The authors thank Prof. Jan Grimm and Mr. Travis Shaffer for their helpful discussions and Mr. David Gregory for editing the manuscript. Technical services provided by the MSK Animal Imaging Core Facility, supported in part by NIH Cancer Center Support Grant P30CA008748-48, are gratefully acknowledged. The authors thank the NIH (K25 EB016673 and R21 CA191679, T.R. and 4R00CA178205-02, B.M.Z.), the MSK Center for Molecular Imaging and Nanotechnology (T.R.), the Tow Foundation (B.C.), and the National Science Foundation Integrative Graduate Education and Research Traineeship (IGERT 0965983 at Hunter College for B.C. and T.M.S.) for their generous support. The research reported in this publication was supported by funding from the King Abdullah University of Science and Technology.

伦理学声明:以上所描述的，根据经批准的协议，并在纪念斯隆-凯特琳癌症中心（MSK）机构动物护理和使用委员会（IACUC）的道德准则进行体内动物实验。 1.一雾化装置的构建<ol><li>木连接部分A（12.5×2.5×1.8 cm 3）的直立用两个螺丝（4×25 平方毫米）B部分（12.7×10.7×1.8 立方厘米 ）的中心。附上木质部-C（11×2.5×1.8 毫升 ），以利用一个螺钉，使得C部分（11×2.5×1.8 毫升 ）仍可被移动部A（12.5×2.5×1.8 毫升 ）的中间。 见图1。 </li><li>钻两个孔通过塑料3盎司微型喷雾器（D）的喷射瓶扳机的下部末端，并按下一个不锈钢棒（10厘米的1/16"钢）（E）通过，以形成两个环，一个在任一触发器的一侧。 </l我&gt; <li>包住喷射瓶用胶带（F）的底部，以防止滑出扎带。附加喷雾瓶使用两个塑料扎带木质部C（11×2.5×1.8 毫升 ）（28厘米）（G）。 </li><li>切断011伺服电机（Ⅰ）中，用伺服控制（H）的松散的电缆重新连接。然后，伺服电机连接至用胶带木质部A（12.5×2.5×1.8 毫升 ）的顶部。 </li><li>附加铅笔（J）使用回形针（K）伺服电机控制杆。紧密连接的铅笔用塑料盖扭线（L）钢棒循环的最外层部分和安全的用胶带铅笔的末端。 </li><li>切断伺服电机控制单元的磁电缆连接器（M），并将其重新连接到扬声器连接线（N）。然后，伺服电机控制部胶带到木质部B（12.7×10.7×1.8 毫升 ）。切断与磁性连接器的W1的电缆在一半和附加一个部分在铜S的松散端调峰电缆（1米）。连接（磁）12拨动开关和P1的电源可用W1电缆和一个9V电池。 </li></ol> 2.方法的灵敏度测定<ol><li>在1.5毫升离心管中，在260微克（347纳摩尔），52微克（69纳摩尔），26微克（34纳摩尔）金额反渗透水（100微升）准备[茹（联吡啶）3] Cl 2的的解决方案，5微克（6.9纳摩尔），3微克（3.5纳摩尔），520纳克（694皮摩尔），260纳克（347皮摩尔），52纳克（69皮摩尔），26纳克（34皮摩尔），5毫微克（6.9皮摩尔）和3纳克（3.5皮摩尔）。 </li><li>混合100μL的每个的[Ru（联吡啶）3] Cl 2中的溶液与100微升硝酸铵铈的水溶液（（NH 4）2的Ce（NO 3）6）在显微镜载玻片上的水（25毫摩尔）。 </li><li> 通过初始化成像软件建立在生物发光读者收购。 <ol><li>登录到用户配置文件，并寻找阿奎习得的控制面板。点击"初始化"，等到仪器准备。 </li><li>寻找"拍摄模式"，并确保"发光"和"照片"被选中，而"荧光"是没有选。 </li><li>改变"曝光时间"为"发光"设置为20秒。设置"发光"其余的设置如下:"分档":中等; "F /停止":1;和"排放过滤器":打开。 注:曝光时间可能需要根据仪器和所用的实验设置，可以适于，如果从呈现的设置不同。 </li><li>对于"照片"，请使用以下设置:"接触时间":自动; "分档":中等;和"F /停止":8.对于"视场"下拉菜单成像target.Look调整"主题高度"。初始设置为"C"。 C在更改为"B"（14厘米的距离相机和样品台）。 </li></ol></li><li> 通过将显微镜载玻片上黑施工纸张上成像室以保护其免受氧化剂的地板的片材设置的喷雾器。混合100μLdroplet的[茹（联吡啶）3] Cl 2的-solution用100微升的水溶液（NH 4）2铈（NO 3）6.观察绿框十字线。 <ol><li>放置在黑色施工纸成像对象，使得所感兴趣的区域是在绿色光盒瞄准器的中心显示在样品台上。由木支持卸下塑料喷雾瓶准备喷雾器。补的三乙胺中的溶液（1:3的水/乙醇）成其塑料容器和其重新安装到木支撑。 </li><li>将生物发光读取器内部雾化器，确保电源线从电源线雾化器断开。确保电源开关是，拨动开关处于关闭状态，红色LED亮起。放置喷雾器，使得喷射流指向朝向上的成像对象感兴趣的区域，同时尽量减少从摄像机朝向由喷嘴头的成像对象的视图阻塞。 </li><li>将在任何潜在的热点黑色小片建筑用纸（如白色的痕迹在显微镜载玻片或注射部位），从喷雾保护他们。地点的至少40厘米的成像室内部的喷雾器遥控线的，使得它不与成像对象，喷雾器，或磁性门闩干扰。关闭成像系统的门。 注意:十字线将根据"视场"改变大小设置"生活图片;"确保此设置为"B"。 </li></ol></li><li>通过启动成像序列获取图像。点击"获取的"中"采集控制面板"。在第一摄像色曲ENCE，如果需要的话（推荐）使自动保存，并选择一个数据文件夹。忽略"编辑影像标签"对话直到序列的结束。 注:控制软件显示仪表的行动一步一步的实时性。制备测量和移动所述样本台，以合适的位置之后，它打开相机快门和计数测量时间。活门开启也可以通过由机器产生的咔嗒声听到。 </li><li>通过切换切换开关三次，以产生化学发光:在快门打开时，喷三乙胺的溶液的三连发（在水/乙醇3，每喷突发0.24±0.04毫升1）。 注:样品阶段将在测量过程中移动。留出足够的（最少40厘米）的电缆在仪器内部，让这一点。确保该溶液通过喷雾器喷洒可由上升管被渴望并有在管道中没有气泡。有几个备用BATTERIES为喷雾器准备需要的情况下。 </li></ol> 3. 在体内成像全身静脉注射后<ol><li>在1.5毫升微量离心管中，制备100微升含有的[Ru（联吡啶）3] Cl 2中的8和33之间纳摩尔磷酸盐缓冲盐水（PBS）溶液。制备的水溶液（NH 4）2的Ce（NO 3）中的同时用水（25毫摩尔）6。 </li><li>静脉内注射100微升的[Ru（联吡啶）3] Cl 2中成健康小鼠的尾静脉中（N = 5）。 </li><li> 通过CO 2窒息注射后安乐死的小鼠10分钟。 <ol><li>从躯干Y切割取下皮肤，然后取出肋弓的U形，露出心脏和肺部。通过一个24号针头在右心房切割的出口和注入20毫升的PBS到左心室18灌注小鼠。通过腹部经过精心切割皮肤和揭露肾脏和肝脏。纵向切开通过机关创建一个可见的削减。 </li></ol></li><li> 如步骤2.3-2.6中所述，做出了以下改变设置的采集。 <ol><li>彻底冲洗喷雾器塑料储之后，具有的（NH 4）2的Ce（NO 3）6在水代替三乙胺溶液（25毫摩尔）填充。 注意:要彻底冲洗每次使用后喷嘴雾化器，因为结晶是很重要的（NH 4）2铈（NO 3）6可经过多次使用破坏喷嘴。 </li></ol></li><li> 使用整个动物或器官样品进行成像。 <ol><li>对于全腹成像，定位与开腹面对相机，指着仪器的后脑勺鼠标尸体。居中机关在绿色灯箱十字线成像（ 例如，肝脏或肾脏）。 </li><li> F或单个器官成像和量化，从成像仪器中取出鼠标和针下来。从已经开体腔开始，切除内部器官（ 例如，肾，肝，肺，肌肉，脾脏，脑，和心脏）。通过后腿皮肤切开切除的肌肉组织。小心打开颅骨用手术刀切除大脑。 <ol><li>如果感兴趣的器官是肝脏，肾脏，脾，切成两半所有器官纵向，将每个器官的培养皿或一块黑色的建设纸。 </li></ol></li><li>按照步骤2.3-2.6建立单个器官的化学发光示踪剂的相对发射描述的过程。 </li></ol></li></ol> 4.淋巴结体内成像<ol><li>制备含有80纳摩尔的[Ru（联吡啶）3] Cl 2中的一个的PBS溶液10μL。制备的水溶液（NH 4）2的Ce（NO 3）6在华之三（25毫米）在同一时间。 </li><li>注入溶液10μL皮下到健康小鼠的后爪（N = 5）。作为阴性对照，注射用纯净的PBS10μL的对侧爪。注射后通过二氧化碳窒息牺牲小鼠15分钟。在两条后腿去除皮肤暴露淋巴管至腘窝淋巴结。 </li><li>如步骤3.4所述设置了收购。 </li><li>从两个后腿除去腘淋巴结，切断他们在一半，并与氧化剂喷他们上的培养皿，如之前（步骤3.5.3）中描述的，用于定量的目的。 </li></ol>

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Protoc. 9 (5), 1146-1159 (2014).</li><li>B&#339;uf, G., 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=Encapsulated+Ruthenium(II)+Complexes+in+Biocompatible+Poly(d,l-lactide-co-glycolide)+Nanoparticles+for+Application+in+Photodynamic+Therapy.">Encapsulated Ruthenium(II) Complexes in Biocompatible Poly(d,l-lactide-co-glycolide) Nanoparticles for Application in Photodynamic Therapy.</a> ChemPlusChem. 79 (1), 171-180 (2014).</li><li>Loizidou, M., Seifalian, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotechnology+and+its+applications+in+surgery.">Nanotechnology and its applications in surgery.</a> Brit. J. Surgery. 97 (4), 463-465 (2010).</li><li>Barry, N. P. E., Sadler, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+for+Metals+in+Medicine:+How+Nanotechnology+May+Help+To+Shape+the+Future.">Challenges for Metals in Medicine: How Nanotechnology May Help To Shape the Future.</a> ACS Nano. 7, 5654-5659 (2013).</li><li>Hasan, K., Bansal, A. K., Samuel, I. D. W., Rold&#225;n-Carmona, C., Bolink, H. J., Zysman-Colman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+the+Emission+of+Cationic+Iridium+(III)+Complexes+Towards+the+Red+Through+Methoxy+Substitution+of+the+Cyclometalating+Ligand.">Tuning the Emission of Cationic Iridium (III) Complexes Towards the Red Through Methoxy Substitution of the Cyclometalating Ligand.</a> Nat.Sci. 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The authors have nothing to disclose.

这里，我们已经提出了一种技术，能够通过由化学发光记者创建的光子的发射光学划定组织。相对于其他，更成熟的，技术4，5，6，7，8，9，此化学发光报道系统采用了成像探针是无放射性和在非常高的灵敏度水平便于检测。或许更重要的是，化学发光成像不需要入射光源（如在光学荧光成像）20，即最大限度地减少自体荧光和大大减少背景信号的性状。 钌记者的[Ru（联吡啶）3] Cl 2中有一个体内 毒性容忍的成像目的（INTraperitoneal小鼠的LD 50:20毫克/公斤）21，是水溶性的（高达8毫摩尔），并且是稳定的血液中的。金属络合物的物理化学性质是充分表征的，并且已经被研究用于癌症22,23的光动力学治疗。氧化剂（NH 4）2的Ce（NO 3）6已经报道具有非常低的毒性（大鼠口服LD 50:1600-3200毫克/千克）24和可溶于水的浓度高达2.57 M达20 °ç25。在这篇文章中，对于一个远程操作喷雾装置的结构的视觉显示以及基于文本的指导介绍。此外，我们还提供了在一个标准的生物发光成像设备中执行的化学发光成像健壮的协议。我们说明了他们对visualizati使用[茹（联吡啶）3] Cl 2的中对小鼠静脉注射两种皮下和注射后的组织中。 然而，与其他任何新生的成像技术，有改善的余地的我们的协议。我们相信，验证的原则，本研究可以刺激多种化学发光应用的发展为生命系统。以下几点是可以解决的，进一步提高了技术和扩大其范围。 一个较小的第二代远程触发喷射装置的将允许试样以更接近相机，从而改善空间分辨率。改进的光学设备可能进一步提高了该方法的检测限。该协议也可以扩展到成像活体动物。转矩（由电流和电压）的精确控制允许试剂与每个喷释放的量的更精确的控制。重要的是保持良好的维护，雾化器是很重要的。不冲洗雾化器可能会破坏nozz勒。新电池是雾化器的正常性能是至关重要的。然而，用于该雾化器的所有材料是便宜的和容易购得。以下成立合成方案中，使用[Ru（联吡啶）3] 2+复合物可以很容易地与各种接头修饰，包括马来酰亚胺26，胺27和NHS酯28，29。这将使生物缀小分子，肽，或抗体，并因此将有利于特定分子靶向30，31，32，33。最终，有针对性的探针递送可以使外科医生来确定小病灶并准确地描绘在手术室手术切缘具有非常高的特异性。另外，高度水溶性的[Ru（联吡啶）的封装3] 2+在纳米材料，有针对性和不相关的，也可以允许病变的可视化，而他们是被手术切除34，35，36。最后，修改所述金属络合物记者的协调范围和/或改变的过渡金属中心本身代表吸引力路线内的可见调制和微调发射波长和近红外范围37,38。 术中的化学发光成像需要化学发光记者和，在我们的情况下，氧化剂，这只能它们的毒性和溶解度的范围内使用。组织膜可以表示为氧化剂的扩散进入组织的屏障，因此，信号生成。由于化学发光报道只产生每个周期一个光子，所生成的信号是相当弱。因此在手术室环境光将必须从进入照相机，而技术是使用防止。这可能使ICI特别有趣的腹腔镜应用中，环境光自然排除的发展。 我们希望，这种方法可能会变成在手术室外科医生的宝贵工具。没有放射性的是病人和运营团队都有益，使更少的安全防护措施，有可能使这一技术成为一个更具吸引力的选择。 雾化器的运行平稳，其定位发挥获得了良好的效果至关重要的作用。次优的角度和领域可能会导致信号变化。控制电缆必须通过门放小心，和足够电缆必须保持在生物发光读者内部，使得它不拥挤或撕掉。 最终，chemiluminescence成像是一个极具吸引力的新方法，分子成像。它是基于既定化学的基础上，采用了价格低廉，容易获得的材料，并避开辐射和激发光源。这样一来，我们都希望并相信在未来，化学发光成像可能对手术治疗的疾病产生深远的影响。

在近几十年中，成像技术已经彻底改变，医生诊断和监测疾病的方法。这些成像技术，然而，已经在很大程度上仅限于全身成像系统，例如正电子发射断层扫描（PET），单光子发射计算机断层摄影（SPECT），计算机断层扫描（CT）和磁共振成像（MRI）。特别注意了癌症，而技术突破成像有很大的提高，这种病诊断和治疗的方式。尽管有这些进展，但是一个地方，这些成像技术只是不适合:手术室。虽然全身成像技术可在手术计划帮助下，他们通常缺乏空间分辨率足够高，以帮助医生实时确定是否所有的肿瘤组织已被删除或残留的肿瘤组织仍然在手术切缘1隐藏。确保无浸润肿瘤边缘被留下是最重要的手术目标之一，和外科医生必须走严格而谨慎的组织切除之间的紧绳子。如果太多被去除，不需要的副作用对患者正在加剧;如果太少被删除，复发率增加2，3。因此，划定精确的肿瘤边缘是至关重要的，而且我们相信，化学发光成像术可通过帮助医生形象化恶性组织，否则会没有被发现已建立的技术，以提高肿瘤边缘的识别精度。 目前正在调查其可能的实用程序，术中成像系统的许多影像技术。这些包括β-和γ射线发射探头4，光学荧光5，拉曼光谱6 </sup&gt;，7和切伦科夫发光8,9。迄今为止，然而，没有这些都成为确立为标准的临床工具。光学荧光成像迄今被证明是最有前途的这些技术，因此是最探讨。虽然它已经被证明是对许多应用的有价值的工具，它也不是没有局限性。事实上，它的主要缺点是由固有的自发荧光的生物组织所产生的背景荧光。这样的背景自发荧光信号是周围组织的激励的产物，除了荧光团，通过所需要的荧光信号的产生的外部光源。从实践的角度来看，这自发荧光可以潜在地导致低信号对噪声比，它可以限制在手术室这种技术的效用。 校长优势化学发光成像在荧光成像是没有激发光是必要的。其结果是，没有背景自发荧光。在化学发光成像，代替化学生成的激发能量。这一过程不会产生意外的背景信号，因此可以导致更高的信号 - 噪声比。这可能最终导致手术切缘的更精确和准确的检测。出人意料的是，这种方法作为一种手术成像技术的效用仍然未开发的10。事实上，最近的例子，这种技术是鲁米诺由髓过氧化物酶的小鼠11，12，13的氧化。因此化学发光生物医学成像是研究的一个，而未开发地区，可以具有以下优点:（1）导致与喜低背景信号最小自发荧光gher信号 - 噪声比; （2）化学发光发射范围从可见光到近红外的波长可调波长; （3）官能化的化学发光复合物，当与连接器的技术相结合，有针对性已经存在的生物分子，靶向分子成像探针14的整个库提供接入。 验证的原理该研究说明了化学发光成像在使用基于钌 - 成像剂的生物医学设置的潜在效用。该化合物的化学发光特性很好的研究，以调查可追溯至60年代中期15。经化学活化，所述试剂产生在约600毫微米16的光，这是非常适合用于医疗成像目的。活化能是由导致激发态-其具有650纳秒寿命在水中17 -foll的氧化还原反应提供在此激发态的松弛由光子的产生欠款。通过使用一个特别设计的远程喷雾器的，我们能够检测到化合物既体外 和体内 。最初的实验的结果是非常有前景的，这表明该技术的进一步调查。

<table border="0" cellpadding="0" cellspacing="0" width="542">
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		</tr>
		<tr height="112">
			<td height="133" rowspan="2" style="height:133px;width:215px;">Tris(2,2&#8242;-bipyridyl)dichlororuthenium(II) hexahydrate</td>
			<td rowspan="2" style="width:119px;">Sigma-Aldrich Inc.</td>
			<td rowspan="2" style="width:87px;">224758</td>
			<td rowspan="2" style="width:121px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Ammonium cerium(IV) nitrate</td>
			<td style="width:119px;">Sigma-Aldrich Inc.</td>
			<td style="width:87px;">22249</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:215px;">Isofluorane</td>
			<td style="width:119px;">Baxter Healthcare</td>
			<td style="width:87px;">1001936060</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">PBS</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:87px;">PBS1</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Ethanol</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:87px;">2854</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Triethylamine</td>
			<td style="width:119px;">Sigma-Aldrich Inc.</td>
			<td style="width:87px;">T0886</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="97">
			<td height="97" style="height:97px;width:215px;">Water</td>
			<td style="width:119px;"></td>
			<td style="width:87px;"></td>
			<td style="width:121px;">Water was purified using a Milipore Mili-Q (R &#8805; 18 M&#937;)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Female nude (outbred) mice</td>
			<td rowspan="2" style="width:119px;">Jackson Laboratories</td>
			<td rowspan="2" style="width:87px;">1929</td>
			<td rowspan="2" style="width:121px;">age 5 - 6 weeks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Strain C57BL/6J&#160;&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:215px;">NU/J male mice at&#160;</td>
			<td style="width:119px;">Jackson Laboratories</td>
			<td style="width:87px;">2019</td>
			<td style="width:121px;">age 6 &#8211; 8 weeks</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:215px;">IVIS 200 bioluminescence reader</td>
			<td style="width:119px;">Caliper Live Science</td>
			<td style="width:87px;"></td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Live Image 4.2 software</td>
			<td style="width:119px;">Perkin-Elmer</td>
			<td style="width:87px;">128165</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Microscope slides</td>
			<td style="width:119px;">ThermoScientific</td>
			<td style="width:87px;">4951PLUS4</td>
			<td style="width:121px;"></td>
		</tr>
	</tbody>
</table>

a novel technique for generating and observing chemiluminescence in a biological setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

在协议部分1中所述的喷雾器系统可以从易得的材料以低成本来构造。它旨在是用于远程触发的生物发光读卡器内的还原/氧化剂的喷射（ 图1）的插入。我们的设计允许生物发光读者内的喷雾器在从透镜14厘米的距离的安全运行。在操作过程中，观察到透镜的不起雾或模糊。我们选择了市售化学发光剂的[Ru（联吡啶）3] Cl 2中为我们的方法是根据它的价格低的发展，在水溶液中，以及所述的氧化还原行为，和化学发光特性的稳定性（ 图2）19。可以由氧化[茹（联吡啶）3] Cl 2的（100微升一滴在协议第2节确定的最小检测信号，6.9 pmol- 347纳摩尔在H 2 O）与（NH 4）在显微镜载玻片2的Ce（NO 3）6（100μL，25毫摩尔）。然后，通过使用喷雾器和喷雾上的三乙胺中的溶液（1:3的水/乙醇）中，化学发光信号被触发。在我们的情况下，确定的最小可检测信号为6.9皮摩尔/厘米2（ 图3）。可以想到，虽然，优化反应条件，摄像灵敏度，快门时间，体积和试剂浓度可能导致甚至更低的检测阈值。这些反应条件也可用于探索和测试的金属配合物，氧化剂和还原剂任何给定组合的化学发光。 移动到体内实验在协议第3及4，雌性裸鼠（远交）小鼠5-6周龄和NU / J雄性小鼠6-8周龄使用。对于静脉注射，金额8-33纳摩尔的的[Ru（联吡啶）3] Cl 2中于100微升PBS中每只小鼠（N = 5）被选择。将动物在注射后10分钟处死，腹腔露出。将小鼠放置在生物发光读者用喷雾器向感兴趣的组织指向（ 图4）。用于静脉内注射的成像的[Ru（联吡啶）3] Cl 2中，被主要检测在肾脏的化学发光信号，强烈暗示肾消除亲水小分子的（ 图5）。信号-噪声比与使用[Ru（联吡啶）3] Cl 2中与注射PBS小鼠27/1为肾和21/1为肝脏。对于淋巴结成像，80纳摩尔的[Ru（联吡啶）3] Cl 2中的PBS10μL的被皮下注入小鼠的后足垫（N = 5）。小鼠通过CO 2窒息处死15分钟后注射。涵盖内后腿西澳皮肤š除去以暴露肌肉，淋巴结，和淋巴管。腘淋巴结随后化学发光可视化导致了淋巴结包含节点观察[茹（联吡啶）3] 2+显示出10±4.3倍，比未处理的人高光辉（167000 P /（S×厘米2×SR）和17000 P /（S×厘米2×SR），P &lt;0.028）（ 图6）。 <img alt="图1" src="/files/ftp_upload/54694/54694fig1.jpg" /> 图1:雾化器的照片。使用部分:木结构件 （A，B，C），喷雾瓶（D），弯钢杆（E），胶带（F），塑料扎带（G），011伺服连接器部分（H），伺服电机（I），铅笔（ j），通过弯曲的回形针（K举行），塑料包线扎带（L）W1线连接器（M）和扬声器连接线（N），导致电池。这个数字是基于研究最初发表于裁判erence 19。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54694/54694fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54694/54694fig2.jpg" /> 图2 的性质[茹（联吡啶）3] 2+。结构（A）和激发和发射光谱（B）中的[Ru（联吡啶）3] 2+。氧化/还原基于化学发光催化循环（C）。这个数字是基于最初发表于19参考研究。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54694/54694fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图3:检测阈值的[Ru（联吡啶）3] 2+。在不同浓度的代表信号强度 [茹（联吡啶）3] 2+在显微镜载玻片（A）。成像信号量化与检测阈值（红色虚线）和背景（黑色虚线）（B）中。这个数字是基于最初发表于19参考研究。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54694/54694fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54694/54694fig4.jpg" /> 图4:化学发光成像。鼠标和定位在生物发光读者喷雾器的示意图（ 一 </stron<html> g&gt;的）和喷雾器喷洒上的鼠标的示意图（ 二 ）。这个数字是基于最初发表于19参考研究。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54694/54694fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54694/54694fig5.jpg" /> 图5:检测的[Ru（联吡啶）3] 2+全身给药后。白光，化学发光，和覆盖（左到右）。即用的[Ru（联吡啶）3] 2+和与喷33纳摩尔注射小鼠体腔内的图像（NH 4）2的Ce（NO 3）6。对右肾的白色箭头指向。这个数字是基于最初发表于19参考研究。ftp_upload / 54694 / 54694fig5large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图6" src="/files/ftp_upload/54694/54694fig6.jpg" /> 图6:[茹（联吡啶）3] 2+后皮下管理局的检测。腘淋巴结成像示出用于与注射的小鼠的白色光，化学发光，并合成图片的[Ru（联吡啶）3] 2+在后肢（顶部）和PBS（底）; 80纳摩尔的PBS10μL的，成像的注射液（A）中经过15分钟。白色光并合成图像对的[Ru（联吡啶）3] 2+（顶部）和PBS（底部）处理切下腘淋巴结（B）。对于PBS化学发光信号和[茹（联吡啶）3] 2+ -处理的淋巴结（C）.The数据表示平均值±SD的定量。这个数字是基于最初的研究发表inreference 19。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54694/54694fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting at JoVE.com

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

在生物环境的新技术的产生和观测化学发光

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

a-novel-technique-for-generating-observing-chemiluminescence

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.4K 次观看.  Memorial Sloan Kettering Cancer Center. This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

视频: A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

卵泡在蛋白质降解的3维系统的封装和文化的一种方法

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

科研

生物工程

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

在生物系统中的离子浓度测量荧光纳米粒子

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9. 
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

密度梯度多层聚合（dGMP的）:创建多室的，可定制的一种新技术，组织工程支架

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

观察和量化纤维细胞介导的纤维素凝胶压实

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

在细胞和组织力学生物学应用一种新型的拉伸平台

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

新方法本地化记者荧光珠近细胞培养表面的牵引力显微镜

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

原发性肾组织段的显微解剖与新型无支架结构技术的结合

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...