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

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

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

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

生物工程

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

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

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (&#956;CP) is a versatile technique to pattern such grafted PEG brushes, but manual &#956;CP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-&#956;CP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal &#8216;click&#8217; chemistries. Here, we describe in detail the workflow to manufacture such substrates.

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

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

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

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

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

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

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

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

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

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other 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

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

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

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 context of liver diseases, 3-dimensional (3D) models based on cholangiocytes have emerged such as cysts, spheroids, or tube-like structures to mimic tissue topology for organogenesis, disease modeling, and drug screening studies. These structures have been mainly obtained by embedding cholangiocytes in a hydrogel. The main purpose was to study self-organization by addressing epithelial polarity, functional, and morphological properties. However, very few studies focus on cyst formation efficiency. When this is the case, the efficiency is often quantified from images of a single plane. Functional assays and structural analysis are performed without representing the potential heterogeneity of cyst distribution arising from hydrogel polymerization heterogeneities and side effects. Therefore, the quantitative analysis, when done, cannot be used for comparison from one article to another. Moreover, this methodology does not allow comparisons of 3D growth potential of different matrices and cell types. Additionally, there is no mention of the experimental troubleshooting for immunostaining cysts. In this article, we provide a reliable and universal method to show that the initial cell distribution is related to the heterogeneous vertical distribution of cyst formation. Cholangiocyte cells embedded in hydrogel are followed with Z-stacks analysis along the hydrogel depth over the time course of 10 days. With this method, a robust kinetics of cyst formation efficiency and growth is obtained. We also present methods to evaluate cyst polarity and secretory function. Finally, additional tips for optimizing immunostaining protocols are provided in order to limit cyst collapse for imaging. This approach can be applied to other 3D cell culture studies, thus opening the possibilities to compare one system to another.

Three-dimensional (3D) cellular systems are relevant models for investigating organogenesis. A hydrogel-based method for biliary cysts production and their characterization is proposed. This protocol unravels the barriers of 3D characterization, with a straightforward and reliable method to assess cyst formation efficiency, sizes, and to test their functionality.

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

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

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 ...