13 。 </li><li>移取细胞培养液中的20μL细胞，并用等量的台盼蓝溶液0.4％染色。 </li><li>使用自动细胞计数器或手动血细胞计数器来计数和获得新细胞悬浮液的浓度和细胞活力。 </li></ol> 3.内皮细胞的制备<ol><li>启动人类的伞如静脉内皮细胞（HUVEC）线所述。按照HUVEC培养方案进行传播并分离，如图14所示 。 </li><li>移取细胞培养液中的20μL细胞，并用等量的0.4％台盼蓝溶液进行染色。 </li><li>使用自动细胞计数器或手动血细胞计数器来计数和获得细胞悬浮液的浓度和细胞活力。 </li></ol>共同文化: <ol><li>将三种细胞类型（hiPSC-CM，HCF和HUVEC）混合在RPMI / B-27细胞培养基中，以一个hiPSC-CM:HCF:HUVEC细胞比率在50mL锥形管中产生混合细胞溶液原料70:15:15，浓度为165,000个细胞/ mL。 </li><li>使用多通道移液管将混合细胞溶液分配到每孔200μL或每孔33,000个细胞的超低附着96孔U底板中。 </li><li>将96孔板孵育3天（37℃，5％二氧化碳）ide，95％湿度）。 </li><li>在光学显微镜下检查单个孔的中部和底部混合细胞多细胞球体的存在。 注意:在二维微观投影中，这些球体将呈圆形（ 图1 ）。在细胞接种到超低附着的96孔板后24小时内，球形体应形成。细胞播种到96孔板后48小时内，球体应开始跳动。不跳动的球体不应用于3D生物印刷，以确保最终的3D生物印迹心脏贴片是功能性的。球蛋白对连接蛋白43（Cx43）和肌钙蛋白的免疫荧光可用于评估球状细胞组成和质量15 。 </li></ol> 5.脚手架心脏组织的3D生物印迹<ol><li>将含有球体的板载入无支架的基于球体的3D生物打印机的杂志。 </li><li> 打开3D生物打印机并执行3D生物打印软件。 <ol><li>单击"辅助"，"操作准备"，"检查模式"，然后单击"归位"。 </li><li>单击"开始"下的绿色图标开始球体检查。 </li><li>观察球体直径出现在屏幕上的"（2）Dia（μm）"列下。 </li></ol></li><li>要将平均球体直径估算为最接近的100μm，请单击"初始设置"，并将平均球体直径输入到堆叠Z间距（μm）。 </li><li>单击"总计"，"层"下的数字应自动调整。点击"应用"确认设置。 </li><li>点击"针数组图"，进入"图层编号"，选择所需的3D生物印刷图层，并通过选择单个球体位置，在屏幕左侧的地图上绘制所需的3D生物印刷设计。社区法网k"应用"以确认3D生物印刷设计。 </li><li>点击"针阵检查"开始针阵检查。如果针阵列失焦，则输入值为"OffsetZ（μm）"，范围为0〜500μm，调整摄像机对焦，起始时间为500μm，减少100μm至0μm。 </li><li>单击"检查参数"，"类型1"，并将"直径（μm）"设置为所需的直径范围。将"圆度（％）"和"平滑度（％）"设置为所需的百分比。点击"应用"确认设置。 注意:这里的球体直径为450μm至550μm。 "圆度（％）"和"平滑度（％）"是计算机计算每个球体的评估。这里，用于"圆度（％）"的设置为60％，"平滑度（％）"为70％。 </li><li>单击"存在"并将"直径（μm）"更改为相同的设置在上一步。点击"应用"确认设置。 </li><li>单击"堆叠模式"，然后点击"开始"下的绿色图标，使用3D生物打印机的喷嘴通过真空吸力开始球体拾取，并将球体放置在针阵列中的特定位置。 注意:对于由81个球体（9个球体×9个球体）组成的心脏贴片，3D生物印迹需要约20至30分钟。 </li><li>生物印刷后，使用无菌镊子，拿起包含3D生物印迹贴片的针阵列，并将其转移到含有150 mL RPMI / B-27细胞培养基的250 mL无菌烧杯中。 </li><li>使用针阵列孵育3D生物印迹贴片3天（37℃，5％二氧化碳，95％湿度）。 </li></ol> 6.从针阵列和贴片成熟中去除3D生物印迹贴片<ol><li>孵育3天后，用一只手在针阵列的底部，另一只手在塑料盖上他补丁，轻轻地将塑料盖向上滑动以从针阵列中移除3D生物印迹补丁。 注意:根据需要，可以在本步骤之前将磷酸盐缓冲盐水（PBS）润滑施用于针头，以便更平滑和更容易地去除。 </li><li>使用一对无菌镊子，将3D生物印迹贴片拾起塑料盖，并将贴片和塑料盖转移到含有RPMI / B-27细胞培养基的35 mm培养皿中。 </li><li>使用RPMI / B-27细胞介质中的贴片轻轻摇动塑料盖，镊子仍贴在塑料盖上，将贴片释放到介质中。 </li><li>在光学显微镜下检查3D生物印迹贴片可视化是否打败以确保3D生物印迹贴片功能正常。 </li><li>将三维生物印迹贴片在35毫米的培养皿中孵育3天或更长时间（37℃，5％二氧化碳，95％湿度）。 注意:3天后，球体会熔合在一起，并将孔插入那些针头的补丁不应该再显眼了。 </li></ol>

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作者没有什么可以披露的。

It is important to use beating, functional spheroids for 3D bioprinting. If spheroids are not beating, continuing to use them will invariably result in a non-functional 3D bioprinted patch.
One benefit of this approach is the ability to manipulate the cell content of the patch by varying the total number of cells and the percentage of cardiomyocytes, endothelial cells, and fibroblasts in the spheroids. This allows for many different types of cardiac patches to be printed, with varying histological and mechanical properties.
This approach also allows different types of spheroids to be used to make a patch, as well as the 3D bioprinting of cardiac patches with complex 3D designs. This complexity of the final 3D construct is achievable by 3D bioprinting with precise spheroid positioning, but may be difficult to achieve by gentle rotation of floating spheroids7 or simple molding of spheroids into a cylindrical 3D construct16.
The limitations of this approach are the relatively fixed size of the spheroids between 450 to 550 &#181;m due to the fixed distance of 400 &#181;m between the needles in the needle array, as well as possibly reduced cell viability if the spheroids are too large in diameter. The patches are mechanically fragile immediately after removal from the need array; however, the strength and durability are improved with culture for longer durations of time.
Finally, 3D bioprinting of cardiac patches has promising clinical applications17, in cardiac regeneration18,19 and as 3D models of heart disease. These 3D models can be used for applications in predictive pharmacology/toxicology and development of cell and genome directed therapies. 
In conclusion, it is feasible to 3D bioprint cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids. 3D bioprinting of cardiac tissue has promising clinical applications in cardiac tissue regeneration and as 3D models of heart disease.

3D生物印迹有很多不同的方法， 1，2，3 。 3D生物打印通常通过印刷技术1进行分类，例如喷墨生物印刷，微压缩生物印刷，激光辅助生物印刷，方法的组合或更新的方法。 3D生物印迹也可以分为无支架或支架依赖的方法4 。 3D生物印迹的大多数方法是支架依赖的，其中需要生物材料， 例如生物蛋白5或支架6 。然而，支架依赖的3D生物印迹面临许多问题和局限性，如脚手架材料的免疫原性，专有生物蛋白的成本，降解产物的速度和毒性等。 SCAF已经尝试使用球体的无折叠心脏组织工程8 ，有可能克服支架依赖组织工程的这些缺点。然而，正如作者在该论文中所承认的那样，在生物制造过程中难以强力地处理和定位固定位置的球体。伴随使用3D生物印迹和基于球形的组织工程有可能克服这些困难。在本协议中，我们描述了没有其他生物材料的心脏组织的3D生物印迹，仅使用球形形式的细胞。 基于无支架的基于球体的3D生物打印机9具有使用真空抽吸拾取单个球体的能力，并将其定位在针阵列上。 3D生物印刷中针阵列上的定位球体的概念是从古代日本使用针阵（被称为" kenzan "）的灵感来的nese艺术的花卉安排， ikebana。该系统允许球体精确地定位在任何配置中，并导致单个球体在短时间内熔合在一起以产生3D生物印刷组织。因此，该方法允许容易地操纵球体，对于无支架器官生物制造的未来具有潜在的影响。

<table><tbody><tr><td>Geltrex</td><td>Invitrogen&amp;nbsp;</td><td>A1413202</td><td></td></tr><tr><td>Trypsin/EDTA 0.05%</td><td>Thermo Fisher</td><td>15400054</td><td></td></tr><tr><td>Defined Trypsin inhibitor 0.0125%</td><td>Thermo Fisher</td><td>R007100</td><td></td></tr><tr><td>RPMI Cell Media</td><td>Invitrogen</td><td>11875-093</td><td>RPMI supplemented with B27 constitutes HIPSC-CM culture media</td></tr><tr><td>B-27 Supplement</td><td>Thermo Fisher</td><td>17504044</td><td>RPMI supplemented with B27 constitutes HIPSC-CM culture media</td></tr><tr><td>Countess Automated Cell Counter</td><td>Invitrogen</td><td>C10227</td><td></td></tr><tr><td>Human cardiac fibroblasts (adult ventricular type)</td><td>Sciencell</td><td>6310</td><td></td></tr><tr><td>Human umbilical vein endothelial cells</td><td>Lonza</td><td>CC-2935</td><td></td></tr><tr><td>PrimeSurface ultra-low attachment 96-well U-bottom plates&amp;nbsp;</td><td>Akita Sumitomo Bakelite Co.</td><td>MS-9096UZ</td><td></td></tr><tr><td>Regenova Bio 3D Printer</td><td>Cyfuse Biomedical K.K.</td><td>N/A</td><td>www.cyfusebio.com/en/</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Thermo Fisher</td><td>15250061</td><td></td></tr><tr><td>Troponin T Antibody</td><td>Thermo Fisher</td><td>701620</td><td></td></tr><tr><td>Connexin 43 (Cx43) Antibody</td><td>Chemicon</td><td>MAB3068</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant with DAPI</td><td>Thermo Fisher</td><td>P36935</td><td></td></tr></tbody></table>

creation of cardiac tissue exhibiting mechanical integration of spheroids using 3d bioprinting

该协议描述了仅使用细胞而不使用生物材料的心脏组织的3D生物印迹。首先分离心肌细胞，内皮细胞和成纤维细胞，计数并以所需细胞比例混合。它们在超低附件96孔板中的各个孔中共培养。在3天内，殴打球体。然后使用真空抽吸由喷嘴拾取这些球体，并使用3D生物打印机在针阵列上组装。然后允许球体在针阵列上熔断。三维生物印迹三天后，球状体作为完整的贴片被去除，已经自发地发生跳动。 3D生物印迹的心脏贴片显示组件球体的机械整合，并且在心脏组织再生和心脏病的3D模型中是非常有希望的。

在步骤4.4（共培养）结束时，每个孔中的细胞应在超低附着96孔U底板的底部聚集，通过重力形成球体。这些球体包含hiPSC-CM，HCF和HUVEC，并且可以在光学显微镜下目视检查，其中它们应该通过二维投影呈现圆形（ 图1 ）。在步骤6.3结束时，3D生物印迹的心脏贴片应该包含组织空隙，这是由于针阵列产生的针孔（ 图2左侧）。在这一点上，球体之间的边界应该变得模糊不清，补丁应该已经开始自发地击败，表现出球体的机械整合。在步骤6.5结束时，组织空隙应该被周围的组织填充（ 图2 ，右），而3D生物印迹的心脏贴片应该继续是自发地。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55438/55438fig1.jpg" /> 图1 :创建混合细胞多细胞球体。将hiPSC-CM，HCF和HUVEC以hiPSC-CM:HCF:HUVEC细胞比为70:15:15混合，将细胞混合物分配到超低附着的96孔U底板中。在24小时内，形成混合细胞多细胞球体（左，右），每孔一个。刻度棒=500μm。 <a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55438/55438fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55438/55438fig2.jpg" /> 图2 :表现出球状体机械整合的心脏组织的形成 。一个3D生物打印心脏贴片后立即从具有可见针孔的针阵列中取出（左）。此时，球体之间的边界已经变得模糊，补丁已经开始自发地跳动，因此球体已经变得机械一体化了。从针阵列中取出三天后，由针阵列引起的组织空隙被周围的组织（右）填充，并且贴片继续自发地跳动。比例尺= 1 mm。 <a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55438/55438fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting at JoVE.com

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

该协议描述了不使用生物材料的心脏组织的3D生物印迹。 3D生物印迹的心脏贴片显示组件球体的机械整合，并且在心脏组织再生和心脏病的3D模型中是非常有希望的。

使用3D生物印刷创建心脏组织展示球体的机械整合

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

creation-cardiac-tissue-exhibiting-mechanical-integration-spheroids

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

9.9K 次观看.  Johns Hopkins Hospital. 该协议描述了不使用生物材料的心脏组织的3D生物印迹。 3D生物印迹的心脏贴片显示组件球体的机械整合，并且在心脏组织再生和心脏病的3D模型中是非常有希望的。 

视频: Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

通过微接触印刷的定向功能性心肌组织的产生

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

科研

生物工程

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 ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

微流控生物工程血管化组织和细胞器解体

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

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. ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

心肌梗塞穆林模型中的 3D 生物打印贴片移植

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

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 ...

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 ...

Creation of Patient-Specific Silicone Cardiac Models with Applications in Pre-surgical Plans and Hands-on Training

Three dimensional&#160;models can be a valuable tool for surgeons as they develop surgical plans and medical fellows as they learn about complex cases. In particular, 3D models can play an important role in the field of cardiology, where complex congenital heart diseases occur. While many 3D printers can provide anatomically correct and detailed models, existing 3D printing materials fail to replicate myocardial tissue properties and can be extremely costly. This protocol aims to develop a process for the creation of patient-specific models of complex congenital heart defects using a low-cost silicone that more closely matches cardiac muscle properties. With improved model fidelity, actual surgical procedural training could occur in advance of the procedure. Successful creation of cardiac models begins with the segmentation of radiologic images to generate a virtual blood pool (blood that fills the chambers of the heart) and myocardial tissue mold. The blood pool and myocardial mold are 3D printed in acrylonitrile butadiene styrene (ABS), a plastic dissolvable in acetone. The mold is assembled around the blood pool, creating a negative space simulating the myocardium. Silicone with a shore hardness of 2A is poured into the negative space and allowed to cure. The myocardial mold is removed, and the remaining silicone/blood pool model is submerged in acetone. The described process results in a physical model in which all cardiac features, including intra-cardiac defects, are represented with more realistic tissue properties and are more closely approximated than a direct 3D printing approach. The successful surgical correction of a model with a ventricular septal defect (VSD) using a GORE-TEX patch (standard surgical intervention for defect) demonstrates the utility of the method.

Patient-specific models improve surgeon and fellow confidence when developing or learning surgical plans. Three-dimensional (3D) printers generate adequate detail for surgical preparation, but fail to replicate tissue haptic fidelity. A protocol is presented detailing the creation of patient-specific, silicone cardiac models, combining 3D printing precision with simulated silicone tissue.

创建患者专用硅胶心脏模型，应用于术前计划和实践培训

Three dimensional&#160;models can be a valuable tool for surgeons as they develop surgical plans and medical fellows as they learn about complex cases. In ...

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 ...