作者想感谢他们的宝贵意见成像钛节和双子座生物制品莱卡微系统提供在这项研究中所使用的猪血清。我们也想感谢授予NIH的支持"自体的EPC的衬里，以改善循环系统的生物相容性协助设备"，RC1HL099863 - 01。此外，我们为国家科学基金会研究生研究奖学金的亚历山德拉Jantzen的支持表示感谢。我们还要感谢协助手术过程中的许多方面和我们研究动物的处理，乔治快速，麦克Lowe和Ianthia帕克。史蒂芬欧文一直是无价的，在许多地方我们播种设备的加工和切割钛管。

1。内皮祖细胞的分离<ol><li> EPC种子管植入前三十天，准备与抗凝枸橼酸葡萄糖溶液15毫升60毫升注射器，安全与3路从外围猪血EPC隔离停止公鸡。前24小时抽血，预涂两个12 - 1型大鼠胶原蛋白（50μg/ ml的，0.02 N醋酸溶液溶解）4板。 </li><li>按照国家的关怀和使用实验动物的健康指引研究所和监督机构的动物护理和使用委员会批准后，才进行的所有动物的照顾和实验。 </li><li>稳重的一个女的约克郡猪（45公斤）Acepromazine（1.1毫克/公斤），氯胺酮（22毫克/公斤）通过19政蝴蝶针肌肉注射。 </li><li>插管与气管插管（30厘米长，8毫米ID）的猪，麻醉与异氟醚（4.5％潮气量面具）的猪。 </li><li>在手术过程中，通过测量血氧饱和度，心率和温度监控猪。使用自动恒温手术台和加热毯和气候变暖注入液体，保持体温。 </li><li>猪放置在上手术台的仰卧姿势，确保其后肢caudolaterally，清洁与洗必泰DuraPrep杀菌。然后进行悬垂的骨盆部位。 </li><li>摹一个21 × 7厘米的针插入一个5楼微介绍人套件内侧可触及股动脉搏动的股静脉（股内侧的内侧和外侧的股薄肌）和cannulate静脉使用Seldinger技术。将准备60毫升注射器，血管内导管，并绘制了45毫升血液。 </li><li>保持血管穿刺部位的压力，以达到止血（5分），停止麻醉和恢复猪。动物进行监测，直到完全恢复，并回到笼子里。 </li><li>稀血与汉克的缓冲盐溶液的解决方案1:1（不含氯化钙 ，氯化镁，硫酸镁4）和等量Histopaque创造良好定义的层上的层。离心机（30分钟，740克，打破设置低）和收集的单核细胞（MNC）的层。悬浮和洗跨国公司x 3贝科的磷酸盐缓冲电镀前到两个12孔全面增长介质板（EBM - 2介质与2％猪血清（PS）和EGM - 2盐水（DPBS）（10分钟，515克） SingleQuots在）37℃，5％的CO 2。 </li><li>慢慢地改变中期每24小时第7天，然后隔日。识别后的平均时间的EPC殖民地 7天（ 图2）。 </li><li>扩大在文化的内皮祖细胞，一旦它们涵盖12孔的表面面积的四分之一。用流式细胞仪进行表面标志物CD31和缺乏CD14，CD45存在检测的EPC身份确认。可能进行的其他实验，包括细胞的形态和一氧化氮三活动曝光后流量4。 </li></ol> 2。钛管装配<ol><li>举行的第3等于120度单位（长4.5厘米）钛管纵向与HHS的分切一个72齿锯的地方，看到在一个立式铣床乔木。运行在300转，并保持饱和塔魔术在切割液（ 图3）在任何时候都切的切割面积。 </li><li>波兰与砂轮机和思高金工轮的内表面。然后手动3M砂布进一步畅通，甚至表面波兰删除任何可见的坑。 </li><li> 王水具有很强的腐蚀性，清洁Alconox肥皂溶液的钛片，5分钟，淹没在王水（1:3浓硝酸浓盐酸），水10升冲洗，格外小心潜在爆炸危险的！ </li><li>超声钛节× 16分钟Alconox肥皂溶液，在去离子水上升× 30和超声再次× 16分钟去离子水。允许干层流罩。 </li><li>切PVC热缩管4.5厘米的长度，并彻底清洁每2.4。 </li><li>使用镊子将其放置在一个支持芯棒（加工铝，8.5 mm外径）和地方PVC热缩管（ 图4）围绕钛节套3清洗钛节。收缩管热风枪，同时转动芯轴均匀包裹体部分紧紧地握在一起。 </li></ol> 3。播种设备和部件组装<ol><li>剪下硅胶管长2.5厘米和3.5厘米。 </li><li>第5毫升的塑料注射器0.8毫升，并保持与露儿结束"头件"。 </li><li>彻底清洗2个硅部分，注射器的头片"和根据2.4所述的由超声露儿第。 </li><li>硅胶管组装的钛管的两端，从2.5 EXTEN丁PVC管材超过5毫米。 </li><li>插入剪切注射器的头件"到硅胶管短，所以它紧靠钛管（ 图5）。 </li><li>密封完成露儿在特卫强邮袋和气体消毒与环氧乙烷（18小时在55 ° C）第一个钛管组装。 </li><li>登上一个有机玻璃平台上的同步调速电机（10 RPH）和连接轴注射器持有人（加工铝，适合5毫升注射器）（ 图6） 。 </li></ol> 4。钛管内表面的EPC播种<ol><li>展开的EPCs隔离在1到3融合的T - 75瓶（或至少有9 × 10 6细胞）。 </li><li>在手术当天，长期染料（PKH26）11荧光标签的细胞。开始冲洗两次在无血清培养基培养的EPCs。注意限制光线照射到保护标签，期间和之后细胞。 </li><li>盖细胞与4μm的稀释剂彗星在室温（PKH26染料溶液）4分钟。 </li><li>停止标签加入相同体积的猪血清，染料溶液的反应。一分钟后，稀结合完整的生长介质的解决方案1:1。 </li><li>吸液和冲洗细胞完全生长培养基× 3。 </li><li>洗净的EPCs两次在DPBS（不含氯化钙 ，氯化镁2）。 </li><li>盖胰蛋白酶孵育的细胞在37 ° C，3分钟和光显微镜下确认支队。添加在双重的胰蛋白酶的使用量胰蛋白酶中和解决方案。 </li><li>吹打细胞结合成一个单一的管和混合解决方案。 10μL细胞液添加到每一个血球侧面进行计数。 </li><li>离心机电池解决方案（1500 RPH，5分钟）。计数细胞和重新悬浮颗粒在2 - 2.5 × 10 6内皮祖细胞/ ml的无血清培养。注意:体积最小，以填补管组装为4.5毫升。 </li><li>无菌巾，在无菌领域的生物罩。打开气体消毒的钛管装配和额外的5毫升注射器，进入无菌区。 </li><li>用无菌手套，取出5毫升注射器的柱塞和领域保持供日后使用。鲁尔第贴上这个注射器。 </li><li>吸取4.5 - 5毫升EPC暂停下4.9到开放的针筒。安全地返回到开放末端的注射器插入柱塞。 </li><li>第注射器向上握住并删除第。露儿年底到打开硅胶管，钛管大会第一次插入注射器，直到温暖的;钛管内不提前。 </li><li>锋注射器推杆缓慢，直到细胞溶液达到削减注射器的顶端"的头一件，"从系统中移除的气泡。关闭露儿帽，用胶带密封的开口端插入无菌鞘整个大会。 </li><li>插入整个组装加工注射器持有3.7。在孵化器放置在37 ° C和调整的平台，使直播室的钛管部分是水平的，使用水位计（ 图 6 ）。 </li><li>允许钛管装配旋转植入前30分钟。 </li></ol> 5。钛管植入到猪的下腔静脉<ol><li>二十四小时，在手术前，premedicate猪（从被孤立的EPCs）与芬太尼补丁（100微克/小时透皮;保持到位× 72小时的补丁）。 </li><li>保持隔夜猪非营利组织和管理Baytril（恩诺沙星）术前在手术当天（5毫克/公斤，IM）和7天以下，每24小时为抗生素预防。 </li><li>稳重，插管及麻醉的猪， 根据 1.3所述（潮气量10 -15毫升/公斤）和安全的猪，在手术室表仰卧位。在手术过程中，通过测量血氧饱和度，心率和温度监控猪。使用自动恒温手术台和加热毯和气候变暖注入液体，保持体温。 </li><li>一个18 G静脉导管插入猪的耳静脉和保护猪的眼睛与Vetropolycin眼药水。 </li><li>清洁，准备和悬垂性好猪的腹部在1.6。从乳腺第二盘＃15手术刀刀片颅最后一组第二尾端切割中线。 </li><li>携带的清扫腹部筋膜与电灼。 </li><li>蚊虫钳提起腹膜，并小心地将其与梅岑鲍姆剪刀进入。 </li><li>外部膀胱和膀胱壁放入一个3 - O薇乔荷包缝合。在它的中间放置一个刺切口，然后插入一个16架F尿管。管理静脉注射液（乳酸林格）滴定尿量&gt; / = 1毫升/公斤/小时手术过程中。 </li><li>之后，外部小及大肠癌和地方两个贝尔福手术拉钩暴露腹腔后方面，并确定下腔静脉（IVC）。 </li><li>使用尖锐，钝性分离，仔细<html>免费下腔静脉周围组织，并skeletonize从右侧肾动脉近端的下腔静脉远端的分叉处的船只。练习过程中的下腔静脉夹层格外小心，因为即使是在非常小的下腔静脉的缺陷可能导致出血和动物放血快速。 </li><li>结扎下腔静脉段的所有分支，以确保将有没有被植入的钛管周围出血。注意:通常比较大，需要非常仔细解剖和结扎后腰椎静脉。此外，请注意，立即到下腔静脉分叉处近端，一个大腰静脉经常遇到的后内侧端，必须解剖自由和控制钳安置准备船只循环。 </li><li>继续播种的钛管，同时根据 4 概述 。 </li><li>管理100 USP /公斤肝素立即夹紧下腔静脉前。广场上腔静脉和腰静脉远端呈45度角手术夹具，然后近端。 </li><li>创建一个纵向之间的近端和远端使用＃11手术刀刀片扩展波茨剪刀夹子veinotomy（4厘米）。 </li><li>撤离的下腔静脉血液和无菌DPBS冲洗内腔。 </li><li>现在，下腔静脉插入EPC种子钛管（或裸机控制）和填充DPBS（氯化钙， 氯化镁 2），以防止干燥的细胞和疏散风。 </li><li> 4-0普理灵连续缝合关闭veinotomy和删除一个近端钳通过后出血少量空气的下腔静脉。通过静脉壁的PVC管材放置一个"留缝合"，随着时间的推移，以防止植入迁移。 </li><li> 2 - O薇乔（运行缝线）与O型PDS关闭筋膜的CT针和皮下空间。关闭带有订书钉的皮肤。 </li><li>管理多达20毫升0.25％Marcaine（布比卡因）沿切口部位皮下，覆盖伤口，用纱布和Tegaderm。也给氟尼辛（2.2毫克/公斤Q 24小时）和羟吗啡酮（0.15毫克/公斤问3 - 4小时），皮下注射的痛苦，因为需要。 </li><li>停止麻醉，监测，直到清醒的猪和返回笼。监控猪每日两次遇险/疼痛，站立和行走，粪便和尿液输出，和皮肤的颜色，表明正常灌注的迹象。 </li></ol> 6。 Explantation的钛管<ol><li> 3周后，稳重，插管及麻醉如1.3中所述的猪- 1.4 。 </li><li>继续在5.5描述了剖腹探查术- 5.8。 </li><li>请注意，疤痕会隐瞒植入部位，但你可以触诊刚性钛管在原位。 </li><li>如5.9中所述的下腔静脉- 5.11解剖和植钛管与下腔静脉周围重剪块。 </li><li>动物安乐死Euthasol安乐死的解决方案（390毫克/毫升的戊巴比妥钠和50毫克/毫升的苯妥英钠1毫升/ 10磅）。 </li></ol> 7。固定和成像体的内表面<ol><li>冲洗在DPBS溶液的静脉段切除钛管，并拍摄高分辨率的数码相机（图 7）流明（专利或闭塞）。 </li><li>下面，修复淹没在3.7％多聚甲醛为至少15分钟的标本。在DPBS溶液冲洗标本。 </li><li>非常小心地切开周围静脉和PVC管材打开钛管。与面临的目标/激发波长在550纳米光源和图像可视化PKH26 -标记的EPCs的红色/橙色（图8A）的内表面的荧光显微镜下放置3个部分。如果需要的话，细胞可进一步染色（如血小板内皮细胞粘附的标记（PECAM）染色可视化单元格边框（ 图8B），DAPI染色可视化核等） 。 </li></ol> 8。代表性的成果: 执行这个协议之后，医生和科学家能够与自体血源性内皮祖细胞在一个大的动物模型endothelialize固体筒结构图2可以看出 ，与我们的方法分离出内皮祖细胞与鹅卵石形态的菌落出现后约7天文化。我们的播种设备在图5和 6所示，允许与EPC悬挂和管内表面均匀覆盖的结果充满9无菌条件下的钛管缓慢旋转。 用于测试的生物材料的倾向，如Ti，允许我们的植入手术，血栓形成一个大型的动物模型。我们发现，猪和容忍此过程，这个植入可只有很少的失血和没有EPC层破坏实现。 T"&gt; 图7表明，裸露的钛管完全阻塞，而我们的EPC内衬管仍然专利，即使在低剪切环境下腔静脉血栓。进 ​​一步，一种荧光标记的细胞融合层的存在，证明这种方法的成功， 如图8所示。 <img alt="图1" src="/files/ftp_upload/3197/3197fig1.jpg" /> 图1自体内皮祖细胞（EPC）的播种实验的原理图。首先，外周血取自猪。下一步，内皮祖细胞是从血液中分离和扩大在文化。内皮祖细胞，然后用于行钛（TI）管设备，然后从细胞中分离出相同的猪进入下腔静脉手术植入。 <img alt="图2" src="/files/ftp_upload/3197/3197fig2.jpg" /> 图2，代表文化的EPCs，大约7天的隔离程序（DMIL一个倒置的徕卡显微镜成像相机和QCapture软件成像）殖民地。 <img alt="图3" src="/files/ftp_upload/3197/3197fig3.jpg" /> 图3。钛管段组装前。钛管，切成3纵向平等的部分，然后切成4.5厘米长。使用砂轮机，砂布，以消除可见的坑，钛部分的内表面抛光。 <img alt="图4" src="/files/ftp_upload/3197/3197fig4.jpg" /> 图4。PVC热缩管（蓝色）和热枪钛管段大会。支持TI部分加工的铝芯棒延伸通过TI部分和钛管内径的尺寸匹配。 <img alt="图5" src="/files/ftp_upload/3197/3197fig5.jpg" /> 图5钛管组件，所有组件:露儿帽，注射器的头件，硅胶管，钛管与PVC包装（蓝色）。大会放在一起之前绝育手术使用。 <img alt="图6" src="/files/ftp_upload/3197/3197fig6.jpg" /> 图6。孵化器钛管内播种设置，显示电机，平台，机加工铝，钛管注射器持有人大会，5毫升注射器。注:大会是为可视化的目的无菌无保护鞘所示。 <img alt="图7" src="/files/ftp_upload/3197/3197fig7.jpg" /> 图7。植入手术的代表总成绩（A）控制裸露的金属钛管结束查看后，在植入猪的下腔静脉（IVC） 。管腔完全闭塞了坚实的血块，（二 ）EPC种子钛管植入后的最终视图。管腔是完全的专利和明确的。（三）解剖控制视图（裸）钛管后3天的植入，显示血栓形成的程度（实验相同的结果进行了为期3个星期） 。 <img alt="图8" src="/files/ftp_upload/3197/3197fig8.jpg" /> 图8。钛管表面内皮祖细胞，植入3天的（同一个堂堂正正的徕卡DMRB与QImaging QICAM显微镜成像单色数码相机和图像的Pro Plus软件）。 （一）聚合细胞表面呈现PKH26手术前的标签。 （二）聚合层的EPCs。红:PKH26手术前的标签。绿色:EPC PECAM -染色。

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没有利益冲突的声明。

细胞播种钛管这里介绍的方法，使医生和科学家，以快速和均匀endothelialize血液接触的植入装置的表面。既然我们孤立和扩大从外周血内皮祖，没有大的侵入过程是必需的收获这些细胞。此外，内皮祖细胞是自体，因此，任何免疫反应的细胞种子植入的风险被淘汰。不仅对钛管，但可以利用许多其他的生物材料，心血管内科利用他的协议中所表现出的原则。 在本议定书中的关键步骤是钛管细致的清理，我们发现任何污染物妥协细胞黏附贴壁膜。此外，一个缓慢的旋转速度（管直径成反比），是在播种过程中至关重要，这样内皮祖可以慢慢解决，并坚持到表面钛管。 我们立即播种前植入的方法避免了不切实际的体外培养时间;细胞坚持个别及后在体内形成一个融合的表，避免栓塞的可能性，如纸，紧随流重新建立。我们以前的研究表明，一旦内皮祖已成长为一个融合层，他们使他们坚定不移地坚持一个细胞外基质，另外尽量减少任何可能脱落的内皮细胞表。虽然栓塞脱离的可能性不能完全排除，这似乎是多方面的低比整个设备的表面血栓形成的风险，问题，这种疗法旨在防止。 我们的方法植入到低剪切，下腔静脉血栓环境利用研究血液相容性及血栓形成的设备5,6的一个最值得信赖的大动物模型。请注意，所有的动物护理和实验是在按照国家的关怀和使用实验动物的健康指引研究所和杜克大学机构动物护理和使用委员会的批准后，才进行。 为了成功地利用这种植入方法来测试的生物材料和设备，重要的是要耐心skeletonize下腔静脉段和结扎静脉分支血管都在准备，设备插入，如设备周围出血，没有在一个"假腔"起源。另一个关键的一步是添加到设备管腔内管表面上的细胞保持湿润，在封闭的veinotomy和再灌注前​​发起的DPBS。如果设备不能被发现在植入的位置，它可能已迁移的"上游"的下腔静脉。这是可以预防的配售缝合（4 - O普理灵）通过静脉壁，并通过2 - 3毫米的PVC管材管部分，因此是牢牢固定在其目前的位置。如果研究者有困难找到了荧光标记的细胞后，在其他专利的管explantation图8所示，它很可能作为一个统一的工作表，这些细胞已去皮。这是可以预防的很温柔的清扫周围静脉和显示3钛管段组装，后固定管静脉。 我们的技术提供了概念证明，通过EPC的播种，以防止心血管设备血栓。这项技术可用于在内衬与病人自身内皮祖细胞的"生源"植入发展。在我们的猪动物模型的可行性研究到临床实践这种"个性化药物"朝翻译的第一个步骤提供。

<table border="1"><tbody><tr><td> 试剂 </td><td> 公司名称 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> Acepromazine </td><td>勃林格 - 殷格翰公司</td><td> BIC670025 </td><td>新来港学童＃10280002 </td></tr><tr><td> Alconox供电的精密清洗剂</td><td> Alconox </td><td> 1104 </td><td></td></tr><tr><td>贝尔福手术牵引器</td><td>阿德勒</td><td> N / A </td><td></td></tr><tr><td> Baytril </td><td>拜耳</td><td> APVMA 46028/0705 </td><td></td></tr><tr><td>蝴蝶针（19G） </td><td> Terumo公司</td><td> SV19CLK </td><td></td></tr><tr><td>洗必泰</td><td> 3M </td><td> 9200 </td><td></td></tr><tr><td>夹钳（45度） </td><td>蛇牌</td><td> FC339T </td><td></td></tr><tr><td> DPBS (-/-) </td><td> Gibco公司</td><td> 14190-144 </td><td></td></tr><tr><td> DPBS (+/+) </td><td> Gibco公司</td><td> 14040-133 </td><td></td></tr><tr><td> DuraPrep </td><td> 3M </td><td> 8635 </td><td></td></tr><tr><td> EBM - 2培养基</td><td>龙沙</td><td> CC - 3156 </td><td>基地血清自由和充分的生长介质</td></tr><tr><td>临时股东大会，2 SingleQuots </td><td>龙沙</td><td> CC - 3162 </td><td> EBM - 2用于血清的自由和充分的生长介质</td></tr><tr><td>电灼工具</td><td>的Valleylab </td><td> SurgII - 20 </td><td></td></tr><tr><td>砂布</td><td> 3M </td><td> 60-0700-0425-8 </td><td> 240砂砾</td></tr><tr><td> Euthasol安乐死解决方案</td><td> Virback动物健康</td><td></td><td>穴田＃:200-071 </td></tr><tr><td>芬太尼补丁</td><td>阿特维斯</td><td></td><td> NDC＃67767-120-18 </td></tr><tr><td>氟尼辛</td><td>先灵葆雅</td><td></td><td>新来港学童＃:10470183 </td></tr><tr><td>导尿管（16F） </td><td>诗人</td><td> 730116 </td><td></td></tr><tr><td>的HBSS </td><td>西格玛</td><td> H8264 - 500ML </td><td></td></tr><tr><td>热风枪</td><td>密尔沃基</td><td> 8988-20 </td><td></td></tr><tr><td>肝素</td><td></td><td></td><td> NDC＃:25021 </td></tr><tr><td> Histopaque - 1077 </td><td>西格玛</td><td> H8889 - 500ML </td><td></td></tr><tr><td>插管</td><td> Mallinckrodt </td><td> 86113 </td><td></td></tr><tr><td>异氟醚</td><td> MWI的，经络</td><td></td><td> NDC＃13985-030 </td></tr><tr><td>静脉导管（18G） </td><td>流式细胞 - 迪金森</td><td> 381547 </td><td></td></tr><tr><td>氯胺酮</td><td>道奇堡</td><td></td><td> NDC＃0856-2013 </td></tr><tr><td>等级</td><td>斯旺森</td><td> LLA001 </td><td></td></tr><tr><td>鲁尔锁尖帽</td><td>慢性粒细胞白血病供应</td><td> 909-001 </td><td></td></tr><tr><td> Marcaine </td><td> Hospira公司</td><td></td><td> NDC＃:0409-1560-10 </td></tr><tr><td>梅岑鲍姆剪刀</td><td>阿德勒</td><td> N / A </td><td></td></tr><tr><td>微介绍人（5F） </td><td>高尔特</td><td>套装002-01 </td><td></td></tr><tr><td>蚊钳</td><td>阿德勒</td><td> N / A </td><td></td></tr><tr><td>汽车</td><td> Herbach和Rademan </td><td> H1 - 08 </td><td></td></tr><tr><td>羟吗啡酮</td><td>远藤实验室</td><td></td><td> NDC:63481-624-10 </td></tr><tr><td> PKH26染料套件</td><td>西格玛</td><td> PKH26GL - 1KT </td><td></td></tr><tr><td>猪血清</td><td>双子座生物产品</td><td> 100-115 </td><td>完全生长培养基中的浓度为2％ </td></tr><tr><td>波茨剪刀</td><td>阿德勒</td><td> N / A </td><td></td></tr><tr><td>精密Vista的皮肤订书机</td><td> 3M </td><td> 3998 </td><td></td></tr><tr><td> PVC管材</td><td> McMaster-Carr/Insultab </td><td> 7132K117 </td><td>扩大编号15.88毫米， 回收编号7.95毫米</td></tr><tr><td>直角中等尺寸</td><td>阿德勒</td><td> N / A </td><td></td></tr><tr><td>手术刀刀片（＃10-15） </td><td>诗人</td><td> 373910 </td><td></td></tr><tr><td>硅胶管</td><td>麦克马斯特 - 卡尔</td><td> 51735K26 </td><td>计增长16.64毫米外径，内径9.52 </td></tr><tr><td>注射器（5毫升） </td><td>贝克顿迪金森（BD） </td><td> 309603 </td><td></td></tr><tr><td> Tegaderm </td><td> 3M </td><td> 90001 </td><td></td></tr><tr><td>三路阀</td><td>肯德尔</td><td> 170060 </td><td></td></tr><tr><td>钛管</td><td>天工钛</td><td> N / A </td><td>指定为½"外径，0.065"华尔街，0.370"ID，0.1737磅/英尺</td></tr><tr><td>胰蛋白酶</td><td>龙沙</td><td> CC - 5012 </td><td></td></tr><tr><td>胰蛋白酶中和溶液（TNS） </td><td>龙沙</td><td> CC - 5002 </td><td> 0.03％ </td></tr><tr><td> Vetropolycin </td><td> Pharmaderm动物健康</td><td></td><td>新来港学童＃:12920110 </td></tr><tr><td>薇乔缝合线（3-0） </td><td>爱惜康</td><td> J808T </td><td></td></tr><tr><td>水洗浴Sonicator </td><td>布兰森</td><td> B200 </td><td></td></tr></tbody></table>

autologous endothelial progenitor cell-seeding technology and biocompatibility testing for cardiovascular devices in large animal model

植入式心血管设备制造人造材料（如钛（Ti），膨体聚四氟乙烯），构成了血栓形成 1,2,3的风险的。我们已经开发出一种方法，以符合人类或猪的自体血源性内皮祖细胞（EPCs）4钛管的内表面。通过植入含有猪内皮祖细胞在猪的下腔静脉（IVC）的融合层的钛管，我们测试的种子细胞表面的一个大型的动物模型血栓环境改善的生物相容性和，它比裸露的金属表面未修改5,6,7（ 图1）。这种方法可用于endothelialize分钟内植入的设备和测试他们体内的抗血栓功能。 外周血获得50公斤约克夏猪和单核细胞比例的培养，分离出内皮祖细胞 4,8 。钛管（9.4毫米ID）被预先切分为三个4.5厘米的纵向部分，并用热缩管重组。建一个播种设备，允许钛管缓慢旋转。 我们进行了剖腹探查和外部猪小肠和膀胱。夏普和钝性分离其右侧肾动脉近端远端的分叉skeletonize从下腔静脉。钛管，然后充满10 RPH x 30分钟自体荧光标记EPC悬挂和旋转，实现统一的细胞涂层 9 。 100 USP /公斤肝素管理后，下腔静脉和腰静脉两端被夹住。一个4厘米veinotomy的执行和设备中插入和充满磷酸盐缓冲液。由于veinotomy 4-0普理灵连续缝合关闭，一个钳去除空气的下腔静脉。在该过程结束时，筋膜0 - PDS（polydioxanone缝合）近似，皮下空间封闭2-0薇乔和皮肤装订关闭。 经过3 - 21天，猪安乐死，explanted EN -块和固定设备。被拆卸的钛管，内表面用荧光显微镜成像。 我们发现，裸露的金属钛管完全闭塞，而EPC种子管仍然专利。此外，我们能够证明血液接触的内表面上的内皮祖细胞融合层。 最后，我们的技术可用于endothelialize与自体内皮祖细胞在几分钟内植入的钛管，以防止血栓的设备。我们的手术方法可用于测试改进这种经过修改的设备，用最少的失血和EPC种子表面中断的生物相容性。

Watch this Scientific Journal Video about Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model at JoVE.com

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

播种钛方法的血液接触与自体细胞和生物材料测试的生物相容性描述。此方法使用内皮祖细胞和钛管，手术植入分钟内接种于猪腔静脉。这种技术是适应许多其他的植入式生物医疗设备。

自体内皮祖细胞播种技术和生物相容性在大动物模型的心血管设备的测试

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

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20.3K 次观看.  Duke University. 播种钛方法的血液接触与自体细胞和生物材料测试的生物相容性描述。此方法使用内皮祖细胞和钛管，手术植入分钟内接种于猪腔静脉。这种技术是适应许多其他的植入式生物医疗设备。

视频: Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

通过胎盘的外来物质和纳米材料的运输率测定<em&gt;体外</em&gt;人胎盘灌注型号

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

科研

生物工程

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

电动细胞基质阻抗传感的内皮细胞增殖，屏障功能的定量和能动性

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

对于中吸入有毒化学测试体外细胞培养模型

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

层与层胶原沉积在微流体装置的Microtissue稳定

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. The feasibility of using magnetic forces to capture and retain endothelial outgrowth cells (EOC) labeled with super paramagnetic iron oxide nanoparticles (SPION) has been shown previously. But this technique requires the development of a mechanically functional stent from a magnetic and biocompatible material followed by in-vitro and in-vivo testing to prove rapid endothelialization. We developed a weakly ferromagnetic stent from 2205 duplex stainless steel using computer aided design (CAD) and its design was further refined using finite element analysis (FEA). The final design of the stent exhibited a principal strain below the fracture limit of the material during mechanical crimping and expansion. One hundred stents were manufactured and a subset of them was used for mechanical testing, retained magnetic field measurements, in-vitro cell capture studies, and in-vivo implantation studies. Ten stents were tested for deployment to verify if they sustained crimping and expansion cycle without failure. Another 10 stents were magnetized using a strong neodymium magnet and their retained magnetic field was measured. The stents showed that the retained magnetism was sufficient to capture SPION-labeled EOC in our in-vitro studies. SPION-labeled EOC capture and retention was verified in large animal models by implanting 1 magnetized stent and 1 non-magnetized control stent in each of 4 pigs. The stented arteries were explanted after 7 days and analyzed histologically. The weakly magnetic stents developed in this study were capable of attracting and retaining SPION-labeled endothelial cells which can promote rapid healing.

Our goals were to design, manufacture and test ferromagnetic stents for endothelial cell capture. Ten stents were tested for fracture and 10 more stents were tested for retained magnetism. Finally, 10 stents were tested in-vitro and 8 more stents were implanted in 4 pigs to show cell capture and retention.

铁磁金属裸支架对血管内皮细胞捕获和保留

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

用 3 d-印刷模具制作细胞播种和组织环自组装用的自定义琼脂糖井

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

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

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

逐步在泰瑟尔脚手架上播种，研究发芽血管

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

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

用于刺激生物组织的电动和磁场设备

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

A Large Animal Model for Pulmonary Hypertension and Right Ventricular Failure: Left Pulmonary Artery Ligation and Progressive Main Pulmonary Artery Banding in Sheep

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing novel therapeutics for PH requires a clinically relevant large animal model of increased pulmonary vascular resistance and RVF. This manuscript discusses the latest development of the previously published ovine PH-RVF model that utilizes left pulmonary artery (PA) ligation and main PA occlusion. This model of PH-RVF is a versatile platform to control not only the disease severity but also the RV&#39;s phenotypic response.
Adult sheep (60-80 kg) underwent left PA (LPA) ligation, placement of main PA cuff, and insertion of RV pressure monitor. PA cuff and RV pressure monitor were connected to subcutaneous ports. Subjects underwent progressive PA banding twice per week for 9 weeks with sequential measures of RV pressure, PA cuff pressures, and mixed venous blood gas (SvO2). At the initiation and endpoint of this model, ventricular function and dimensions were assessed using echocardiography. In a representative group of 12 animal subjects, RV mean and systolic pressure increased from 28 &#177; 5 and 57 &#177; 7 mmHg at week 1, respectively, to 44 &#177; 7 and 93 &#177; 18 mmHg (mean &#177; standard deviation) by week 9. Echocardiography demonstrated characteristic findings of PH-RVF, notably RV dilation, increased wall thickness, and septal bowing. The longitudinal trend of SvO2 and PA cuff pressure demonstrates that the rate of PA banding can be titrated to elicit varying RV phenotypes. A faster PA banding strategy led to a precipitous decline in SvO2 &#60; 65%, indicating RV decompensation, whereas a slower, more paced strategy led to the maintenance of physiologic SvO2 at 70%-80%. One animal that experienced the accelerated strategy developed several liters of pleural effusion and ascites by week 9. This chronic PH-RVF model provides a valuable tool for studying molecular mechanisms, developing diagnostic biomarkers, and enabling therapeutic innovation to manage RV adaptation and maladaptation from PH.

This manuscript describes the surgical technique and experimental approach to develop severe right ventricular pressure overload to model their adaptive and maladaptive phenotypes.

肺动脉高压与右心室衰竭的大型动物模型:绵羊左肺动脉结扎与进行性主肺动脉束带

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.