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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<li>Takahashi, K., Okita, K., Nakagawa, M., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+pluripotent+stem+cells+from+fibroblast+cultures%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Induction of pluripotent stem cells from fibroblast cultures.</a> Nature Protocols. 2, 3081-3089 (2007).</li>
<li>McCall, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myocardial+infarction+and+intramyocardial+injection+models+in+swine%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">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/pubmed?term=((Transplantation+of+an+allogeneic+vein+bioengineered+with+autologous+stem+cells%3A+a+proof-of-concept+study%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">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><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&#039;s Modification of Ham&#039;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&amp;nbsp;</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&amp;amp;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>&amp;nbsp;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></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&#039;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&amp;nbsp;</td><td>Jepson Bolton&#039;s, Janke&amp;amp;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&amp;nbsp;</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&amp;nbsp;</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&quot; Tubing, ID 0.01&quot; (250&amp;micro;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&amp;nbsp;</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)&amp;nbsp;</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-Prkdc&lt;sup&gt;scid&lt;/sup&gt;/NcrCrl mouse</td><td>Charles River</td><td></td><td></td></tr><tr><td>Surgical sutures, 8-0&amp;nbsp; 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&amp;nbsp;</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&amp;amp;T</td><td>JFX-7</td><td></td></tr><tr><td>Vessel Dilator</td><td>S&amp;amp;T</td><td>JFL-3dZ</td><td></td></tr><tr><td>Vessel Dilator</td><td>S&amp;amp;T</td><td>D-5aZ</td><td></td></tr><tr><td>Mini applier&amp;nbsp;</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

12.0K 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

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

基于生物工程，人造血管使用脉动生物反应器

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

科研

生物工程

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

原位主动脉瓣移植在小鼠血管疾病的研究

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

医学

Mouse Models for Graft Arteriosclerosis

Graft arteriosclerois (GA), also called allograft vasculopathy, is a pathologic lesion that develops over months to years in transplanted organs characterized by diffuse, circumferential stenosis of the entire graft vascular tree. The most critical component of GA pathogenesis is the proliferation of smooth muscle-like cells within the intima. When a human coronary artery segment is interposed into the infra-renal aortae of immunodeficient mice, the intimas could be expand in response to adoptively transferred human T cells allogeneic to the artery donor or exogenous human IFN-&gamma; in the absence of human T cells. Interposition of a mouse aorta from one strain into another mouse strain recipient is limited as a model for chronic rejection in humans because the acute cell-mediated rejection response in this mouse model completely eliminates all donor-derived vascular cells from the graft within two-three weeks. We have recently developed two new mouse models to circumvent these problems. The first model involves interposition of a vessel segment from a male mouse into a female recipient of the same inbred strain (C57BL/6J). Graft rejection in this case is directed only against minor histocompatibility antigens encoded by the Y chromosome (present in the male but not the female) and the rejection response that ensues is sufficiently indolent to preserve donor-derived smooth muscle cells for several weeks. The second model involves interposing an artery segment from a wild type C57BL/6J mouse donor into a host mouse of the same strain and gender that lacks the receptor for IFN-&gamma; followed by administration of mouse IFN-&gamma; (delivered via infection of the mouse liver with an adenoviral vector. There is no rejection in this case as both donor and recipient mice are of the same strain and gender but donor smooth muscle cells proliferate in response to the cytokine while host-derived cells, lacking receptor for this cytokine, are unresponsive. By backcrossing additional genetic changes into the vessel donor, both models can be used to assess the effect of specific genes on GA progression. Here, we describe detailed protocols for our mouse GA models.

We describe protocols for our mouse graft arteriosclerois (GA) models which involve interposition of a mouse vessel segment into a recipient of the same inbred strain. By backcrossing additional genetic changes into the vessel donor, the model can assess the effect of specific genes on GA.

嫁接动脉硬化的小鼠模型

Graft arteriosclerois (GA),  also called allograft vasculopathy, is a pathologic lesion that develops over  months to years in transplanted organs ...

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 ...

Engineered Vascularized Muscle Flap

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen supply to the cell microenvironment, as passive diffusion is limited to a very thin layer. Although various materials have been described to restore the integrity of full-thickness defects of the abdominal wall, no material has yet proved to be optimal, due to low graft vascularization, tissue rejection, infection, or inadequate mechanical properties. This protocol describes a means of engineering a fully vascularized flap, with a thickness relevant for muscle tissue reconstruction. Cell-embedded poly L-lactic acid/poly lactic-co-glycolic acid constructs are implanted around the mouse femoral artery and vein and maintained in vivo for a period of one or two weeks. The vascularized graft is then transferred as a flap towards a full thickness defect made in the abdomen. This technique replaces the need for autologous tissue sacrifications and may enable the use of in vitro engineered vascularized flaps in many surgical applications.

To date, thick tissue defects are typically reconstructed by applying autologous tissue flaps or engineered tissues. In this protocol, we present a new method for engineering vascularized tissue flap bearing an autologous pedicle, to serve as a substitute to autologous flaps.

工程化血管肌瓣

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

下腔静脉间置移植物在小鼠模型中植入

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

工程血管移植缩放使用3D打印指南和环堆叠方法

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

一种新的显微模型异位，整块胸壁，胸腺，和心脏移植小鼠

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

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 ...

Implantation of Tissue-Engineered Vascular Graft in Mouse Carotid Artery via Cuff Technique

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal experimentation plays a pivotal role in assessing the efficacy and safety of vascular grafts, particularly in the absence of clinical applications. Compared to alternative animal models, the mouse implantation model offers several advantages, including a well-defined genetic background, a mature method for disease model construction, and a straightforward surgical procedure. Based on these advantages, the present study devised a simple cuff technique for the implantation of tissue-engineered vascular grafts in the mouse carotid artery. This technique began with the fabrication of polycaprolactone (PCL) small-diameter vascular grafts via electrostatic spinning, followed by the seeding of macrophages onto the grafts through perfusion adsorption. Subsequently,&#160;the cellularized tissue-engineered vascular grafts were transplanted into the mouse carotid artery using the cuff technique to evaluate patency and regenerative capability. After 30 days of in vivo implantation, vascular patency was found to be satisfactory, with evidence of neo-tissue regeneration and the formation of an endothelial layer within the lumen of the grafts. All data were analyzed using statistical and graphing software. This study successfully established a mouse carotid artery implantation model that can be used to explore the cellular sources of vascular regeneration and the mechanisms of action of active substances. Furthermore, it provides theoretical support for the development of novel small-diameter vascular grafts.

Here, a protocol is presented for the implantation of a tissue-engineered vascular graft into the mouse carotid artery using the cuff technique, providing a suitable animal model for investigating vascular tissue regeneration mechanisms.