Name: a net mold-based method of scaffold-free three-dimensional cardiac tissue creation
Uploaded: 2018-08-05T13:00:00.000+00:00
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作者承认以下资金来源: 为心血管研究提供资金的魔力。

1. 心肌细胞的制备
<ol><li>用基底膜基质和培养人诱导的多能干细胞 (hiPSCs) 6 井板, 如前所述的17。</li>
	<li>使用以前描述的方法18将 hiPSCs 区分为 hiPSC CMs。</li>
	<li>在 16-18 d 的分化后, 通过冲洗每个井与2毫升的1x 磷酸盐缓冲盐水 (PBS), 不含钙或镁, 然后孵化与1毫升/井的胰蛋白酶或细胞离解试剂 (见表材料) 室温下5分钟。</li>
	<li>与胰蛋白酶或细胞离解试剂 (参见材料表) 使用同样数量的罗斯威尔公园纪念协会 (RPMI) 细胞媒介补充与 B-27 (RPMI/B-27 细胞媒介)。吸管向上和向下松开任何粘附细胞。</li>
	<li>用10毫升血清学吸管收集悬浮心肌细胞, 将其转移到50毫升圆锥管。</li>
	<li>离心细胞悬浮在 250 x g 5 分钟室温下获得细胞颗粒。</li>
	<li>并用重悬10毫升 RPMI/B-27 细胞培养基中的颗粒。</li>
	<li>将20µL 的细胞悬架与同等数量的0.4% 台盼蓝溶液结合, 轻轻搅拌。</li>
	<li>使用手动 hemocytometer 计数和获得细胞悬浮液的浓度和细胞活性。</li>
</ol>2. 成纤维细胞的制备
<ol><li>启动一个人心脏成纤维细胞 (HCF) (成人心室类型) 单元格线的培养, 如前所述16。</li>
	<li>在室温16下, 用适当数量的胰蛋白酶或细胞离解试剂 (见材料表) 将 HCFs 悬浮在5分钟内。对于 T175 瓶, 使用10毫升的胰蛋白酶或细胞离解试剂。</li>
	<li>用同等体积的培养基中和胰蛋白酶或细胞离解试剂 (见材料表), 然后将样品转移到50毫升锥形管中, 离心机在室温下以 250 x g为5分钟的细胞悬浮液, 以获得颗粒。</li>
	<li>并用重悬10毫升成纤维细胞生长介质中的颗粒 (见材料表)。</li>
	<li>将 HCF 细胞悬浮液的20µL 与同等量的0.4% 台盼蓝溶液结合, 轻轻搅拌。</li>
	<li>使用自动单元格计数器或手动 hemocytometer 计数并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>启动人类脐静脉内皮细胞 (HUVEC) 线的培养, 如前所述17。在室温17下, 用适当数量的胰蛋白酶或细胞离解试剂 (见材料表) 将血管内皮细胞悬浮在3分钟内。中和胰蛋白酶或细胞离解试剂 (见材料表) 使用同等体积的介质, 然后转移到一个50毫升圆锥管和离心机的细胞悬浮在 250 x g 5 分钟在室温下获得颗粒。 注: 对于 T175 瓶, 使用10毫升的胰蛋白酶或细胞离解试剂。</li>
	<li>并用重悬10毫升内皮细胞生长培养基中的颗粒 (见材料表)。</li>
	<li>结合20µL 的 HUVEC 悬浮液, 并将其与同等数量的0.4% 台盼蓝溶液进行染色, 并轻轻搅拌。</li>
	<li>使用自动单元格计数器或手动 hemocytometer 计数和获得细胞悬浮液的浓度和细胞活力。</li>
</ol>4. 创建吊滴球体
<ol><li>将包含850孔 (每个直径350µm) 的悬挂放置装置放入无菌6井板中。</li>
	<li>隔离上述三种类型的细胞: hiPSC-CMs、HCFs 和血管内皮细胞。将它们与 70% iPSC CMs、15% HCFs 和15% 血管内皮细胞的比例结合在一个50毫升圆锥管中, RPMI/B-27 细胞介质的浓度为每毫升247.5万个细胞。</li>
	<li>将4毫升的细胞悬浮液 (每毫升247.5万个细胞) 分配给一个超低附着物悬挂系统的每一个井 (其中微孔直径为350µm), 坐在6井板上。</li>
	<li>球体将自发地在12小时内将细胞悬浮液放入吊滴装置中。继续养殖为共 72 h (3 d) 在37°c, 5% CO2, 和95% 湿气为成熟的球体。</li>
	<li>经过72小时的养殖, 通过将设备放入介质中, 并轻轻旋转, 将球体从悬挂装置中释放出来, 在悬挂系统底部的毛孔中收获球体。</li>
</ol>5. 使用新颖的网模创建3维补丁
<ol><li>将底座、底板、底网和10层侧网组装起来, 用一副无菌钳来制造新型模具。首先, 使用拐角柱堆叠并对齐底座、底方板和底部网。然后, 堆叠和层10侧网在交替的方向, 以创建一个良好的不锈钢齿网。</li>
	<li>用 PBS 或介质冲洗填充底座, 以减少表面张力, 然后将装配模转移到填充底座上。</li>
	<li>用同一方法将网模腔与 PBS 或介质一起湿。将球体慢慢填入所需尺寸的预设腔体 (2 x 2 x 1 毫米, 4 x 4 x 1 毫米, 或 6 x 6 x 1 毫米)。 注: 确保球体的预期体积的120% 被送入腔内, 使细胞聚集紧密连接并保持静止。</li>
	<li>将顶部网和顶方板装配到拐角的柱子上, 并确保塞子和保温管的安全, 防止球体的冲刷。</li>
	<li>将填充的网模系统转移到6井板上, 6 毫升的 RPMI/B-27 细胞介质, 或进入一个无菌容器, 有足够的介质覆盖整个组装模具。</li>
	<li>在 7-10 天, 在 3000-4000 x g上, 在摆动的振动筛上孵化6井板, 并装满模具系统。 注意: 孵化的持续时间取决于心脏斑块的大小。随着斑块的增大, 心脏斑块成熟时需要增加潜伏期。</li>
</ol>6. 从新型网模中去除修补程序
<ol><li>将模具系统从介质中取出, 并将其放在无菌的10厘米盘中灭菌处理垫上。</li>
	<li>卸下保温管、塞子、顶方板和顶网。小心地滑出分层的侧网一个一个用一对消毒钳。全麻拔管后, 在底部网上获得完整的心脏修补。</li>
	<li>用无菌钳把底网接上, 用5毫升的 RPMI/B27 介质将底网转移到35毫米的盘子里。在盘子里慢慢地旋转网, 或者用不育的细胞刮刀轻轻地松开底部网的补丁。从底网分离后, 可以用 RPMI/B27 培养基培养心脏组织。</li>
	<li>继续在35毫米的菜肴中孵化游离漂浮的3维心脏补丁。功能同步跳动可以观察早在24小时后, 从模具去除。</li>
	<li>从网模中去除修补后, 观察其形状保持完整性, 同步跳动强度随时间增加。 注: 3 维网状模心片在全麻拔管后表现为 cardiospheres 元件的电集成。我们观察到每分钟60到80次的跳动频率, 持续时间超过60天。</li>
</ol>

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作者没有什么可透露的。

该方法的意义在于其重现性和结果多层心肌组织的有效性。在心脏组织工程领域, 目前的目标之一是确定一种方法来构建跳动, 多层和功能3维心脏补丁。我们报告了一个有效的和可重复的方法创建多层心脏组织的直接人工播种的球体, 由心肌细胞, 内皮干细胞, 成纤维形成一个新的网霉菌。本方法所用的网模有各种不同的尺寸, 可用于从 2 x 2 x 1 毫米到 6 x 6 x 1 毫米不等的组织的创建。我们所描述的?...

目前心脏组织工程的目的是开发一种替代或修复损伤心肌组织的结构和功能的治疗方法1。方法建立3维心脏组织模型, 显示本机心脏组织的重要收缩和电生理特性, 迅速扩大2,3。在研究4、5中探索和使用了各种战略。这些方法包括使用特定的合成和天然生物活性水凝胶, 如明胶, 胶原蛋白, 纤维蛋白和肽6, 生物墨水沉积技术2和生物打印技术7。
研究表明, 无支架的方法可以产生可比的组织作为生物材料的方法, 而不存在的缺点, 纳入外国脚手架材料8。奥伦 Caspi等人证明, 结合各种类型的细胞, 使生成高血管化的人类工程心脏组织9。下巴等人开发了一种3维印刷方法的心脏补丁创建从球体。所产生的斑块由心肌细胞、纤维细胞和内皮干细胞组成, 比例为 70:15:15.10。球体已被证明是有效的 "积木" 的无支架的心脏组织创建, 因为他们耐缺氧, 并拥有足够的机械完整性, 植入11,12。以前的研究已经展示了几种制造球体的方法, 包括使用吊滴法、微调烧瓶13、微流控系统14和非粘附的培养表面, 不涂或涂有琼脂糖微模15。在本协议中, 我们使用悬挂放置装置, 其中包含孔, 以凝聚数以百计的球体一次。
本研究提出了一种新的、高效的无支架的心脏组织创建方法, 包括将球体人工播种到方形模腔中, 并在振动筛上孵化组织以进行成熟。在通常的静态培养条件下, 氧扩散仅限于组织结构的外部, 导致中心坏死。然而, 随着网模, 所有的球体种子进入模具都沉浸在介质中, 不断的射流运动, 使养分和氧气的扩散增加。此外, 这种基于模具的方法允许同时创建不同大小的组织补丁, 用最少的人工努力, 由此产生的组织可以很容易地从模具中删除。这种新的方法允许有效和可重复的创建无支架, 多层心脏补丁。

<table><tbody><tr><td>Human Cardiac fibroblasts (HCF)</td><td>Sciencell</td><td>6310</td><td></td></tr><tr><td>FM-2 Consists of Basal Medium</td><td>Sciencell</td><td>2331</td><td>HCF culture medium</td></tr><tr><td>Human umbilical vein endothelial cells (HUVEC)</td><td>Lonza</td><td>CC-2935</td><td></td></tr><tr><td>EGM+Bullet Kit&amp;nbsp;</td><td>Lonza</td><td>CC5035</td><td>HUVEC culture medium</td></tr><tr><td>E8 media&amp;nbsp;</td><td>Invitrogen</td><td>A1517001</td><td>HiPSC culture medium</td></tr><tr><td>Geltrex&amp;nbsp;</td><td>Invitrogen</td><td>A1413202</td><td></td></tr><tr><td>TrypLE Express Enzyme (1X)</td><td>Thermo Fisher</td><td>12604013</td><td>Trypsin and Cell dissociation reagent</td></tr><tr><td>RPMI media</td><td>Invitrogen</td><td>11875093</td><td>RPMI media with B-27 supplement is hiPSC-CM culture medium</td></tr><tr><td>B-27 supplement (50x)</td><td>Thermo Fisher</td><td>17504044</td><td>RPMI media with B-27 supplement is hiPSC-CM culture medium</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Thermo Fisher</td><td>15250061</td><td></td></tr><tr><td>Novel net mold&amp;nbsp;</td><td>TissueByNet Co.,Ltd</td><td>NM25-1</td><td></td></tr><tr><td>Hanging drop plate</td><td>Kuraray Co.,Ltd</td><td>MPc350</td><td></td></tr><tr><td>6 well plates&amp;nbsp;</td><td>Sigma-Aldrich</td><td>CLS-3516</td><td></td></tr></tbody></table>

a net mold-based method of scaffold-free three-dimensional cardiac tissue creation

该协议描述了一种新的和简单的基于网络模型的方法, 以创建三维 (3 维) 心脏组织没有额外的支架材料。人诱导的多潜能干细胞衍生心肌细胞 (iPSC cms), 人心脏成纤维细胞 (HCFs) 和人脐静脉内皮干细胞 (血管内皮细胞) 被隔离, 并用于产生细胞悬浮与 70% iPSC CMs, 15% HCFs, 15% 血管内皮细胞。他们是共同培养的超低附件 "悬挂下降" 系统, 其中包含孔, 以凝聚数以百计的球体一次。在共培养3天后, 细胞聚集并自发形成跳动球体。球体的收获, 种子成一个新的模具腔, 并培养在一个振动筛在孵化器。球体成为成熟的功能组织大约7天后播种。所合成的多层组织包括融合球体, 结构完整性良好, 同步跳动行为。这种新方法具有很好的潜力, 可以作为一种重现性和成本效益高的方法, 为将来的心力衰竭治疗创造出工程组织。

在我们的实验中, 我们利用细胞悬浮 70% iPSC CMs, 15% HCFs, 15% 血管内皮细胞在 RPMI/B-27 细胞介质中浓度247.5万细胞每毫升。在建立细胞悬浮液后, 我们将4毫升的细胞悬浮液分配给超低附着悬挂系统的每个井, 如协议步骤4.3 所述。在3天的文化在37°c、5% CO2和95% 湿气以后, 使用垂悬的下落系统导致了数以百计的跳动的球体的自发形成。球体很容易被观察到在光显微镜下跳动...

Watch this Scientific Journal Video about A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation at JoVE.com

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

基于网模的无支架三维心脏组织创建方法

本协议描述了一种基于净模的方法来创建三维无支架的心脏组织, 结构完整性和同步跳动行为令人满意。

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

a-net-mold-based-method-scaffold-free-three-dimensional-cardiac

Research

JoVE Journal

Bioengineering

8.8K 次观看.  The First Hospital of Jilin University. 本协议描述了一种基于净模的方法来创建三维无支架的心脏组织, 结构完整性和同步跳动行为令人满意。

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

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

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. ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

组织工程：建设多细胞三维支架的层状细胞片交货

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

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 ...

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 ...

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, structures, phenotypes, and performance depending on each specific perfused tissue. The field of tissue engineering, which aims to repair or replace damaged or missing body tissues, relies on controlled angiogenesis to create a proper vascularization within the engineered tissues. Without a vascular system, thick engineered constructs cannot be sufficiently nourished, which may result in cell death, poor engraftment, and ultimately failure. Thus, understanding and controlling the behavior of engineered blood vessels is an outstanding challenge in the field. This work presents a high-throughput system that allows for the creation of organized and repeatable vessel networks for studying vessel behavior in a 3D scaffold environment. This two-step seeding protocol shows that vessels within the system react to the scaffold topography, presenting distinctive sprouting behaviors depending on the compartment geometry in which the vessels reside. The obtained results and understanding from this high throughput system can be applied in order to inform better 3D bioprinted scaffold construct designs, wherein fabrication of various 3D geometries cannot be rapidly assessed when using 3D printing as the basis for cellularized biological environments. Furthermore, the understanding from this high throughput system may be utilized for the improvement of rapid drug screening, the rapid development of co-cultures models, and the investigation of mechanical stimuli on blood vessel formation to deepen the knowledge of the vascular system.

Engineered tissues heavily rely on proper vascular networks to provide vital nutrients and gases and remove metabolic waste. In this work, a stepwise seeding protocol of endothelial cells and support cells creates highly organized vascular networks in a high-throughput platform for studying developing vessel behavior in a controlled 3D environment.

逐步在泰瑟尔脚手架上播种，研究发芽血管

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, ...

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 ...