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>Technetium99m&#160;</td>
			<td></td>
			<td></td>
			<td>Please enquire with local ionisation radiation supplier in accordance with legal requirements.&#160; The isotope must be used within 2 h of elution from the molybdenum-99 generator</td>
		</tr>
		<tr>
			<td>Ceretec - Hexamethylpropyleneamine oxime (HMPAO)&#160;</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>18G and 19G Needles</td>
			<td>Terumo Medical</td>
			<td>NN-1838R (18G);&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; NN1938R (19G)</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

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

酪氨酸激酶抑制剂的临床前评价急性白血病的治疗

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

科研

医学

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

在体内 19女性 MRI对细胞跟踪

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

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 ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

股骨骨髓的愿望在活体小鼠

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

从小型猪关节液和骨髓间充质干细胞的分离和细胞表型

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Nerve Stimulator-guided Injection of Autologous Stem Cells Near the Equine Left Recurrent Laryngeal Nerve

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent laryngeal nerve shows lesions of demyelination. The benefit of applying stem cells to demyelinated nerves has been demonstrated in various animal models. The aim of the study was to test the feasibility and safety of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to the left recurrent laryngeal nerve in healthy horses by using an electrical nerve stimulator.
Muscle-derived stems cell are obtained from five healthy Standardbred horses by sampling 20 mg of muscle tissue with a semi-automatic 14 G biopsy needle from the triceps muscle. Movements of the larynx are monitored via upper-airway video endoscopy. The left recurrent laryngeal nerve is approached with an insulated nerve block needle. Nerve stimulation is applied, starting at 2 mA, and the successful abduction of the left arytenoid is monitored. The stimulation intensity is reduced progressively. When a loss of the motor response is observed at 0.5 mA, 107 autologous muscle-derived stem cells are injected. Two examiners, who are blinded to the time point, score the laryngeal function of the horses prior to the treatment and at day 1, day 7, and day 28 after the injection of the cells. In a sixth horse, 1 mL of 2% lidocaine is injected to further confirm the correct positioning of the needle. This leads to a temporary paralysis of the left arytenoid cartilage.
This study proves that the recurrent laryngeal nerve can be approached with the help of an electrical nerve stimulator and that the electrical stimulation of the nerve is well tolerated by the horses. No modification of the laryngeal function was observed in any of the horses after the injection of the stem cells. Further studies should be conducted to describe the effects of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to horses suffering from RLN.

Here, we present a protocol to inject autologous muscle-derived stem cells in the proximity to the recurrent laryngeal nerve with the help of an electrical nerve stimulator. This novel technique may become useful for the treatment of equine recurrent laryngeal neuropathy.

神经刺激器引导注射自体干细胞近马左喉返神经

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent ...

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing continuous remodeling with the competing activity of osteoblasts, which produce the new bone matrix, and osteoclasts, whose function is to eliminate mineralized bone. [18F]-NaF is a positron emission tomography (PET) radiotracer that enables visualization of bone metabolism. [18F]-NaF is chemically absorbed into hydroxyapatite in the bone matrix by osteoblasts and can thus noninvasively detect osteoblastic activity, which is occult to conventional imaging techniques. Kinetic modeling of dynamic [18F]-NaF-PET data provides detailed quantitative measures of bone metabolism. Conventional semi-quantitative PET data, which utilizes standardized uptake values (SUVs) as a measure of radiotracer activity, is referred to as a static technique due to its snapshot of tracer uptake in time.&#160; Kinetic modeling, however, utilizes dynamic image data where tracer levels are continuously acquired providing tracer uptake temporal resolution. From the kinetic modeling of dynamic data, quantitative values like blood flow and metabolic rate (i.e., potentially informative metrics of tracer dynamics) can be extracted, all with respect to the measured activity in the image data. When combined with dual modality PET-MRI, region-specific kinetic data can be correlated with anatomically registered high-resolution structural and pathologic information afforded by MRI. The goal of this methodological manuscript is to outline detailed techniques for performing and analyzing dynamic [18F]-NaF-PET-MRI data. The lumbar facet joint is a common site of degenerative arthritis disease and a common cause for axial low back pain.&#160; Recent studies suggest [18F]-NaF-PET may serve as a useful biomarker of painful facetogenic disease.&#160; The human lumbar facet joint will, therefore, be used as a prototypical region of interest for dynamic [18F]-NaF-PET-MRI analysis in this manuscript.

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. We present detailed methodologies for performing and analyzing dynamic [18F]-NaF-PET-MRI data in a patient with facetogenic low back pain using the lumbar facet joints as a prototypical region of interest.

定量 =18F_-Naf-PET-MRI 分析,用于评估面部腰痛患者的动态骨质周转率

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.