Name: positron emission tomography imaging of cell trafficking: a method of cell radiolabeling
Uploaded: 2023-10-27T14:20:00
Description: Watch this Scientific Journal Video about Positron Emission Tomography Imaging of Cell Trafficking: A Method of Cell Radiolabeling at JoVE.com

这项工作得到了 NIH 5R21HL127389-02、NIH 4T32HL007111-39、NIH R01HL134664 和 DOE DE-SC0008947 资助、维也纳国际原子能机构、妙佑医疗国际核医学科放射科和明尼苏达州罗切斯特梅奥诊所再生医学中心的支持。所有数字都是使用 BioRender.com 创建的。

树突状细胞和黑色素瘤细胞在商业上获得18.腹腔镜肝部分切除术后从猪肝脏中分离肝细胞22,24。从骨髓抽吸物中分离干细胞18,19,26。脂肪组织来源的干细胞购自梅奥诊所罗切斯特院区的人类细胞治疗实验室23。从妙佑医疗国际罗切斯特输血医学科采?...

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以下是方案中需要优化以实现有效细胞放射性标记的关键步骤。在方案步骤1.2和1.3中，根据所采用的[89Zr]Zr（HPO4）2或[89Zr]ZrCl4的体积，必须使用适当体积（微升）的碱;1.0 M K 2CO 3 溶液必须用于中和 [89 Zr]Zr（HPO 4）2,1.0 M Na2CO 3 溶液用于中和 [89Zr]ZrCl4，以达到 7.5-8.0 的 pH 范围。在步骤2...

干细胞和嵌合抗原受体 （CAR） T 细胞疗法越来越受欢迎，并正在积极研究用于治疗各种疾病，例如心肌衰竭1,2、视网膜变性 2、黄斑变性 2、糖尿病 2、心肌梗塞3、4、5 和癌症6、7、8、9，10.在干细胞疗法的两种合理方法中，干细胞可以直接移植到疾病部位引起治疗反应，也可以引起疾病部位微环境的变化而不粘附在疾病部位以启动间接治疗反应。间接治疗反应可以通过释放修复或治疗疾病的因子来引起疾病部位微环境的变化5.这些干细胞治疗方法可以通过放射性标记干细胞的无创成像来评估。无创成像可以将疾病部位放射性标记细胞的摄取与治疗反应相关联，以破译直接与间接治疗反应。
此外，基于免疫细胞的疗法正在开发中，以使用 CAR T 细胞6、7、8、9、10 和树突状细胞免疫疗法11,12 治疗各种癌症。从机制上讲，在CAR T细胞免疫疗法6,7,8,9,10中，T细胞被设计为表达与需要治疗的肿瘤上的特定抗原结合的表位。这些工程化的CAR T细胞在给药时，通过表位-抗原相互作用与肿瘤细胞上存在的特异性抗原结合。结合后，结合的CAR T细胞发生激活，然后增殖并释放细胞因子，细胞因子向宿主的免疫系统发出信号，攻击表达特异性抗原的肿瘤。相反，在树突状细胞疗法11,12的情况下，树突状细胞被设计成在其表面呈递特定的癌症抗原。这些经过工程改造的树突状细胞在给药时是淋巴结的家园，并与淋巴结中的T细胞结合。T细胞在与施用的树突状细胞上的特定癌症抗原结合后，发生激活/增殖，并启动宿主对表达该特定抗原的肿瘤的免疫反应。因此，通过对放射性标记的CAR T细胞和树突状细胞进行成像来评估给药的CAR T细胞向肿瘤部位的运输9,10以及树突状细胞归巢到淋巴结11,12以确定免疫疗法的疗效。此外，非侵入性细胞运输可以帮助更好地了解治疗潜力，阐明直接与间接治疗反应，并预测和监测干细胞和基于免疫细胞的疗法的治疗反应。
已经探索了细胞运输的不同成像方式 3,4,9,10,12，包括光学成像、磁共振成像 （MRI）、单光子发射计算机断层扫描 （SPECT） 和正电子发射断层扫描 （PET）。这些技术中的每一种都有自己的优点和缺点。其中，PET因其定量性和高灵敏度而成为最有前途的方法，这对于基于成像的细胞运输中的细胞可靠定量至关重要3,4,9,10。
正电子发射放射性同位素89Zr，半衰期为78.4 h，适用于细胞标记。它允许对细胞运输进行 PET 成像超过 1 周，并且很容易由广泛使用的低能量医用回旋加速器13、14、15、16、17 产生。此外，市售的对异硫氰酸基苄基去铁胺 （DFO-Bn-NCS） 螯合剂可用于合成 89 Zr 标记的即用型细胞标记合成子 [89 Zr]Zr-p-异硫氰酸基苄基去铁胺，也称为 [89Zr]Zr-DBN 18,19,20,21,22,23,24，25.[89Zr]Zr-DBN 介导的细胞标记原理基于细胞膜蛋白的伯胺与 [89Zr]Zr-DBN 的异硫氰酸酯 （NCS） 部分之间的反应，以产生稳定的共价硫脲键。
[89锆]基于 Zr-DBN 的细胞标记和成像已发表，用于追踪各种不同的细胞，包括干细胞 18、23、25、树突状细胞18、造心干细胞19、蜕膜基质细胞 20、骨髓来源的巨噬细胞 20、外周血单核细胞 20、Jurkat/CAR T 细胞 21、肝细胞22、24 和白细胞 25.以下方案提供了使用[89Zr]Zr-DBN制备和细胞放射性标记的分步方法，并描述了特定细胞类型的放射性标记方案中可能需要的变化。为了更清楚起见，这里介绍的细胞放射性标记方法分为四个部分。第一部分涉及通过将 89Zr 与 DFO-Bn-NCS 螯合来制备 [89Zr]Zr-DBN。第二部分描述了[89Zr]Zr-DBN的生物相容性制剂的制备，该制剂可以很容易地用于细胞放射性标记。第三部分介绍了用于放射性标记的细胞预处理所需的步骤。细胞的预处理包括用无蛋白磷酸盐缓冲盐水 （PBS） 和 HEPES 缓冲的 Hanks 平衡盐溶液 （H-HBSS） 洗涤细胞，以去除外部蛋白质，这些蛋白质可能会干扰或竞争 [89Zr]Zr-DBN 与细胞表面蛋白质上存在的伯胺的反应在放射性标记过程中。最后一部分提供了细胞实际放射性标记和质量控制分析所涉及的步骤。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetonitrile</td>
			<td>Thermo Fisher Scientific, Inc., Waltham, MA, USA</td>
			<td>A996-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha Minimum Essential Medium</td>
			<td>Thermo Fisher Scientific, Inc., Waltham, MA, USA</td>
			<td>12571063</td>
			<td></td>
		</tr>
		<tr>
			<td>Anion exchange column</td>
			<td>Macherey-Nagel, Inc., D&#252;ren, Germany</td>
			<td>731876</td>
			<td>Chromafix 30-PS-HCO3&#160;SPE 45 mg cartridge</td>
		</tr>
		<tr>
			<td>Conical centrifuge tubes (15 mL)</td>
			<td>Corning Inc., Glendale, AZ, USA</td>
			<td>352096</td>
			<td>Falcon 15 mL high-clarity polypropylene (PP) conical centrifuge tubes</td>
		</tr>
		<tr>
			<td>Dendritic cells</td>
			<td>The American Type Culture Collection, Manassas, VA, USA</td>
			<td>CRL-11904</td>
			<td></td>
		</tr>
		<tr>
			<td>DFO-Bn-NCS</td>
			<td>Macrocyclics, Inc., Plano, TX, USA</td>
			<td>B-705</td>
			<td>p-SCN-Bn-Deferoxamine</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich, Inc., St. Louis, MO</td>
			<td>276855</td>
			<td></td>
		</tr>
		<tr>
			<td>Dose calibrator</td>
			<td>Mirion Technologies (Capintec), Inc., Florham Park, NJ, USA</td>
			<td>5130-3234</td>
			<td>CRC -55tR Dose Calibrator&#160;</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium&#160;</td>
			<td>The American Type Culture Collection, Manassas, VA, USA</td>
			<td>30-2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>The American Type Culture Collection, Manassas, VA, USA</td>
			<td>30-2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt solution (HBSS)</td>
			<td>Thermo Fisher Scientific, Inc., Waltham, MA, USA</td>
			<td>14025092</td>
			<td>For preparation of H-HBSS</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid (trace metal basis grade)</td>
			<td>Thermo Fisher Scientific, Inc., Waltham, MA, USA</td>
			<td>A508P212</td>
			<td></td>
		</tr>
		<tr>
			<td>Melanoma cells</td>
			<td>The American Type Culture Collection, Manassas, VA, USA</td>
			<td>CRL-6475</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich, Inc., St. Louis, MO</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>30108442</td>
			<td>Protein LoBind microcentrifuge tube</td>
		</tr>
		<tr>
			<td>Murine GM-CSF</td>
			<td>R&#38;D Systems, Inc., Minneapolis, MN USA</td>
			<td>415-ML-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fisher Scientific, Inc., Waltham, MA, USA</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline without Ca2+ and Mg2+</td>
			<td>Thermo Fisher Scientific, Inc., Waltham, MA, USA</td>
			<td>10010023</td>
			<td>For washing cells</td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Covidien LLC, Mansfield, MA, USA</td>
			<td>1020</td>
			<td>0.9% Sterile Saline Solution</td>
		</tr>
		<tr>
			<td>Shaker&#160;</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>T1317</td>
			<td>Thermomixer</td>
		</tr>
		<tr>
			<td>Silica gel-rad-TLC paper sheet&#160;</td>
			<td>Agilent Technologies Inc., Santa Clara, CA, USA</td>
			<td>SGI0001</td>
			<td>iTLC-SG</td>
		</tr>
	</tbody>
</table>

positron emission tomography imaging of cell trafficking: a method of cell radiolabeling

干细胞和嵌合抗原受体（CAR）T细胞疗法正在成为器官再生的有前途的疗法和各种癌症的免疫疗法。尽管在这些领域取得了重大进展，但仍需学习更多知识，以更好地了解生命系统中施用治疗细胞的药代动力学和药效学。对于使用正电子发射断层扫描 （PET） 对细胞进行非侵入性体内追踪，已经开发了一种利用 89 Zr （t1/2 78.4 h） 的新型 [89Zr]Zr-p-异硫氰酸基苄基-去铁胺 （[89Zr]Zr-DBN） 介导的细胞放射性标记方法。本方案描述了一种[89Zr]Zr-DBN介导的即用型放射性标记合成子，用于直接放射性标记各种细胞，包括间充质干细胞、谱系引导的造心干细胞、肝脏再生肝细胞、白细胞、黑色素瘤细胞和树突状细胞。所开发的方法能够在给药后长达 7 天内对细胞运输进行无创 PET 成像，而不会影响放射性标记细胞的性质或功能。此外，该协议描述了[89Zr]Zr-DBN的放射合成，[89Zr]Zr-DBN的生物相容性制剂，用于放射性标记的细胞的制备，以及最后用[89Zr]Zr-DBN对细胞进行放射性标记的逐步方法，包括成功放射性标记细胞所需的所有复杂细节。

本手稿中提出的代表性结果来自先前的 [89Zr]Zr-DBN 合成和细胞放射性标记研究 18,19,22,23,24,25。简而言之，使用7.5-15μgDFO-Bn-NCS，在25-37°C下，89Zr可以在~30-60分钟内与DFO-Bn-NCS成功络合（表2）。细胞放射性标记效率在洗涤后?...

Watch this Scientific Journal Video about Positron Emission Tomography Imaging of Cell Trafficking: A Method of Cell Radiolabeling at JoVE.com

Positron Emission Tomography Imaging of Cell Trafficking: A Method of Cell Radiolabeling

这里介绍的是使用即用型放射性标记合成子[89Zr]Zr-p-异硫氰酸基苄基-去铁胺（[89Zr]Zr-DBN）用正电子发射断层扫描（PET）放射性同位素89Zr（t1/2 78.4 h）对细胞进行放射性标记的方案。用 [89Zr]Zr-DBN 放射性标记细胞允许在给药后长达 7 天内对体内 PET 中施用的放射性标记细胞进行无创跟踪和成像。

细胞运输的正电子发射断层扫描成像：一种细胞放射性标记方法

Stem cell and chimeric antigen receptor (CAR) T-cell therapies are emerging as promising therapeutics for organ regeneration and as immunotherapy for ...

该协议描述了一种用于放射性标记细胞和锆-89-DBN的逐步方法及其通过PET进行的非侵入性跟踪。该技术还提供了一种可靠且稳定的方法，通过将锆-89-DBN共价偶联到细胞表面蛋白的主要手段来放射性标记细胞。从事神经系统疾病和各种癌症细胞疗法的医生和科学家会发现该协议有助于通过非侵入性PET成像了解基于细胞的疗法的药代动力学。详细的协议为新用户学习和采用这种放射性标记技术提供了指导。关键步骤中包含的其他节点将有助于为新用户解决问题。首先，用大约 100 毫克羟肟酸盐树脂制备色谱柱，然后用 8 毫升纯无水乙腈洗涤色谱柱，然后用 5 至 6 毫升空气冲洗来激活它。将 2 毫升 0.5 生盐酸通入色谱柱。再冲洗五到六毫升空气后，将含有锆-89和天然钇的溶液缓慢加载到异羟肟酸盐树脂中。用 20 毫升两种普通盐酸洗涤异羟肟酸盐树脂中未结合的天然钇，然后用 10 毫升去离子水洗涤。为了以磷酸氢锆-89 的形式逸出锆，向色谱柱中加入 0.5 毫升 1.2 摩尔磷酸二钾磷酸二氢钾缓冲液，并使其在色谱柱上静置 30 分钟。再加入 1.5 毫升 1.2 摩尔缓冲液，从色谱柱中洗脱锆。接下来，取约120微升在缓冲液中配制的磷酸锆-89磷酸氢锆，并用HEPES-KOH和碳酸钾中和溶液，使pH值达到7.528。将 4 微升新鲜制备的 5 毫摩尔 DFO-Bn-NCS 加入约 285 微升中和的磷酸锆 89 氢盐中。并通过移液混合溶液。从钇中分离出锆后，向色谱柱中加入0.5毫升一摩尔草酸，让其在色谱柱上静置一分钟，以草酸锆-89的形式洗脱锆。向色谱柱中再加入 2.5 毫升一摩尔草酸。要将草酸锆-89转化为氯化锆-89，请用6毫升乙腈清洗阴离子交换柱，然后用5至6毫升空气冲洗。然后，用 10 毫升生理盐水清洗色谱柱，然后再次冲洗空气。慢慢地，将含有草酸锆-89的溶液加载到活化的阴离子交换柱上，然后用空气重新洗涤。然后，用 50 毫升去离子水洗涤色谱柱以除去未结合的草酸根离子。为了以氯化锆-89的形式逸出锆，向色谱柱中加入0.1毫升一种普通盐酸，并使其在色谱柱上静置一分钟。用另外 0.4 毫升的一种普通盐酸从色谱柱中洗脱锆。将逸出的氯化锆-89放入V形小瓶中，在65摄氏度的加热块中，在稳定的氮气流下干燥10至30分钟，然后在水中重新配制干燥的氯化锆-89。在 1.5 毫升微量离心管中，在 500 微升 HEPES 缓冲的 HBSS 中制备 500 微升细胞悬液，其中约 600 万个细胞，并加入约 100 微升配制的锆-89-DBN。用微量移液管轻轻上下移液溶液，混合锆-89-DBN和细胞悬浮液。将细胞和锆-89-DBN 混合物在振荡器上以约 550 RPM 的速度在 25 至 37 摄氏度下孵育 30 至 45 分钟以进行细胞标记。使用新鲜制备的放射性TLC溶剂对细胞放射性标记反应进行放射性TLC。使用屏幕上显示的公式计算 Rf 等于 0.01 至 0.02 时的放射性百分比。在单独的 1.5 毫升微量离心管中，将约 100 微升配制的锆-89-DBN 与约 500 微升 HEPES 缓冲的 HBSS 混合，将其用作背景校正的无细胞对照。使用屏幕上显示的公式计算 Rf 等于 0.01 至 0.02 处的放射性百分比，然后通过减去两个计算出的放射性百分比来计算有效的细胞放射性标记。确认放射性标记完成后，加入 600 μL 冷冻细胞适当完全培养基以执行放射性标记反应的淬灭。将细胞在 96G 下在 4 摄氏度下离心 10 分钟。并弃去上清液。用微量移液管上下移液培养基，将沉淀的细胞轻轻重悬于500微升的冷冻培养基中。将细胞在4摄氏度下以96G离心10分钟，弃去上清液。接下来，使用屏幕上显示的公式计算所有洗涤后的最终放射性标记效率。为确保放射性标记细胞的质量，目视检查放射性标记细胞的最终悬浮液，如果不存在团块，则在放射性标记和洗涤步骤后一小时内使用在PBS中制备的0.4%台盼蓝溶液进行台盼蓝排阻性试验。DFO-Bn-NCS使用磷酸氢锆-89和氯化锆-89对锆-89的螯合效率是不同的，通过放射性TLC测量。这里显示了用锆-89放射性标记的各种细胞在7天内的稳定性。发现放射性标记细胞对锆-89放射性的保留在所有研究的细胞中都是稳定的，在标记后7天内观察到的外排可以忽略不计。下表显示了使用锆-89-DBN的不同细胞类型中的细胞放射性标记效率。细胞放射性标记效率从 20% 到 50% 不等，因为洗涤后在放射性标记的细胞沉淀中观察到未校正的产量。该协议最关键的步骤是将DFO-Bn-NCS添加到螯合混合物中以及放射性标记细胞的质量评估。该技术的应用将有助于了解基于细胞的疗法的药代动力学。它还会引发关于改变细胞疗法清除率的任何临床需求的问题。

Research

JoVE Journal

Cancer Research

A Method of Targeted Cell Isolation via Glass Surface Functionalization

通过玻璃表面功能靶细胞提取方法

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual ...

科研

癌症研究

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

肿瘤微环境的模仿：产生富集的细胞群及侦查间通讯的简单方法

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

TIP60 短暂耗竭后乳腺上皮细胞迁移的活体影像学研究

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the ...

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

长期活细胞成像对紫杉醇反应细胞命运的评估

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

胶质瘤移植中体内成像的荧光分子层析成像研究

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

nog 小鼠多发性骨髓瘤异种移植的多模态生物发光和正电子发射成形断层扫描成像

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Management of Respiratory Motion Artefacts in 18F-fluorodeoxyglucose Positron Emission Tomography using an Amplitude-Based Optimal Respiratory Gating Algorithm

Management of Respiratory Motion Artefacts in 18F-fluorodeoxyglucose Positron Emission Tomography using an Amplitude-Based Optimal Respiratory Gating Algorithm

使用基于振幅的最佳呼吸门控算法对 18个F-氟二氧氧葡萄糖正电子发射断层扫描中的呼吸运动人工制品进行管理

Positron emission tomography (PET) combined with X-ray computed tomography (CT) is an important molecular imaging platform that is required for accurate ...

Quantifying the Brain Metastatic Tumor Micro-Environment using an Organ-On-A Chip 3D Model, Machine Learning, and Confocal Tomography

使用有机体片 3D 模型、机器学习和共声断层扫描量化脑转移肿瘤微环境

Brain metastases are the most lethal cancer lesions; 10-30% of all cancers metastasize to the brain, with a median survival of only ~5-20 months, ...

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

使用[18F]四氟硼酸盐正电子发射断层扫描/计算机断层扫描对嵌合抗原受体T细胞进行动态成像

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B ...