Name: in vivo imaging and tracking of technetium-99m labeled bone marrow mesenchymal stem cells in equine tendinopathy
Uploaded: 2015-12-09T13:00:00.000+00:00
Duration: 7 min 52 s
Description: Watch this Scientific Journal Video about In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy at JoVE.com

The authors would like to acknowledge funding from the Horserace Betting Levy Board U.K. (grant number 721) and VetCell BioScience Ltd, U.K. and by Consejer&#236;a de Innovaci&#242;n, Ciencia y Empresa, Junta de Andaluc&#236;a, Spain.

下面授予的存留Veterinarios马拉加，西班牙的动物伦理和福利委员会，以及皇家兽医学院，北Mymms，英国的马匹使用的程序伦理许可本文所描述的情况下，进行基于批准的方案是在临床上使用接收的干细胞为基础的治疗，其包括镇静，骨髓抽吸内，腱注射，局部灌注，静脉注射，后处理程序和疼痛管理和监测植入后马。 1.重点安排，事先提出<ol><li>寻求制度伦理和动物福利的批准，并坚持在开始工作之前当地法律要求。 </li><li>寻求机构的批准与电离辐射工作作为规定由地方和法律法规，包括人员保护的适当水平，环境及处置CONTAM的inated浪费。 </li><li>使用下列入选标准:存在有非创伤性过度紧张损伤（腱炎或desmopathy）的前肢和病变导致肌腱内的缺陷和缺陷的浅屈肌数字肌腱马匹由完整肌腱和/或腱旁组织包围。 注意:检查，诊断为这种伤害和临床准备的马细胞移植已在别处6,15详细说明。 注意:从马的骨髓来源在BMMSC的培养的分离和扩增先前已6,16详述。目前的协议注入BMMSC的SDFT受伤的马每使用自体骨髓上清液（BMS）16毫升4 10万个细胞。 </li><li>使用已经历不超过4通道，用于植入体内的细胞，以避免与高传代数字c相关的潜在的细胞表型或遗传改变ELLS。 </li><li>递送的细胞在BMS所需数量（在24小时内以寒意盒在4 - 8℃）的兽医诊所，其中细胞植入将被执行。 注意:在骨髓上清液中的临床使用间充质干细胞是一种专利程序由ReCellerate公司（美国）和它的使用资可能需要授权。 注意:锝- 99m的是γ发射体（140千电子伏）具有相对短的半衰期（6.0058小时，因此近94％的它衰减到其稳定99锝形式中24小时）。伽玛能量使得它适合于检测通过γ相机（显像）和短半衰期的结果在非常有限的辐射暴露时间既马和动物处理人员。这个协议描述了使用锝 - 99m的-HMPAO [HMPAO标记锝 - 99m的（如高锝酸钠）]细胞内的标记。 </li><li>订购锝 - 99m的从供应商新鲜使得其被输送，并用于标记细胞机智欣2小时洗脱从TC - 99m的发电机。确保锝 - 99m的是作为1 GBq在1ml溶液（供应商的标准缓冲液）。 </li><li>使用所有的缓冲区在室温。执行标记过程中的同位素封闭的房间与酒精清洗所有设备单元和成像（γ闪烁）植入湿巾工作开始之前。 </li></ol> 2.准备前肢肌腱的马和超声<ol><li>记录超声波扫描在伤害第一入场（当骨髓吸出），然后在时间点时的放射性标记的细胞注射。 </li><li>与马承重量均匀地在一档和下站在镇静（而非全身麻醉）进行超声和细胞移植。使用detomidine HCl和酒石酸布托啡诺的混合物中，每个在0.01到0.02毫克/公斤静脉施用镇静剂。 注意:恢复是快速的和手术后的治疗，无需特殊注意事项。 </li><li>经过周围神经麻醉适用于患肢（步骤2.6），让马在车站镇静下减少治疗过程中的马的运动。目视检查镇静从降低头部位置和马的神态，而马是细胞注射和伽玛相机采集核素图像在运动过程中的舒适判断的适当水平。 注意:这是没有必要适用兽医软膏上的眼睛（防止干燥），因为下镇静闪烁不受影响，马是能够保持对眼睛的水化。细胞的植入后的监视是非侵入性（超声和γ闪烁）。 </li><li>紧紧夹在头发上覆肌腱（马理发器），然后清除使用洗必泰外科洗手和组合终于酒精剪切区域。 </li><li>应用声耦合凝胶的面积和扫描有条不紊地记录完整的手掌掌骨区域的扫描，依次标有等级1 - 7月17日，具有线性传感器（12 MHz或类似）来获取序列上发生横向和纵向的图像。数字存储图像，使得截面积和最大伤区 4可以与图像分析软件的帮助而获得。 </li><li>准备覆掌的神经立刻远端腕骨无菌注射麻药，以保证皮肤覆盖在肌腱和表面数字屈肌腱的完全脱敏的区域。注入2 ml的mepivicaine的屈肌腱之间既皮下和相邻的掌侧神经（在它们的子筋膜位置）和悬韧带立即远端在肢体上的两侧腕骨。 </li><li>一旦初步扫描已经执行通过用氯己定外科擦洗小号的区域准备植入部位至少5分钟crub溶液，最后用浸泡的纱布醇。请在无菌时尚所有后续步骤。 </li></ol>马间充质干细胞3锝 - 99m的标签注:可以在前肢的超声检查作为细胞标记和标记细胞植入之间，这样可以降低同位素衰变为肌腱期间进行的细胞标记的步骤。 <ol><li>从细胞中除去在BMS因为它与同位素的吸收干扰。要做到这一点，检索BMMSC从寒意箱小瓶（10百万个细胞在1毫升BMS）和颗粒细胞在离心350 XG 5分钟，在室温。小心取出用18G或19G针（连接到2毫升注射器）上清，留下一小滴（≤0.1毫升）在管，以帮助细胞悬浮。保存上清液（在BMS）在无菌的离心管并保持在冰上购买再利用。 </li><li>由FL悬浮细胞培养液中残余的液滴伊京管轻轻地用手指（而不是涡旋）和离开所述管在机架在RT。确保将细胞重悬于完全并且不存在任何可见的细胞团块。 </li><li>准备锝 - 99m的如下。传送1 ml的锝高锝成HMPAO的小瓶用1ml注射器和针头。轻轻混匀但彻底离开背后的铅屏蔽5分钟。注意:此步骤可以在将细胞在纺丝步骤2.1来完成。 </li><li>将所有使用1ml注射器和针头对细胞的锝 - 99m的-HMPAO混合物轻轻混匀。下室温温育30分钟，后面铅屏蔽。 </li><li>在接下来的步骤中，使用钳子（或一块薄纸）打开的离心管的盖，以尽量减少（戴手套）拇指同位素污染并随后其他设备。用2ml的注射器和21g的针来添加或删除的洗涤缓冲液。 注意:使用一个字段同位素监视器的鼓励监视表面和设备的污染。 </l我&gt; <li>加入1毫升的PBS的锝 - 99m的-HMPAO细胞混合，盖上盖子，轻轻混匀沉淀细胞如步骤3.1。 </li><li>小心取出PBS到一个新的管（保存此背后铅屏蔽和标签为W1）。重悬细胞沉淀在1ml新鲜的PBS。离心管，去除上清如上（这和标签保存为W2）。 </li><li>通过测量计数W1和W2管和在细胞沉淀计算标记的效率。通过将各管在一个良好的同位素剂量校准仪和记录的放射性每分钟（CPM）计数做到这一点。计算标记效率（如放射性细胞的CPM）/（放射性细胞+上清）×100。 </li><li>在剩余的PBS彻底重悬细胞，然后加入BMS保存步骤3.1。这些细胞是准备病灶内植入。 </li></ol> 4.注入锝 - 99m的-HMPAO标记的细胞<ol><li>病灶内植入为超声引导下的肌腱<ol><li>慢慢引入1.5或2英寸（50毫米）20G的针头插入病变的纵向方向与超声换能器，盖在无菌披盖的最大伤区，在线路与针，使针的长度可被超音波识别和尖端如果针头被准确地定位在病变的中心。 </li><li>用适当的屏蔽，收集细胞到2ml注射器（Luer锁紧套口，以尽量减少外部污染的风险，并在铅屏蔽注射器套）用20G的针。丢弃的针小心，不要失去任何液体​​。现在附加注射器与标记的细胞到针预先位于肌腱。 </li><li>放置下针的超声波探头，以便在所述组织的针道是清晰可见。注入细胞处于缓慢但稳定的步伐进入病灶。确保有注射无阻力。如果此遇到仔细重新定位超声引导下针。检查流体喷射到病灶的超声图像作为包含高回声气泡消声流体上可见。 </li><li>拔出针头，并立即放置压力在针孔，以防止注射细胞的损失，并尽量减少在肢的外表面同位素的传播。马上装扮肢体一层自粘合绷带。马现在已经准备好了γ闪烁。立即执行此。 注意:注射细胞进入腱后，取得空的注射器和针头的计数，以评估可能的直接损失细胞可能残留在注射器中。 </li></ol></li><li>锝 - 99m的-HMPAO标记的细胞局部灌注<ol><li>按照步骤2.4。 </li><li>在近端掌骨有两个百毫米棉卷纱布包扎定位内侧和外侧采用100mm的橡皮止血带包扎。 </li><li>无菌准备日e皮肤上在横近端籽骨的水平的横向侧指静脉通过用至少5分钟的洗必泰手术擦洗溶液擦洗区，最后用纱布浸泡于酒精。请在无菌时尚所有后续步骤。 </li><li>填写肝素化无菌生理盐水准备100毫米扩展设置了21国祥¾"（0.8×19毫米），蝴蝶针静脉穿刺，然后引入针头插入侧掌侧静脉。一旦静脉进入血液将部分填补扩展集。在鲁尔锁连接器连接鲁尔锁定橡胶帽便于以后的注射标记的细胞。 </li><li>通过收集所述细胞在一个20毫升注射器预装的PBS稀释19毫升的PBS中的MSC。 （ - 40秒30），通过使用18克（1.2×40 MM）皮下注射针在缓慢而稳定的速度皮碗应用细胞。冲洗导管用1毫升的PBS预装在一个syring即</li><li>取出针，并立即将压力施加在皮肤上有一层5 4×4棉花纱布针孔。然后将一个光绷带用弹性粘结剂在近端籽骨面积和留在原地止血带为注射后30分钟。 </li><li> 30分钟后，松开止血带。马现在已经准备好了γ闪烁。 注意:注射细胞后，将获得的空的注射器和针头的计数，以评估可能的直接损失细胞可能残留在注射器中。 </li></ol></li></ol> 5.γ闪烁<ol><li>显像图像<ol><li>获得平面闪烁图像用γ照相机（设定为140千电子伏光电峰使用20％对称窗口）配备了低能量，平行孔准直仪。 <ol><li>获取时间为0后，所有图像上站在马注射镇静剂如步骤2.2。时间0闪烁图后，更换绷带光在植入SI忒带衬垫的绷带。删除之前，每个显像检查绷带。 </li><li>获取静态图像使用处理软件的"取得"60秒（每256 256矩阵） - "研究的选择" - "骨静态"的选项。至关重要的是，马并不在图像采​​集期间移动，因为静态模式选项没有用于校正运动的模块。 </li><li>为病灶内注射，获得从病变区域从腕骨横向图像到远侧末端，对侧肢体，左肺字段和左甲状腺的等效面积。在采集前肢图像，定位两腿之间铅屏，以避免肢体对侧的成像。获得在5分钟的喷射图像（时间0）后，再在1，3，6，12，24，36和48小时。 </li><li>对于局部灌注，获得外侧和足背伽玛图片为5.1.1.3。获取图像5分钟自动对焦之三止血带释放。注入细胞，以评估止血带释放之前计数后立即这将是时间0。它可以是有用的图像的肢体。得到的γ图像在细胞给药后1，3，6，12，24，36和48小时。 </li></ol></li><li>图像已被获取后，过程中手动使用具有选择为"Sokoloff"色彩和"反转"颜色选项软件的"处理"选项的图像。接下来选择"感兴趣区域"（ROI）和"椭圆形"，因为这可以让选择掩码和运动在病变区的重塑。通过选择"添加到统计"选项获得了投资回报率的计数。确保的感兴趣区（ROI），该相同大小的区域记录每个动物在不同时间点。 </li><li>接着，校正锝的在每个时间点所预测的衰变计数。使用以下方程来校正衰减:A = A 出 O E - （0.693吨/ T 1/2），其中A o为同位素的初始活性，A是衰减校正的放射性在零时间，t是经过时间和T 1/2是半 - 生命的同位素。然后表示在每个时间点作为ROI的一个百分比的校正计数在时间0制定出剩余的，使用下面的公式细胞的百分比: </li></ol></li></ol> ％细胞其余（吨）= 100 - {[（预测衰变（吨） - 衰变（吨））/预测衰变（吨）]}×100 衰减（T）=投资回报率（T）/ ROI（0）×100。

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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=Evaluation+of+mesenchymal+stem+cell+migration+after+equine+tendonitis+therapy.">Evaluation of mesenchymal stem cell migration after equine tendonitis therapy.</a> Equine Veterinary Journal. 46, 635-638 (2014).</li><li>Welling, M. M., Duijvestein, M., Signore, A., van der Weerd, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+biodistribution+of+stem+cells+using+molecular+nuclear+medicine+imaging.">In vivo biodistribution of stem cells using molecular nuclear medicine imaging.</a> Journal of Cellular Physiology. 226, 1444-1452 (2011).</li><li>Dowling, B. A., Dart, A. J., Hodgson, D. R., Smith, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superficial+digital+flexor+tendonitis+in+the+horse.">Superficial digital flexor tendonitis in the horse.</a> Equine Veterinary Journal. 32, 369-378 (2000).</li><li>Kasashima, Y., Ueno, T., Tomita, A., Goodship, A. E., Smith, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimisation+of+bone+marrow+aspiration+from+the+equine+sternum+for+the+safe+recovery+of+mesenchymal+stem+cells.">Optimisation of bone marrow aspiration from the equine sternum for the safe recovery of mesenchymal stem cells.</a> Equine Veterinary Journal. 43, 288-294 (2011).</li><li>Avella, C. S., 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=Ultrasonographic+assessment+of+the+superficial+digital+flexor+tendons+of+National+Hunt+racehorses+in+training+over+two+racing+seasons.">Ultrasonographic assessment of the superficial digital flexor tendons of National Hunt racehorses in training over two racing seasons.</a> Equine Veterinary Journal. 41, 449-454 (2009).</li><li>de Vries, E. F., Roca, M., Jamar, F., Israel, O., Signore, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+labelling+of+leucocytes+with+(99m)Tc-HMPAO.+Inflammation/Infection+Taskgroup+of+the+European+Association+of+Nuclear+Medicine.">Guidelines for the labelling of leucocytes with (99m)Tc-HMPAO. Inflammation/Infection Taskgroup of the European Association of Nuclear Medicine.</a> European Journal of Nuclear Medicine and Molecular Imaging. 37, 842-848 (2010).</li><li>Trela, J. 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=Scintigraphic+comparison+of+intra-arterial+injection+and+distal+intravenous+regional+limb+perfusion+for+administration+of+mesenchymal+stem+cells+to+the+equine+foot.">Scintigraphic comparison of intra-arterial injection and distal intravenous regional limb perfusion for administration of mesenchymal stem cells to the equine foot.</a> Equine Veterinary Journal. 46, 479-483 (2014).</li><li>Barbash, I. 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=Systemic+delivery+of+bone+marrow-derived+mesenchymal+stem+cells+to+the+infarcted+myocardium:+feasibility,+cell+migration,+and+body+distribution.">Systemic delivery of bone marrow-derived mesenchymal stem cells to the infarcted myocardium: feasibility, cell migration, and body distribution.</a> Circulation. 108, 863-868 (2003).</li><li>Heckl, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Future+contrast+agents+for+molecular+imaging+in+stroke.">Future contrast agents for molecular imaging in stroke.</a> Current Medicinal Chemistry. 14, 1713-1728 (2007).</li></ol>

JD and RKS are Scientific Advisory Board members to ReCellerate Inc.

除了骨髓，干细胞等来源的脂肪组织中分离适合与此协议标签。此外，从冷冻状态的细胞可以恢复并在培养中扩增到所需的号码标记研究12。 用于确定标记所述BMMSC的效率的一个关键因素是锝 - 99m的从钼发生器的洗脱之间的时间在放射性药物，制备锝 - 99m的-HMPAO和使用在临床的放射性药物。有从发电机，使得超出此期间延迟可显著损害细胞标记的效率锝 - 99m的洗脱?...

对于患病或受损组织的修复再生策略基于来自于多种组织和注入到患部的多能干细胞。自体BMMSC的肌腱和韧带受伤的马治疗应用的最新进展表明在这两个实验1-5改善成果的措施和临床研究6。马是一个特别有吸引力的模型来评估干细胞的相关治疗的功效，因为它遭受年龄和过度紧张有关的伤害到远侧前肢的筋，它是一个运动动物，而且它是一个大的，促进骨髓恢复和准确植入。肌腱损伤自然愈合与纤维化，但愈合肌腱功能逊色7并且有再度受伤8的高风险。浅数字屈肌腱（SDFT）最常受到影响，因为它已演变作为弹性能存储和经验的高负荷强调，实现节能和高速运动。伤后恢复功能，因此，至关重要。这些损伤是类似于那些影响跟腱在人中它执行类似的功能9。有没有好的治疗方案治疗或达到良好的修为这种伤害，因此基于细胞的再生战略提供了一个有吸引力的机会，改善预后，减少再损伤。 在大多数研究中5年 - 2000万元自体BMMSC直接注入这通常发生在腱体，其因此用作容器的细胞的芯内的病变。一旦注入细胞的命运不明确和不同的细胞标记方法来跟踪单元已经被最近描述。细胞标记有荧光标记示生存只在相对 ​​较小的数量（&lt;10％）10,11。荧光标记必要组织提取和切片用于组织学分析这是耗时和不容易促进在大型动物模型或在临床病例时间分析。在最近的工作中，我们使用了放射性同位素99m锝标记细胞，并按照他们的命运由γ闪烁1。此方法允许细胞递送的不同途径，包括病灶内，经颈静脉或1区域性灌注静脉内经由动脉12或静脉内注射1,12之间进行快速比较。细胞的持久性和分布然后通过各个器官的γ闪烁成像。这已经证明，只有24％的细胞注射的病灶内留在病变24小时1，这是支持通过使用实验产生的损伤，并使用相同的放射性标记5的另一项研究。此外，细胞表现出的能力，家里有限的肌腱病变时delivere改发局部灌注或静脉内而是被分散进入肺部受到后者的路线4。 BMMSC标记铁纳米颗粒是一种替代的方法来跟踪细胞植入到前肢筋13。虽然铁纳米颗粒标记的细胞允许在体内通过MRI细胞跟踪，在大型动物颞研究是由麻醉可以在用于执行磁共振成像扫描每个时间点进行给药的次数的限制。此外，铁纳米颗粒是低信号的MRI这限制了对标记细胞的迁移进入腱体中的信息。可以使用的其它放射性同位素包括铟-111但这遭受更长的半衰期比锝 - 99m的的缺点（2.8天比6.0小时）和较高的γ射线发射能量。此外，细胞存活率已报道可以减少当与铟-111 14标记。锝 - 99m的，另一方面，是经常在两个马和人核用药品标签外周血单个核细胞，并按照其在体内的分布显像。它可以相对容易地采取由使用HMPAO作为连接分子结合的锝细胞，在Tc-99米-HMPAO，至细胞。锝- 99m的-HMPAO标记BMMSC显示出良好的可行性和可在体外增殖4。这个协议的细节注入自然发生病变的前肢SDFT标签和马自体BMMSC的跟踪。 要注意，该协议仅用于被用作研究工具是重要的。其作为临床治疗方法不推荐使用作为上的细胞表型的放射性标记还没有得到完全阐明的效果。

<table><tbody><tr><td>Technetium&lt;sup&gt;99m&lt;/sup&gt;&amp;nbsp;</td><td></td><td></td><td>Please enquire with local ionisation radiation supplier in accordance with legal requirements.&amp;nbsp; The isotope must be used within 2 hr of elution from the molybdenum-99 generator</td></tr><tr><td>Ceretec - Hexamethylpropyleneamine oxime (HMPAO)&amp;nbsp;</td><td>GE HealthCare</td><td></td><td>Please enquire directly with GE HealthCare</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Microfuge, Minispin/Minispin Plus</td><td>Ependorf</td><td>22620100</td><td></td></tr><tr><td>18 G and 19 G Needles</td><td>Terumo Medical</td><td>NN-1838R (18 G);&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; NN1938R (19 G)</td><td></td></tr><tr><td>Syringes 1 ml and 2 ml</td><td>Scientific Laboratory Supplies Ltd</td><td>SYR6200 (1 ml); SYR6003 (2 ml)</td><td></td></tr><tr><td>Microcentrifuge tubes 1.5 ml</td><td>Greiner Bio-One Ltd</td><td>616201</td><td></td></tr><tr><td>PBS - Phosphate-Buffered Saline</td><td>LifeTechnologies</td><td>14190</td><td></td></tr><tr><td>Sterile Gauze Swabs</td><td>Shermond Ltd</td><td>UNG602</td><td></td></tr><tr><td>CoflexVet self adhering bandage</td><td>Andover Healthcare, Inc.</td><td>3540RB-018</td><td></td></tr><tr><td>Ultrasound imaging software</td><td>Scion Image, Scion Corporation, USA</td><td></td><td></td></tr><tr><td>MicasXplus Scintigram processing software</td><td>Bartec Technologies Ltd</td><td></td><td>http://www.bartectechnologies.com/veterinaryscintigraphy.html</td></tr><tr><td>Field isotope counter for monitoring isotope</td><td>John Caunt U.K.</td><td>GMS1800a</td><td>http://www.johncaunt.com/</td></tr><tr><td>Well counter for isotope measurements, dose calibrator</td><td>Capintec Southern Scientific</td><td>CRC-25R</td><td></td></tr></tbody></table>

in vivo imaging and tracking of technetium-99m labeled bone marrow mesenchymal stem cells in equine tendinopathy

骨髓间充质干细胞（BMMSC）的肌腱和韧带受伤的马的治疗应用的最新进展表明，在实验和临床研究改善结果的措施。虽然BMMSC注入到肌腱病变大量（通常为10 - 20个百万细胞），只有相当少数存活（&lt;10％），尽管这些能持续植入后长达5个月。这似乎是在其他物种的共同观测其中BMMSC已注入到其它组织和重要的是要理解，当这个损失发生时，有多少生存最初植入过程和细胞是否被清除到其他器官。跟踪细胞的命运可以通过放射性标记的BMMSC在植入前，允许非侵入性的细胞的位置和细胞数的定量体内成像来实现。 本协议描述的小区labeliNG过程使用锝-99m（TC - 99m的），而这些细胞植入后追踪到马受伤的肌腱。锝- 99m的是一个短暂的（吨1/2 6.01的小时）同位素发射的γ射线，并且可以通过细胞的亲脂性化合物hexamethylpropyleneamine肟（HMPAO）的存在下内化。这些特性使其非常适合在核医学诊所使用了很多不同的疾病的诊断。标记的细胞的命运可以遵循在短期内（长达36小时）通过γ闪烁来量化细胞保留在病灶和细胞分布到肺，甲状腺和其它器官的两个数。该技术适于从血液中的白细胞的标签和可用于图像中的其它器官植入BMMSC。

锝- 99m的-HMPAO掺入BMMSs并不其粘附到组织培养塑料能力产生不利影响，而它们显示出扩散以形成单层的能力（图1），我们还没有完全确定的增殖率或其他细胞表型是否会受到影响。其形态类似于未标记细胞具有典型的纺锤形。蜂窝标记效率（即，摄取标签）通常变化约1.5％至25％。的主要原因的低标记率与在交付99m锝的诊所从其中的Mo-99 /锝- 99m的发电机已洗脱站点的延?...

Watch this Scientific Journal Video about In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy at JoVE.com

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

在锝-99m在马腱末端标记的骨髓间充质干细胞的体内成像和跟踪

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

in-vivo-imaging-tracking-technetium-99m-labeled-bone-marrow

Research

JoVE Journal

Medicine

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

9.7K 次观看.  The Royal Veterinary College. This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

视频: In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

氧化铁标记的胚胎干细胞和的hMSCs纳米粒子在体内的跟踪与非侵入性磁共振成像

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

科研

生物学

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

利用磁共振成像，容积电脑断层扫描及超音波的裸鼠乳腺癌骨转移大鼠模型血管生成的多模态成像

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

医学

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the efficacy of cell persistence and biodistribution of haMSCs in animal models. We demonstrated a method to label the cell membrane of haMSCs with lipophilic fluorescent dye. Subsequently, intra-articular injection of the labeled cells in rats with surgically induced KOA was monitored dynamically by an in vivo imaging system. We employed the lipophilic carbocyanines DiD (DilC18 (5)), a far-red fluorescent Dil (dialkylcarbocyanines) analog, which utilized a red laser to avoid excitation of the natural green autofluorescence from surrounding tissues. Furthermore, the red-shifted emission spectra of DiD allowed deep-tissue imaging in live animals and the labeling procedure caused no cytotoxic effects or functional damage to haMSCs. This approach has been shown to be an efficient tracking method for haMSCs in a rat KOA model. The application of this method could also be used to determine the optimal administration route and dosage of MSCs from other sources in pre-clinical studies.

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

This protocol describes an efficient way to monitor the cell persistence and biodistribution of human adipose-derived mesenchymal stem cells (haMSCs) by far-red fluorescence labeling in a rat knee osteoarthritis (KOA) model via intra-articular (IA) injection.

在体内荧光亲脂膜染料对大鼠膝骨关节炎模型中人脂肪间充质干细胞的追踪

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

发育生物学

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105 cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

小动物的肌肉植入动态跟踪在体内荧光成像

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

顺序<em&gt;在体内</em&gt;成骨干/祖细胞在骨折修复的影像学

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

跟踪小鼠骨髓单核细胞<em&gt;在体内</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

免疫与感染

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.&#160; Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.

This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy.

CCL5诱导小鼠骨膜骨骼干细胞迁移的实时成像

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

超声引导下人骨髓间充质干细胞在实验性肠炎小鼠模型中的应用

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical hurdles such as invasive cell delivery and tracking with low transplantation efficiency remain challenges in translational stem-based therapy. This article describes an emerging technique for stem cell labeling and tracking based on the labeling of the mesenchymal stem cells (MSCs) with superparamagnetic iron oxide (SPIO)&#160;nanoparticles, as well as intranasal delivery of the labeled MSCs. These nanoparticles are fluorescein isothiocyanate (FITC)-embedded and safe to label the MSCs, which are subsequently delivered to the brains of TBI-induced mice by the intranasal route. They are then tracked non-invasively in vivo by real-time magnetic resonance imaging (MRI). Important advantages of this technique that combines SPIO for cell labeling and intranasal delivery include (1) non-invasive, in vivo MSC tracking after delivery for long tracking periods, (2) the possibility of multiple dosing regimens due to the non-invasive route of MSC delivery, and (3) possible applications to humans, owing to the safety of SPIO, non-invasive nature of the cell-tracking method by MRI, and route of administration.

Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide&#160;nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

跟踪超副磁性氧化铁标记的美感血干细胞使用MRI后，创伤性脑损伤Murine模型内伤分娩

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...