这部分工作是在纳米系统（CNS），在该中心的成员，这是由美国国家科学基金会的支持下，美国国家科学基金会奖没有国家纳米技术基础设施网络（NNIN）。 ECS-0335765。 CNS是哈佛大学的一部分。

生成对齐的功能性心肌组织的协议可分为三个主要部分组成。使用软光刻技术制造的微图案主不考虑了以下协议的一部分，但可以根据建立的方法6。 1。微接触印刷PDMS基板上的纤维连接蛋白<ol><li>混合SYLGARD184（道康宁）PDMS弹性体固化剂的比例和脱气组件，使用干燥器，以消除任何气泡在10:1的基础。 </li><li>对先前制造的主站与宽为20μm和2μm的高大脊，分离了20微米的间距为要么是2天，在室温下或4小时，在65℃，得到的微织构的PDMS邮票PDMS固化。 </li></ol> *固化在干燥器中强烈建议，以消除任何气泡。 <ol start="3"><li>到盖玻片（ 例如旋涂PDMS22x22毫米）在5,000 rpm下，持续2分钟，使用也是Headway自旋涂布机产生薄而均匀的厚度为15μm的PDMS基板。 </li><li>去除灰尘和污垢颗粒，用70％乙醇清洁邮票。让它们空气干燥。 </li></ol> *邮票可以多次使用，但是，定期更新PDMS邮票的建议因表面磨损随着时间的推移。 <ol start="5"><li>将邮票在一个SPI等离子准备II等离子清洗机和1分钟，在275毫呈现PDMS表面亲水性。 </li></ol> *氧等离子体处理应关注。较长的处理时间可能会导致弹性体开裂。 <ol start="6"><li>消毒，用70％乙醇中1分钟的邮票。用压缩空气干燥迅速。 </li></ol> *采取进一步的措施，应在生物安全柜中进行，保持无菌。 <ol start="7"><li>盖邮票表面与Fibronectin溶液（DDH 2 O中的50微克/毫升）。让它吸附的至少10分钟。 </li><li>从邮票甩掉纤维连接蛋白的解决方案和快速干燥压缩空气。 </li><li>建立适形邮票和PDMS基板之间的接触2分钟，然后用力按压。 </li><li>冲洗DDH 2 O的微图案化基板它们存储在DDH 2 O 2周，在4°C，除非最初用于最大。商店的PDMS邮票在DDH 2 O。 </li></ol> 2。维护的双转基因胚胎干细胞系首先，需准备以下媒体培养小鼠胚胎干细胞: 小鼠胚胎成纤维细胞（MEF）媒介:Dulbecco改良的Eagle培养基（DMEM），10％胎牛血清（FBS）的胚胎干细胞或分化级，1％青霉素-链霉素（P / S） 胚胎干细胞（ESC）的媒介:DMEM，15％FBS的ES细胞级1％非必需氨基酸（NEAA），0.5％（P / S），0.2％，白血病抑制因子（LIF），0.0007％2 - 巯基乙醇（2-ME） <ol><li>涂层6孔板用无菌的0.1％明胶DDH 2 O中，并让它吸附15分钟，在37℃下</li><li>从解冻的等分试样添加的MEF在锥形管中的50毫升PBS和离心机在1,000 rpm离心5分钟。重悬MEF中在MEF介质。 </li><li>经过吸附，吸出多余的明胶和MEF中添加入井。允许的MEF 1日至连接。 </li></ol> *等分千万的MEF足以3 ​​6 -孔板。 <ol start="4"><li>附上的MEF时，添加从解冻的等分试样，以50毫升PBS ES细胞在一个锥形管和离心机在1,000 rpm离心5分钟。的重悬ES细胞在ESC介质，并将其添加MEF中的孔中。融合文化ES细胞ESC介质，直到达到。 </li></ol> *一旦ES细胞达到汇合，传代的热曲红外发光二极管，以避免过度生长和维持ES细胞集落。 <ol start="5"><li>准备的ES细胞的传代。吸液ESC介质，并用无菌PBS洗一次。 </li><li>吸出PBS，加入500μl胰蛋白酶。流程3.5分钟，37°C。 </li><li>移液器的胰蛋白酶溶液，向上和向下几次打破ES细胞集落的中和用500μl的ESC介质。 </li><li>通过ES细胞根据您所需的比率， 例如转移33μl到另一家与事先准备好的MEF中通过1/30。维持ES细胞在ESC中。 </li></ol> *一个通道的1/30的比率允许ES细胞在3天内达到汇合。根据通道ES细胞在体外分化时间表。 （3） 在体外分化的双转基因胚胎干细胞系和心脏祖细胞的分离和幼苗生长的首先，准备followi纳克媒体的胚胎干细胞在体外分化为: 适应媒介:Iscove氏改良的Dulbecco氏培养基（IMDM），15％FBS的ES细胞级，1％NEAA，0.5％，P / S，0.2％LIF，0.0007％2-ME的 分化中:IMDM，15％FBS级分化。 1％NEAA，P / S，0.35％，0.0007％的0.1M抗坏血酸0.5％2-ME的<ol><li> ES细胞在体外分化开始时已经达到汇合。外套10厘米的培养皿中，用无菌的0.1％明胶DDH 2 O中，并让它吸附15分钟，在37℃下</li></ol> *成为知道在体外分化过程的持续时间。需要8天，直到心脏祖细胞可以分离。相应地设计您的实验。 <ol start="2"><li>像以前一样的收获ES细胞。吸附后，吸出物过量的明胶，约1/3-1/2的ES细胞悬浮液添加到制备的培养适应介质的培养皿中。培养细胞2天。 </li></ol> *选择适应的胚胎干细胞的数量根据你的实验。 <ol start="3"><li>收获适于ES细胞入50毫升含锥形管中的PBS中，用1。胰蛋白酶5毫升。离心离解的ES细胞以1,000 rpm离心5分钟，和他们在分化培养基中重悬。 </li><li>进行胚状体（EB），约1,000个细胞每10微升在15厘米的培养皿中的下拉使用一个多通道移液管。反转的盘子和文化的EB挂滴2.5天，在37°C。 </li><li>后2.5天，池类胚体的6-815厘米培养皿到一个使用分化介质并进行培养在37℃下进一步3.5天中</li><li>经过3.5天，收集的EB，在一个圆锥形50毫升的试管中，并让他们沉到了底部约5分钟。吸取上清液，用无菌PBS洗胚体。让我们再次胚体下沉的底部为APPRoximately 5分钟。 </li><li>游离的EB处理他们2。 5毫升的胰蛋白酶中2分钟，在水浴中，在37℃下振荡管轻声。然后，添加2。 5毫升的无菌PBS中的胰蛋白酶溶液，向上和向下与10毫升的血清学吸管吸取试剂约8-10倍打破细胞团。中性化胰蛋白酶与10毫升的FACS缓冲液中含有7.5％FBS的ES细胞或分化级，并在PBS中的0.01％的DAPI。 </li><li>解离胚体在1,000 rpm离心5分钟，它们在约1ml的FACS缓冲液中重悬。 1毫升移液管移液管向上和向下分解细胞团约8-10倍。 </li></ol> * FACS缓冲液中添加游离的EB估计金额。过于高度集中的细胞可能会堵塞在FACS，，也低浓度细胞可能会不必要地延长FACS时间。 <ol start="9"><li>转移细胞到无菌的圆底管与一个35微米的细胞过滤器，以获得单细胞悬浮液。 </li><li>排序分化的胚胎干细胞的使用FACSAria流式细胞仪，并获得纯化群体中的CVP。 </li></ol> *我们开发了一个多步骤的选通策略，以隔离高度纯化的着色细胞。简单地说，我们第一门上的前向散射振幅（FSC-A）和侧向散射光的幅度（SSC-A）（ 图1A）。这让我们分开整个细胞从细胞碎片。然后，我们门上的FSC-A和前向散射宽度（FSC-W）（ 图1B）。这使我们能够分离细胞汗衫，双峰和其他细胞聚集。然后，我们门上SSC-A和侧向散射光宽度（SSC-W）（ 图1C）。这增加了细胞汗衫纯度。然后，我们栅极上的DAPI染色，隔离活细胞（DAPI染色阴性）从非存活细胞（ 图1D）。然后，我们门上Phycoeryhthrin幅度（PE-A）和PE-菁染料幅度（PE-CY7-A），以获得真正的红色正相关（r + </sup&gt;的）和真正的红色阴性（R - ）细胞，并排除自身荧光的红色阴性细胞（ 图1E）。 R +，R -细胞，然后选通分别对异硫氰酸荧光素的幅度（FITC-A）和PE-A排序真正的绿色环保阳性（G+）和真正的绿色阴性（G - ）细胞在这些人群中（ 图1F + 1G）。这样的结果在4的细胞群体，如下所示:R + G +，R + G - ，R - G +和R - G - （ 图1H）。 <ol start="11"><li>钝化微模式基片，用1％的Pluronic F-127 DDH 2 O中的10分钟。然后，用无菌PBS洗3倍。 </li></ol> *的Pluronic F-127是一种表面活性剂，阻止细胞粘附。它支持禁闭的细胞粘附到微图案的面积。 <ol start="12"><li>附加的图案化区域周围的PDMS垫片然后用力按压。然后，种子CVP纤维连接蛋白微图案基板上。 </li></ol>

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作者宣称，他们没有竞争的金融利益。

在这个协议中，我们提出了一个方法，心源性祖细胞分离纯化的人口和种子微图案纤连蛋白底物是什么让他们调整和采取心肌细胞杆形。正常的细胞组织是正常组织的功能8,10，特别是用于心肌组织的关键。已经示出心肌细胞发展改进的机械11,12和形成各向异性单元阵列时的电生理特性11,13。由于微图案纤连蛋白底物上培养的心脏祖细胞棒状的心肌细胞在体外分化成?...

尽管在药物治疗的最新进展，先进的心脏衰竭的发病率和死亡率在发达国家仍然是一个领先的原因。出现临床综合征，功能性心肌组织的亏损，随后衰竭的心脏无法满足代谢的需求受影响的个人。由于心脏的再生能力是有限的，自体心脏移植是目前唯一临床接受治疗的直接目的是补充失去功能的心脏组织。心脏移植的显着的缺点，包括有限数量的供心和长期免疫抑制治疗，需要排除这种疗法的广泛普及使用。因此，与心脏疾病的患者，药物治疗是主要的治疗方法。在药物开发的一个主要挑战是确定的细胞分析，准确地概括正常和病变心肌生理特性的影响 。 <html>最近收敛的干细胞与组织工程技术提出了新的心肌再生的可行途径。从可再生的病人特定的源生成功能的心肌组织将在该领域的一大进步。这将允许特定疾病的细胞实验药物开发和发现的发展，并会为心脏再生医学奠定了基础。人类胚胎干细胞（ES）和，更重要的，人类诱导多能干细胞（iPS）细胞代表一个潜在的可再生室祖细胞和心室肌细胞的成熟病人的具体来源。干细胞生物学和组织工程战略的组合解决了这个问题，通过产生用于药物开发的或先进的心脏衰竭的治疗在心脏再生的方法的发展中的细胞分析，可以使用的功能在体外的心脏组织。 _content"&gt; A的主要挑战一直在心肌再生的最佳细胞类型的鉴定。一个广泛的细胞进行了研究，但到今天为止，已经发现虽然不同从各种来源具有多源性前体，一个细胞的识别源，以满足如承诺生肌细胞命运的关键要求，维修能力扩大在体内或在体外和组合物的功能性心肌组织已被证明是一个中心问题2。我们以前曾描述了一种双转基因小鼠系统承诺心室祖细胞（CVP）， 在体外由ES细胞分化的高度纯化的种群的隔离，使我们产生了一种新的双转基因小鼠表达红色荧光蛋白DsRed的ISL1依赖增强剂的MEF2C基因的控制下，并增强型绿色荧光蛋白的控制下心脏特异性NKX2.5增强。基于这两种颜色的荧光报道系统，第一和第二心脏领域祖细胞从显影的ES细胞中，可以通过荧光激活细胞分选术（FACS）根据它们的表达MEF2C和Nkx2.5基因分离。 CVP类似心肌细胞，心肌标志物及结构与功能品质，提供了巨大的潜力心脏组织再生的目的的表达模式的基础上。 虽然已经有许多组织工程领域的进步，模仿原生细胞结构仍然是一个关键的挑战。传统的方法来应对这一挑战，包括种子细胞在体外的生物或人工合成的支架。由于一些弊端的棚架，包括迅速降解，有限的物理和机械稳定性和低细胞密度1,4,5，我们试图工程支架的组织。 Alt键尽管心肌细胞可以修改自己的局部微环境细胞外基质蛋白的分泌，他们有一个更有限的能力，组织起来，杆状心肌细胞在没有外线索。因此，需要一个模板，提供适当的空间和生物细胞的线索，组成功能性心肌组织。微接触印刷解决这个难题，提供一种简单和廉价的技术，能够精确地控制细胞的形状，组织，和功能6-8，所有这一切都是至关重要的对齐的功能性心肌组织的生成。它包括使用的微织构的聚二甲基硅氧烷（PDMS）的特征尺寸范围下降至2μm9，使胞外基质蛋白的沉积在精确的图案的PDMS基板上，从而影响细胞的粘附空间的邮票。 在此，我们建议组织生物工程技术相结合的干CEL升生物产生各向异性功能的心肌组织。因此，我们在这里展示我们最近发表的方法为如下:（1）生成的微图案蛋白质表面上的PDMS基板通过微接触印刷的模板的对齐的心肌组织的生成，（2）的高度纯化的心脏祖细胞从隔离ES细胞分化在体外的 ，和（3）这两种技术的组合，以产生对准功能性心肌组织。

<table border="1"><tbody><tr><td> 材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> L-抗坏血酸</td><td> Sigma-Aldrich公司</td><td> A4544 </td><td>准备0.1M溶液在DDH 2 O </td></tr><tr><td>干燥器，真空10英寸</td><td> NOVA科技国际有限公司</td><td> 55206 </td><td></td></tr><tr><td> 4&#39;-2,6 - 二脒-2 - 苯基吲哚二盐酸盐（DAPI） </td><td> Life Technologies公司</td><td> D1306 </td><td></td></tr><tr><td>的Dulbecco改良的Eagle培养基</td><td> Thermo Scientific的</td><td> SH30022.01 </td><td></td></tr><tr><td> DPBS </td><td> Gibco公司</td><td> 14190 </td><td></td></tr><tr><td> FACSAria II流式细胞仪</td><td> BD Biosciences公司</td><td></td><td></td></tr><tr><td>胎牛血清ES细胞级</td><td>双子座生物制品</td><td> 100-106 </td><td></td></tr><tr><td>胎牛血清的分化等级</td><td>双子座生物制品</td><td> 100-500 </td><td></td></tr><tr><td>从牛血浆纤维粘连蛋白表达</td><td> Sigma-Aldrich公司</td><td> F4759 </td><td>溶解到1毫克/毫升DDH 2 O </td></tr><tr><td> Fisherfinest高级封面玻璃22x22mm </td><td> Fisher Scientific则</td><td> 12-548-B </td><td></td></tr><tr><td>猪皮明胶从</td><td> Sigma-Aldrich公司</td><td> G1890 </td><td>准备在DDH 2 O 0.1％的溶液</td></tr><tr><td>班次旋转涂布机</td><td>车头时距研究公司</td><td> PWM32-PS-CB15 </td><td></td></tr><tr><td> Iscove氏改良的Dulbecco的中</td><td> Thermo Scientific的</td><td> SH30228.01 </td><td></td></tr><tr><td>白血病抑制因子（LIF） </td><td>自准备从LIF-分泌细胞系</td><td></td><td>准备好500倍的股票解决方案</td></tr><tr><td> MEM非必需氨基酸</td><td> Gibco公司</td><td> 11140-050 </td><td></td></tr><tr><td> 2 - 巯基乙醇</td><td> Sigma-Aldrich公司</td><td> M6250 </td><td></td></tr><tr><td>小鼠胚胎成纤维细胞</td><td>采收期12-15天小鼠胚胎</td><td></td><td></td></tr><tr><td>青霉素 - 链霉素</td><td> Gibco公司</td><td> 15140-122 </td><td></td></tr><tr><td>聚醚F-127 </td><td> Sigma-Aldrich公司</td><td> P2443 </td><td>准备1％水溶液在DDH 2 O </td></tr><tr><td>圆底管为35μm的细胞过滤器</td><td> BD Biosciences公司</td><td> 352235 </td><td></td></tr><tr><td> SPI等离子Prep II等离子清洗机</td><td> SPI用品</td><td> 11005-AB </td><td></td></tr><tr><td> SYLGARD184有机硅弹性体套件</td><td>道康宁</td><td> 184 SIL ELAST KIT 0.5KG </td><td></td></tr><tr><td> 0.25％胰蛋白酶-EDTA </td><td> Gibco公司</td><td> 25200-056 </td><td></td></tr><tr><td>真空计</td><td> SPI用品</td><td> 11019-AB </td><td></td></tr></tbody></table>

generation of aligned functional myocardial tissue through microcontact printing

充血性心脏衰竭是一个重大的未满足临床的挑战，可行的和/或功能齐全的心肌细胞所产生的损失。尽管最佳的药物治疗，心脏衰竭的发病率和死亡率在发达国家的首要原因。在药物开发的一个主要挑战是确定的细胞分析，准确地概括了人类正常和病变心肌生理特性的影响 。同样，心脏再生生物学的主要挑战围绕特定病人的心脏祖细胞的数量在临床相关的识别和分离。这些细胞，然后组装成类似于天然心脏组织架构允许创建精确的微图案的的蛋白质形状类似的组织结构的心。微接触印刷的功能性组织，从而提供了控制细胞黏附空间的几何线索。于此我们描述我们的方法隔离的高度纯化的心肌细胞在体外 ，微图案与胞外基质蛋白的细胞的生长表面的生成由多能干细胞分化的干细胞来源的心肌细胞的到各向异性心肌组织中的组装。

FACS纯化在体外分化的ES细胞中显示的前体细胞（ 图1）的四个不同的种群。微图案纤连蛋白的存在下，确认通过免疫荧光显微镜连续纤维粘连蛋白线（ 图2）显示一个完整的转印。到纤维连接蛋白微图形电镀的的FACS隔离祖导致调整R + G +的R - G +人口。的Pluronic F-127虽然作为一个支持细胞粘附阻断剂使用，在R + G -和R - G -</s...

Watch this Scientific Journal Video about Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing at JoVE.com

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

对齐心肌组织的产生是一个关键的要求，适应干细胞生物学的最新进展，临床上有用的目的。在这里，我们描述了一个微接触印刷方法的精确控制细胞的形状和功能。使用高度纯化群体中的胚胎干细胞源性心肌祖细胞，然后产生各向异性功能的心肌组织。

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. ...

generation-aligned-functional-myocardial-tissue-through-microcontact

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

11.1K 次观看.  Massachusetts General Hospital and Harvard Medical School. 对齐心肌组织的产生是一个关键的要求，适应干细胞生物学的最新进展，临床上有用的目的。在这里，我们描述了一个微接触印刷方法的精确控制细胞的形状和功能。使用高度纯化群体中的胚胎干细胞源性心肌祖细胞，然后产生各向异性功能的心肌组织。

视频: Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

毛细力光刻技术在心脏组织工程

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

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 ...

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

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

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 ...

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. ...

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 ...

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 ...

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 ...

A Cardiac Microphysiological System for Studying Ca2+ Propagation via Non-genetic Optical Stimulation

In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely accepted by the scientific community, these systems are still limited in longevity due to the absence of non-invasive stimulation techniques. Phototransducers provide an efficient stimulation method, offering a wireless approach with high temporal and spatial resolution while minimizing invasiveness in stimulation processes. In this manuscript, we present a fully optical method for stimulating and detecting the activity of an in vitro cardiac microphysiological model. Specifically, we fabricated engineered laminar anisotropic tissues by seeding human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated in a 3D bioreactor suspension culture. We employed a phototransducer, an amphiphilic azobenzene derivative, named Ziapin2, for stimulation and a Ca2+ dye (X-Rhod 1) for monitoring the system&#39;s response. The results demonstrate that Ziapin2 can photomodulate Ca2+ responses in the employed system without compromising tissue integrity, viability, or behavior. Furthermore, we showed that the light-based stimulation approach offers a similar resolution compared to electrical stimulation, the current gold standard. Overall, this protocol opens promising perspectives for the application of Ziapin2 and material-based photostimulation in cardiac research.

Using light to control cardiac cells and tissue enables non-contact stimulation, thereby preserving the natural state and function of the cells, making it a valuable approach for both basic research and therapeutic applications.

一种通过非遗传光刺激研究 Ca2+ 传播的心脏微生理系统

In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely ...