Name: generation of aligned functional myocardial tissue through microcontact printing
Uploaded: 2013-03-19T13:00:00
Description: Watch this Scientific Journal Video about Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing at JoVE.com

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?...

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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. ...

Research

JoVE Journal

Bioengineering

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

心肌组织工程生物衍生的注射材料的制备

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

科研

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Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

人体软骨组织制作采用三维喷墨打印技术

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R-Ãƒâ€šÃ‚ÂµCP and Sequential Nucleophilic Substitution

制造复杂的栽培基质使用机器人微接触印刷（R-μCP）和顺序亲核取代

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

组织工程血管在小鼠模型的生成和嫁接

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

管状结构组织工程的核心/壳印刷支架

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

用于打印 3D 细胞-拉丹胰腺组织构造的胰腺组织衍生细胞外基质生物墨水

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

功能性与极化胆囊肿的生成与定量特征

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

使用高通量微流体设备生成动态环境条件

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...