This work was supported by The British Heart Foundation and The Oak Foundation.

根据批准的机构委员会使用实验动物和护理协议执行所有的动物实验。 1.准备文化传媒<ol><li>使培养基为CCL-153人成纤维细胞系:F-12K培养基，10％胎牛血清（FBS）和100单位/毫升青霉素和链霉素。 </li><li>使重新编程媒体为PiPSC代:敲除Dulbecco改良的Eagle培养基（DMEM）中含有20％敲除血清替代品，0.1mM的β巯基乙醇，0.1mM的极限必需培养基（MEM）非必需氨基酸，10纳克/毫升碱性成纤维细胞生长因子2（bFGF的-2）和100U / ml的青霉素和链霉素。新鲜每次使用媒体之前添加的bFGF-2。 </li><li>使分化培养基（DM），以诱导平滑肌细胞（SMC）的分化:α-MEM中含有10％FBS，0.1mM的β巯基乙醇，100U / ml青霉素和链霉素和25毫微克/毫升的血小板源生长因子的媒体（PDGF-ββ）。 </li><li>使内皮分化培养基（EC-DM）诱导内皮细胞（EC）的分化:内皮生长培养基-2（EGM-2）含有50纳克媒体/ ml的血管内皮生长因子（VEGF）和100U / ml的青霉素和链霉素。 </li></ol> 2.重新编程人类成纤维细胞进入部分诱导的多能干细胞（PiPSC） <ol><li>培养人成纤维细胞系:CCL-153上的0.04％的培养基中的明胶溶液涂布烧瓶中。 </li><li>传代细胞每3天以1:1的比例:6。通过9 PiPSC一代最佳效率之前使用细胞。细胞是准备在到达80转 - 90％汇合。 </li><li>线性化含有4重编程因子多顺反子质粒pCAG2LMKOSimO八聚体结合转录因子4（OCT4），SOX2，Kruppel样因子4（KLF4）和c-Myc（pCAG-OSKM）。消化5微克的质粒的5个单位的PvuⅠ的限制性内切酶为3小时，在37℃。净化该质粒具有根据制造商的协议商业试剂盒。 </li><li>转染2×10 6人成纤维细胞用4微克线性化的pCAG-OSKM质粒通过电穿孔具有根据制造商的协议的人真皮成纤维细胞（NHDF）核转染试剂盒。轻轻种子转染的细胞上的预0.04％明胶涂布含有5ml预热的重编程媒体的T25培养瓶中。 </li><li> 24小时后，改变培养基的25微克/ ml的新霉素补充来选择转染细胞的纯群体。 </li><li>改变中的每一天，直到第4天。 注:人类成纤维细胞后第4天重编程成为PiPSC。 </li></ol> 3.分化PiPSC成内皮细胞和平滑肌细胞<ol><li>种子PiPSC上含DM 4天IV型胶原预涂菜以诱导分化成平滑肌细胞。改变中的每一天，直到第4天。 </li><li>种子PiPSC胶原IV预涂菜çontaining内皮-DM 6天以诱导分化为内皮细胞。改变中的每一天，直到6天。 </li></ol> 4.脱细胞移植主动脉准备注:所有的解决方案和设备应是无菌的。 <ol><li>牺牲小鼠颈椎脱位并修复鼠标在解剖显微镜下仰卧位。 </li><li>通过胸骨和周围的肋骨切开以打开胸腔。取出心脏，肺和食管暴露主动脉。轻轻地用钳子取出围主动脉脂肪。 </li><li>切从前端用剪刀主动脉，轻轻按住结束钝钳。分离从后面用钝性分离脊椎柱的主动脉。小心关闭所有分支动脉从主动脉结扎用双极electrocoagulator并从用剪刀结扎的远端切割。 </li><li>使用5ml的注射器冲洗主动脉管腔带3米升的含有肝素的100单位，以防止血液凝块的形成盐溶液。 </li><li>削减它分支成肾前主动脉的后端。保持主动脉的完整性是在整个过程中非常重要的。 </li><li>冲洗主动脉管腔用5ml稀释在磷酸盐缓冲盐水（PBS）0.075％十二烷基硫酸钠（SDS）的溶液中。 </li><li>浸泡在0.075％SDS溶液胸主动脉在10cm培养皿。将培养皿在定轨振荡器2小时以150rpm在室温。 </li><li>冲洗主动脉管腔用5ml PBS中。 </li><li>浸泡在PBS主动脉在一个10cm培养皿。放置在轨道摇床培养皿2小时以150rpm在室温。 PBS刷新每隔20分钟。 </li><li>冲洗主动脉管腔用5ml PBS中。 </li><li>请PBS脱细胞移植主动脉在4℃下长达一个星期。 </li></ol> PiPSC和生物反应器调理5.双播种注意:所有的溶液s和设备应是无菌的。在组织培养罩进行所有操作。 <ol><li>组装的生物反应器流动回路， 如图1，连接孵育室，媒体库，蠕动泵和柔性小室与管。需要流通中的最小体积为40毫升</li><li>前提条件，通过浸泡在DM介质主动脉在一个10cm培养皿的脱细胞的容器中的培养基中。放置在定轨摇床上1小时培养皿在50rpm在RT。 </li><li>插入1cm长的尼龙管（外径0.9毫米，ID 0.75mm）组合成显微镜下对脱细胞的容器的两端。领带的容器中，并用8-0丝缝线在管。 </li><li>通过连接尼龙管具有从腔室壁的入口和出口端口（1/32"管）组装在孵育室的脱细胞的主动脉移植物。保持孵育室与介质的每次5mls。 </li><li> Trypsinize PiPSC通过加入预热的胰蛋白酶以覆盖培养皿，并轻轻摇动培养皿10倍。弃去胰蛋白酶和保留在孵化器的菜2 - 3分钟。添加预热的培养基在盘中混合并与该细胞的培养基。 </li><li>计数使用血球细胞数。离心机含有每5×10 5细胞5分钟，在300×g下的细胞悬液的2等分。完全吸出上清液。 </li><li>完全吸出上清液后，重悬沉淀1的PIPS细胞沉淀中加入50μl的DM。这种注入细胞悬液到脱细胞血管腔。 </li><li>然后，悬浮等PIPS细胞沉淀在100微升基质胶。认真吸取到脱细胞移植主动脉混合物。 </li><li>等待10 - 15分钟，直到混合物变成凝胶状态和均匀环绕该容器的外部表面。填写孵化室DM。 </li><li>将整个生物反应器中在5％CO 2培养箱设定在37℃的。手动旋转脱细胞移植物周围的纵向轴线每半小时90°在第2小时。 </li><li>保持静止培养12小时，使细胞粘附。 </li><li>通过内腔递送的DM通过蠕动泵以诱导平滑肌细胞的分化。启动初始流速为5毫升/分钟，并逐步增加至20毫升/分钟以上24小时。 </li><li>保持在流通介质的流速为20毫升/分钟24小时。 </li><li>阻止介质流动和移动的生物反应器设置了孵化器。 </li><li>重种子与PiPSC移植物的内腔。 Trypsinize，计数和离心1×10 6个PiPSC与步骤5.5 - 5.6。重悬在50微升EGM-2培养基的含有50纳克/ ml的VEGF的细胞。注入细胞混合物进入移植物的管腔。 </li><li>改变在孵育室并且整个循环到含有50纳克/ ml的VEGF诱导的内皮分化EGM-2培养基的培养基。 </li><li>将生物反应器设置回THË孵化器。手动旋转接枝90°，每30分钟在第2小时，然后保持静置培养12小时。 </li><li>开始从5毫升/分钟，并逐步增加至35毫升/分钟的循环流量。保持/流速在35毫升分钟5天。改变循环和室中的每一天。 </li><li>收获的设计移植进一步嫁接到鼠标。 </li></ol> 6.嫁接双种子PiPSC移植到小鼠<ol><li>使用NOD.CB17-SCID PRKDC / NcrCrl雄性小鼠血管移植受者。始终确保小鼠约10周龄，体重约25 - 35克，并处于良好的健康状况。 </li><li>通过施用Hypnorm（25毫克/千克）和Hypnovel（25毫克/千克）经腹膜内的组合麻醉受体小鼠。取适量涂抹于眼睛凡士林眼药膏，以防止干燥，而在麻醉下。通过确保小鼠有放松肌肉，并呼吸平稳确认有效的麻醉。 <ol><li>解决鼠标在仰卧位，其颈部剃光和扩展。施用阿托品硫酸盐（1.7毫克/千克）与麻醉剂组合，以确保鼠标呼吸道保持清亮。 </li></ol></li><li>准备从下颌骨中线切口鼠标的胸骨。在解剖显微镜下用5〜10倍的放大，解除右唾液腺横向并取出右cleidomastoid肌肉以暴露右颈总动脉。 </li><li>轻轻除去附着组织，从朝向近端的远端动员的右颈总动脉。用8-0丝线缝合结扎颈总动脉的中间部分的两倍，并在两个关系之间解剖。 </li><li>通过在每个容器最终由高压灭菌尼龙管袖口和修复两端与microhemostat夹。 </li><li>去除缝线领带一端并申请下降肝素（100单位/毫升）。紧接着，反转次的前端ë动脉以覆盖整个箍套主体和固定倒置容器用8-0丝线缝合到箍。 <ol><li>重复同样的步骤，以在动脉的其它部分。同花顺动脉生理盐水，以消除血凝块。 </li></ol></li><li>通过在动脉袖口滑动的脱细胞容器端部，并与8-0丝线缝合将其固定植入颈动脉的两端之间的脱细胞的容器接枝。除去血管钳从任一评估移植物的脉动之前结束。 </li><li>将右唾液腺回其原来的位置。使用6-0聚乳糖的缝线间断缝合关闭皮肤上的手术位置。 </li><li>持续观察小鼠生命体征的程序完成后，直到它恢复意识。颈椎脱位和收获血管移植牺牲受体小鼠无论是24小时或更高版本以供进一步分析3周。 </li></ol>

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+infarction+and+intramyocardial+injection+models+in+swine.">Myocardial infarction and intramyocardial injection models in swine.</a> Nature Protocols. 7, 1479-1496 (2012).</li><li>Olausson, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+an+allogeneic+vein+bioengineered+with+autologous+stem+cells:+a+proof-of-concept+study.">Transplantation of an allogeneic vein bioengineered with autologous stem cells: a proof-of-concept study.</a> Lancet. 380 (9838), 230-237 (2012).</li></ol>

作者有没有冲突的财务权益。

当前协议指示声音，快速，简单，高效和可重复性，其中功能性的组织工程血管可使用PiPSC从人成纤维细胞产生的策略。这种技术代表了用于再生医学，组织工程和潜在的患者特异性细胞治疗在不久的将来的宝贵工具。关键步骤，以确保协议的功效包括PiPSC的制备中，制备无菌的，且完全脱细胞主动脉移植物，PiPSC在支架成功播种和分化和主动脉移植物保持无菌在血管的生物反应器。 <p class="jove...

血管管道的建设是由心血管疾病导致受损和损伤血管修复手术的基本策略。迄今为止，在外科手术中使用的接枝物质包括生物相容的合成聚合物（聚四氟乙烯[聚四氟乙烯]，膨胀的聚四氟乙烯[膨体聚四氟乙烯;奥亚特克斯]或聚对苯二甲酸乙酯[涤纶]），同种异体移植物，自体组织（心包或隐静脉）和异种移植物1。虽然人工移植物（ 例如，戈尔特斯和涤纶）是最常用的，这些材料可能会导致大量的短期和长期的并发症，包括狭窄，钙沉积，血栓栓塞和感染。尽管患者目前生物移植减少血栓栓塞事件，他们还是遇到限制，如二次移植失败，缩短耐久性由于钙化退化2。因此，尽管在手术吨显著改善被echniques多年来，研究人员和临床医生仍然背负着需要用于识别提供了理想的导管用于血管疾病。最近，血管组织工程的研究领域已产生了在其中细胞掺入可生物降解的支架，以创建缩影成功移植1的官能容器仿生环境的目的的概念。根本的是，血管构建体的成功取决于三个主要组分;细胞组成的支架， 即，内皮细胞内层和平滑肌细胞层，含有合适的细胞外基质，以提供相当的机械性能与天然血管的支架，并且所需要用于启动/调节分子/细胞信号修理。 长期移植通畅和新组织的持续发展在很大程度上取决于支架的有效细胞接种，日ereby渲染的至关重要的细胞类型决定。一些报告展示了利用成熟的内皮细胞，并从各种来源的平滑肌细胞的发育小口径管道3-6。尽管有希望，缺乏足够的自体血管，以获得成熟的内皮细胞和平滑肌细胞保持一个相当大的负担。最近，从各种来源的干细胞已被开发用于血管组织工程的应用程序。确实，多种干细胞类型，包括胚胎干细胞7，诱导多能干细胞（iPS细胞）8,9，PiPSC 10,11，骨髓来源的单核细胞12，间质干细胞13，内皮祖细胞和成血管壁衍生干细胞抗原-1（SCA-1）+干/祖细胞14,15都被证明是能够分化成任何官能内皮或平滑肌细胞响应于确定的培养基和培养条件。此外，干细胞的无限自我更新能力使得它们不同于成熟的内皮细胞和平滑肌细胞可以经历生长停滞和衰老之前仅划分为次数有限数量的更好的候选。 支架材料的选择，以产生成功的组织工程血管接枝取决于几个因素，例如生物相容性，生物力学性能，和生物降解的速率。从根本上说，用料打造的支架移植物应该是可生物降解，​​不会安装不必要的接受者的免疫反应。此外，它必须包括合适的孔隙度和微结构用于细胞附着和随后的存活。迄今为止，用于在血管组织工程支架中最常见的材料包括聚乙醇酸，聚乳酸和聚ε己内酯16的聚合物。最近，脱细胞的生物材料具有也已应用取得了一些成功。几个实验室证明，脱细胞播种人，犬或猪血管自体细胞提供了一个生物学的移植物的抵制凝血和内膜增生17-19。在血管组织工程等战略包括细胞外基质蛋白为基础的血管移植物如种子细胞的纤维蛋白胶13，并产生细胞片无支架支持20，21。 当前协议证明人类PiPSC分化为功能性内皮细胞和平滑肌细胞中，生成的组织工程血管的生物反应器，包括一个脱细胞血管支架怀有官能PiPSC源性血管细胞的，和接枝到重症联合免疫缺陷病（SCID ）小鼠。 PiPSC是一个最佳的细胞类型以用于容器的移植物的组织工程，因为这些细胞不形成在小鼠中的肿瘤或提高的伦理和同种异体的免疫应答。此外，我们已经表明，该策略用于产生PIPS内皮细胞和PIPS平滑肌细胞是有效的和可再现的10,11。此后，我们设计用于PiPSC源性血管细胞密切模仿天然容器内存在的基质蛋白播种去细胞的容器中，从而提高了移植存活和功效。此外，前PiPSC播种血管的脱细胞防止安装在由免疫细胞类型的炎症反应，如巨噬细胞的产生。更重要的是，该协议不只是代表了一种方法，以产生希望的血管导管翻译成人类，而且还提供了学习和理解，通过小鼠模型支配的血管组织再生的分子机制的有价值的手段。

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Human Fibroblasts CCL-153</td>
			<td >ATCC</td>
			<td >CCL-153</td>
			<td>Prenatal human embryonic fibroblasts</td>
		</tr>
		<tr >
			<td >ATCC F-12K Medium (Kaighn&#39;s Modification of Ham&#39;s F-12 Medium)</td>
			<td >ATCC</td>
			<td >30-2004</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum</td>
			<td >ATCC</td>
			<td >30-2020</td>
			<td></td>
		</tr>
		<tr >
			<td >Knockout DMEM medium optimized for embryonic stem cells</td>
			<td >Life technologies (Gibco)</td>
			<td >12660-012</td>
			<td></td>
		</tr>
		<tr >
			<td >Knockout Serum Replacement</td>
			<td >Life technologies (Invitrogen)</td>
			<td >10828-028</td>
			<td></td>
		</tr>
		<tr >
			<td >Human Basic FGF-2&#160;</td>
			<td >Miltenyi Biotech</td>
			<td >130-093-837</td>
			<td></td>
		</tr>
		<tr >
			<td >alpha-MEM medium</td>
			<td >Life technologies (Invitrogen)</td>
			<td >32571093</td>
			<td></td>
		</tr>
		<tr >
			<td >Human PDGF</td>
			<td >R&#38;D System</td>
			<td >120-HD-001</td>
			<td></td>
		</tr>
		<tr >
			<td >Gelatin Solution 2%</td>
			<td >Sigma</td>
			<td >G1393</td>
			<td></td>
		</tr>
		<tr >
			<td >Plasmid 20866: pCAG2LMKOSimO (SOX2, OCT4, KLF4, C-MYC)</td>
			<td >Addgene</td>
			<td >20866</td>
			<td></td>
		</tr>
		<tr >
			<td >&#160;PvuI Restriction Enzyme</td>
			<td >New England Biolabs</td>
			<td >RO150S</td>
			<td></td>
		</tr>
		<tr >
			<td >SureClean Plus</td>
			<td >Bioline</td>
			<td >BIO-37047</td>
			<td></td>
		</tr>
		<tr >
			<td >Nucelofection Kit (NHDF Kit)</td>
			<td >LONZA</td>
			<td >VPD-1001</td>
			<td></td>
		</tr>
		<tr >
			<td >Neomycin</td>
			<td >SIGMA</td>
			<td >G418</td>
			<td>Selection of&#160;</td>
		</tr>
		<tr >
			<td >KL 1500 LCD, Illumination for Stereo Microscopy</td>
			<td >SCHOTT</td>
			<td >KL 1500 LCD</td>
			<td>Cold light illumination for stereo microscopy</td>
		</tr>
		<tr >
			<td >Nikon Zoom Steromicroscope SMZ800</td>
			<td >Nikon</td>
			<td >SMZ800</td>
			<td></td>
		</tr>
		<tr >
			<td >Heparin sodium salt</td>
			<td >Sigma</td>
			<td >H3393</td>
			<td></td>
		</tr>
		<tr >
			<td >10% SDS Stock Solution Molecular Biology Reagent</td>
			<td >Severn Biotech</td>
			<td >CAS 151-21-3</td>
			<td></td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td >Sigma</td>
			<td >D8537</td>
			<td></td>
		</tr>
		<tr >
			<td >Matrigel (10mg/ml)</td>
			<td >BD</td>
			<td >A6661</td>
			<td></td>
		</tr>
		<tr >
			<td >Shaker IKA Vibrax with Shaking platform VX 7&#160;</td>
			<td >Jepson Bolton&#39;s, Janke&#38;Kunkel</td>
			<td >S32-102</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex L/S Digital Pump Drive</td>
			<td >Cole-Parmer</td>
			<td >WZ-07523-80</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex L/S 6-channel, 6-roller cartridge pump head</td>
			<td >Cole-Parmer</td>
			<td >EW-07519-15</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex L/S large cartridges for pump head</td>
			<td >Cole-Parmer</td>
			<td >EW-07519-75</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex platinum-cured silicone pump tubing, L/S 14, 25 ft</td>
			<td >Cole-Parmer&#160;</td>
			<td >WZ-96410-14</td>
			<td >Tubing goes through the peristaltic pump</td>
		</tr>
		<tr >
			<td >0.5mm ID, 0.8 mm OD Silicone Tubing</td>
			<td >SILEX</td>
			<td >N/A</td>
			<td>Tubings connect incubation chamber, media reservoir and compliance chamber&#160;</td>
		</tr>
		<tr >
			<td >Fitting Reducer 0.5 to 1.6, natural Polypropyline</td>
			<td >Ibidi</td>
			<td >10829</td>
			<td>Adapter connect above two types of tubings</td>
		</tr>
		<tr >
			<td >1/32&#34; Tubing, ID 0.01&#34; (250&#181;m) Material: PEEK</td>
			<td >LabSmith</td>
			<td >T-132-010P</td>
			<td>Tubing through the incubation chamber wall which connects the graft with outside tubing</td>
		</tr>
		<tr >
			<td >One-Piece Fittings&#160;</td>
			<td >LabSmith</td>
			<td >T-132-100</td>
			<td>Fix the above tubings through the incubation chamber wall</td>
		</tr>
		<tr >
			<td >Nylon tubes (OD 0.9mm, ID 0.75mm)&#160;</td>
			<td >Smiths Medical</td>
			<td >N/A</td>
			<td>Tubings insert into two ends of the aorta graft</td>
		</tr>
		<tr >
			<td >NOD.CB17-Prkdcscid/NcrCrl mouse</td>
			<td >Charles River</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Surgical sutures, 8-0&#160; silk</td>
			<td >ETHICON</td>
			<td >W819</td>
			<td ></td>
		</tr>
		<tr >
			<td >Hypnorm</td>
			<td >Vetapharm</td>
			<td >Vm21757/4000</td>
			<td >Neuroleptanalgesic for use in mice</td>
		</tr>
		<tr >
			<td >Hypnovel (Midazolam)</td>
			<td >Roche</td>
			<td >59467-70-8</td>
			<td >Induction of anaesthesia</td>
		</tr>
		<tr >
			<td >Dissecting microscope</td>
			<td >Carl Zeiss</td>
			<td >Stemi 2000</td>
			<td ></td>
		</tr>
		<tr >
			<td >Nylon Tubing</td>
			<td >Portex LTD</td>
			<td >800/200/100/200</td>
			<td >0.65 mm in diameter and 1 mm in length; to make artery cuff</td>
		</tr>
		<tr >
			<td >Electrocoagulator</td>
			<td >Martin&#160;</td>
			<td >SN 54.131</td>
			<td >Ligation of artery branches on aorta</td>
		</tr>
		<tr >
			<td >Bipolar micro hemostat forceps</td>
			<td >Martin</td>
			<td >80-91-12-04</td>
			<td >Fixation of vessel ends</td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >JFX-7</td>
			<td ></td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >JFL-3dZ</td>
			<td ></td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >D-5aZ</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mini applier&#160;</td>
			<td >AESCULAP</td>
			<td >FE572K</td>
			<td ></td>
		</tr>
		<tr >
			<td >Micro hemostats clips</td>
			<td >AESCULAP</td>
			<td >FE720K</td>
			<td ></td>
		</tr>
		<tr >
			<td >Surgical sutures, 6-0 VICRYL</td>
			<td >ETHICON</td>
			<td >V489</td>
			<td ></td>
		</tr>
	</tbody>
</table>

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.

成功代PiPSC的确认4天nucleofecting人成纤维细胞用线性pCAG2LMKOSimO质粒携带4转录因子OCT4，SOX2，KLF4和c-Myc（OSKM）之后。 PiPSC显示明显不同的表型相比，成纤维细胞（ 图2A），并表示4程序重编因子在mRNA（ 图2B）和蛋白质（ 图2C）的水平10。一个PiPSC基于血管移植物的效力是高度依赖于PiPSC的分化为内皮细胞和平滑肌细胞系的能力。因此，至关重要的，利用它...

Watch this Scientific Journal Video about Generation and Grafting of Tissue-engineered Vessels in a Mouse Model at JoVE.com

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

在这里，我们提出了一个协议，以产生组织工程血管移植物中有功能的移植入小鼠双播部分诱导多能干细胞（PiPSC） - 来源的平滑肌细胞和PiPSC - 在脱细胞血管支架的生物反应器来源的内皮细胞。

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-and-grafting-of-tissue-engineered-vessels-in-a-mouse-model

Research

JoVE Journal

Bioengineering

11.9K 次观看.  King's College London BHF Centre. 在这里，我们提出了一个协议，以产生组织工程血管移植物中有功能的移植入小鼠双播部分诱导多能干细胞（PiPSC） - 来源的平滑肌细胞和PiPSC - 在脱细胞血管支架的生物反应器来源的内皮细胞。 

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

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

协议相对液力评估的三单张聚合物阀

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

科研

生物工程

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

手术技术组织工程血管移植和后续的植入<em&gt;在体内</em&gt;监控

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

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 ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

三维组织工程人食管黏膜型号生产，表征和潜在用途

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

一种混合的3D组织工程<em&gt;体外</em&gt; /<em&gt;在硅片</em在具体的突变背景预测药物的有效性&gt;肺肿瘤模型

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

中的应用<em&gt;在体内</em&gt;评定组织的鼠胫骨前的功能测试工程化骨骼肌修复

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

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 ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

一种新型的腱绦缝合技术，采用组织工程胶原蛋白移植物修复大肌腱缺损

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

小鼠模型中生物合成组织工程气管移植的育苗与植入

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

用于大鼠圆周食管重建的组织工程移植

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...