Name: real-time monitoring of human glioma cell migration on dorsal root ganglion axon-oligodendrocyte co-cultures
Uploaded: 2019-12-13T12:00:00
Description: Watch this Scientific Journal Video about Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures at JoVE.com

这项工作得到了布朗大学神经外科内部基金的支持。

罗德岛医院IRB委员会批准了患者源性人类胶质瘤细胞的收集、分离和繁殖方案。所有动物都按照NIH《实验室动物护理和使用指南》进行维护。所有动物使用协议都通过了罗得岛医院机构动物护理和使用委员会的批准。
1. 媒体和缓冲准备
<ol><li>制备50 mL的神经球培养基：1x神经基底培养基培养基，不含维生素A，1x血清免费补充剂，不含维生素A，2 mM L-谷氨酰胺，20纳克/mL表皮生?...

<ol>
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K., 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=Identification+of+human+brain+tumour+initiating+cells.">Identification of human brain tumour initiating cells.</a> Nature. 432 (7015), 396-401 (2004).</li><li>Aubert, M., Badoual, M., Christov, C., Grammaticos, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+glioma+cell+migration+on+collagen+and+astrocytes.">A model for glioma cell migration on collagen and astrocytes.</a> Journal of the Royal Society Interface. 5 (18), 75-83 (2008).</li><li>Jia, W., 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=Effects+of+three-dimensional+collagen+scaffolds+on+the+expression+profiles+and+biological+functions+of+glioma+cells.">Effects of three-dimensional collagen scaffolds on the expression profiles and biological functions of glioma cells.</a> International Journal of Oncology. 52 (6), 1787-1800 (2018).</li><li>Kaphle, P., Li, Y., Yao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+and+pharmacological+regulation+of+glioblastoma+cell+migration+in+3D+matrices.">The mechanical and pharmacological regulation of glioblastoma cell migration in 3D matrices.</a> Journal of Cellular Physiology. 234 (4), 3948-3960 (2019).</li><li>Gritsenko, P., Leenders, W., Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recapitulating+in+vivo-like+plasticity+of+glioma+cell+invasion+along+blood+vessels+and+in+astrocyte-rich+stroma.">Recapitulating in vivo-like plasticity of glioma cell invasion along blood vessels and in astrocyte-rich stroma.</a> Histochemistry and Cell Biology. 148 (4), 395-406 (2017).</li><li>Gritsenko, P. 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可以使用博伊登腔室系统或划痕检测来执行 hGC 的迁移研究。然而，虽然这些实验未能提供有关肿瘤细胞与其他周围组织相互作用的任何信息，但目前的系统可以重述GC与骨髓和非骨髓化纤维的相互作用。此外，为了研究肿瘤的形成和端点迁移，啮齿动物大脑的器官切片培养物或胶质瘤细胞在体内植入啮齿动物大脑或侧翼之前已被利用23、24、25。</sup...

胶质母细胞瘤是人脑中最具攻击性和致命性的肿瘤之一。目前的护理标准包括肿瘤的手术切除，然后是辐射1加上伴随的特莫佐洛米德2的辅助和辅助。即使采用这种多疗法，肿瘤复发也是不可避免的。这部分是由于肿瘤细胞的广泛迁移性质，它侵入大脑胸痛，在大脑4内产生多个手指状的投影，使得完全切除的可能性不大。
近年来，胶质细胞瘤的侵略性明显增强，部分原因在于肿瘤质量5、6中存在一群癌症干细胞，其中7、8、抗化疗和放疗9、10和形成继发性肿瘤11的能力具有很高的迁移潜力。当异种移植到裸鼠5时，GsCs能够重述原始的多克隆肿瘤。
尽管关于胶质细胞瘤遗传背景的知识丰富，但缺乏有效的体外或体内迁移模型目前阻碍了对胶质瘤细胞（GC）迁移的研究。值得注意的是，虽然由细胞和环境因素调节的胶质瘤细胞-斧理相互作用是胶质瘤入侵的核心组成部分，但据我们所知，目前没有实验系统能够模拟这些相互作用12、13、14。为了解决这一缺陷，我们开发了一种与纯化DRG的腺苷酸联合培养的原生hG的体外培养系统，该系统导致分化肿瘤标记物的表达升高，以及hGCs与骨髓化和非骨髓化纤维的广泛迁移和相互作用。这个外体平台由于其条块分割的布局，适合测试新疗法对hGC迁移模式的影响。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mm Suspension Culture Dish</td>
			<td>Corning</td>
			<td>430591</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5S NGF</td>
			<td>ENVIGO</td>
			<td>B.5025</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Suspension Culture Dish</td>
			<td>Corning</td>
			<td>430589</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>Thermo Fisher</td>
			<td>A1049201</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Hydroxide Solution</td>
			<td>Fisher Scientific</td>
			<td>A669-500</td>
			<td>Concentrated</td>
		</tr>
		<tr>
			<td>Animal-Free Recombinant Human EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal-Free Recombinant Human FGF-basic (154 a.a.)</td>
			<td>Peprotech</td>
			<td>AF-100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-A2B5 MicroBeads, human, mouse, rat</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-392</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>Thermo Fisher</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoMACS Rinsing Solution (PBS, pH 7.2)</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-222</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Thermo Fisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement, minus vitamin A</td>
			<td>Thermo Fisher</td>
			<td>12587001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteriological Plate</td>
			<td>BD Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Sigma</td>
			<td>B4639</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Campenot Chamber</td>
			<td>Tyler Research</td>
			<td>CAMP-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td>35mm X 10mm</td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td>70 uM, Nylon</td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td>30 uM, Nylon</td>
		</tr>
		<tr>
			<td>Collagenase/Dispase</td>
			<td>Roche</td>
			<td>11097113001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex Rat Collagen I</td>
			<td>Trevigen</td>
			<td>3440-100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma</td>
			<td>G5146</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher</td>
			<td>10313021</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>D7291</td>
			<td></td>
		</tr>
		<tr>
			<td>Dow Corning High-Vacuum Grease</td>
			<td>Fisher Scientific</td>
			<td>14-635-5D</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Roboz</td>
			<td>RS-5045</td>
			<td></td>
		</tr>
		<tr>
			<td>E16 Timed Pregnant Sprague Dawley Rat</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EBSS</td>
			<td>Sigma</td>
			<td>E7510</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Hyclone</td>
			<td>SH30070.02</td>
			<td></td>
		</tr>
		<tr>
			<td>FUDR</td>
			<td>Sigma</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Thermo Fisher</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 Nutrient Mix</td>
			<td>Thermo Fisher</td>
			<td>11765054</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Thermo Fisher</td>
			<td>14175095</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostatic Forceps</td>
			<td>Roboz</td>
			<td>RS-7035</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Sodium Salt, 0.2% in PBS</td>
			<td>Stem Cell Technologies</td>
			<td>07980</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic Needle, 18G</td>
			<td>BD</td>
			<td>511097</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium G</td>
			<td>Thermo Fisher</td>
			<td>41400045</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma</td>
			<td>C7477</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Thermo Fisher</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium</td>
			<td>Thermo Fisher</td>
			<td>11415064</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS BSA Stock Solution</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM</td>
			<td>Thermo Fisher</td>
			<td>1190081</td>
			<td></td>
		</tr>
		<tr>
			<td>Mg2SO4</td>
			<td>Sigma</td>
			<td>M2643</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Columns plus tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-041-301</td>
			<td></td>
		</tr>
		<tr>
			<td>NAC</td>
			<td>Sigma</td>
			<td>A8199</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Thermo Fisher</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal-A Medium</td>
			<td>Thermo Fisher</td>
			<td>10888022</td>
			<td></td>
		</tr>
		<tr>
			<td>Ordinary forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P2 Sprague Dawley Rat Pups</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>LS003126</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr>
			<td>Pin Rake</td>
			<td>Tyler Research</td>
			<td>CAMP-PR</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>Thermo Fisher</td>
			<td>A1110501</td>
			<td></td>
		</tr>
		<tr>
			<td>Syrine Grease Applicator</td>
			<td>Tyler Research</td>
			<td>CAMP-GLSS</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Sigma</td>
			<td>T2036</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma</td>
			<td>U3003</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time monitoring of human glioma cell migration on dorsal root ganglion axon-oligodendrocyte co-cultures

胶质母细胞瘤是最具攻击性的人类癌症之一，由于广泛的细胞异质性和hGCs的迁移特性。为了更好地了解胶质瘤细胞迁移背后的分子机制，研究hGC和斧子在肿瘤微环境中的相互作用的能力至关重要。为了模拟这种细胞相互作用，我们开发了一个混合培养系统，由hGC和背根神经结膜（DRG）的斧状-寡核苷酸共培养物组成。选择DRG文化是因为它们可以有效地隔离，并可以形成长期、广泛的投影，非常适合这种性质的迁移研究。然后，在纯化大鼠DRG斧子上加入纯化大鼠寡核苷酸，并诱导至骨髓酸。在确认形成紧凑型骨髓后，hGCs最终被添加到共培养物中，并使用延时显微镜实时监测它们与DRG斧子和寡核苷酸物的相互作用。在这些条件下，hGCs形成肿瘤状的聚合结构，表达GFAP和Ki67，沿着骨髓和非骨髓化的斧道迁移，并通过伪波迪亚的形成与这些斧子相互作用。我们的前体共培养系统可用于识别hGC迁移的新型细胞和分子机制，并可能用于体外药物疗效测试。

为了研究hGCs与斧子的相互作用，我们产生了纯化的DRG斧子，如前面描述的15，16，17，18。这些纯化的DRG斧子然后用hG进行种子，形成GFAP+/Ki67+肿瘤状结构，这些结构集成在斧网中，而单个hGCs在联结或斧子之间迁移（图2）。为了确定hGCs如何与髓化的斧子相互作用，我们用纯化的大鼠寡核苷?...

Watch this Scientific Journal Video about Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures at JoVE.com

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

在这里，我们提出了一个前活体混合单层培养系统，用于实时研究人类胶质瘤细胞（hGC）迁移。该模型提供了观察hGCs与隔板腔内骨髓和非骨髓化斧子之间的相互作用的能力。

多端根结核细胞迁移的实时监测

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better ...

这种体外共培养系统可用于识别人类胶质瘤细胞迁移的新细胞和分子机制，并可能用于体外药物疗效测试。该技术的主要优点是能够实时研究人类胶质母细胞细胞和轴子之间的相互作用。演示这个程序的将是约翰·泽佩奇，一个研究生从我的实验室。为了组装分离的培养皿，将胶原蛋白库存溶液稀释至每毫升500微克，在无菌蒸馏水中彻底混合。使用无菌转移移液器，用稀释的胶原蛋白溶液填充 35 毫米培养盘。然后稍微倾斜盘子，取出溶液，留下一层胶原蛋白薄膜。将溶液分配到下一个 35 毫米的盘中。重复此过程，根据需要添加更多胶原蛋白溶液，直到所有盘子都涂覆。为了聚合胶原蛋白，在层流罩中，用三毫升氢氧化铵的湿纱垫，将托盘盖住15分钟。取下纱布垫，让 35 毫米的盘子干燥。接下来，使用金属文件从 18 量表指针的点上文件，以制作钝尖。将针头浸泡在70%乙醇中进行消毒。将硅胶润滑脂涂抹在隔间底部。确保润滑脂整齐地放置，并在所有角落重叠。这项技术可能非常具有挑战性，因此我们建议您在设置隔离室之前，花额外的时间练习涂抹正确数量的润滑脂，以确保良好的生长。慢慢工作也很重要。设置更多的房间，超过你认为你将需要，所以你有额外的，如果出了问题。从 35 毫米的盘子上取下盖子，反转盘子，将划痕放在室内。用一对钳子轻轻敲下盘子的底部。将润滑脂室涂在盘子上时，不要用太硬的水龙头。否则，轴子将无法在下方生长到相邻的腔室。使用止血钳轻轻翻转盘子。松开钳子。将一堆润滑脂放在中心隔间底部。用补充的神经元基础介质填充每个腔室。根据需要检查有无泄漏和密封泄漏与硅胶润滑脂。继续组装所有文化菜肴，并在 37 摄氏度和 5% 的二氧化碳下过夜储存。隔离室允许使用不同的药理试剂，以及比较和对比同一板上两个孔的结果的能力。要播种含有GBM神经球的已准备培养物，首先用每个直肠分室中含有10%FBS的补充NBF替换培养物。接下来，将 P20 移液器设置为 10 微升，从培养皿中绘制一个 GBM 神经球。将移液器的尖端放在离中心室最近的离心室中，而不接触轴孔，然后缓慢排出 GBM 神经球，使移液器轻轻地落在轴上。将培养物留在生物安全柜中，在室温下保持一小时，以便连接 GBM 神经球。该方法可用于测试治疗脑肿瘤的药理化合物。该方法也适用于外周和中枢神经系统的其他疾病，如脱髓性神经病变和多发性硬化症。在该协议中，纯化的DHG轴突与HCG一起播种，形成双阳性GFAP和KI67肿瘤样结构，这些结构集成在红色肿瘤标记GFAP指示的轴突网络中，而表达绿色荧光蛋白的单个HGCC则结合或结合轴突迁移。在骨髓DHG oligodend细胞联合培养物上添加HGC表明，HBC与红色染色的骨髓轴子一起迁移，并通过伪波迪亚的形成远离肿瘤质量。一旦这项技术得到完善，我们能够利用它实时研究人类GBM干细胞的迁移。我们还能够研究在添加新的治疗性小分子抑制剂后对细胞迁移的影响。此处显示了入侵骨髓的骨髓细胞 Drg 轴龙区域性的 Gbm 球体。此处描述的协议可以作为其他仿生三维外活体系统的基础，旨在评估新药物治疗的效果。在处理人源细胞时，应始终小心谨慎。应佩戴适当的 PPE，并完成血液传播的病原体培训，以便了解使用患者衍生组织的风险。氢氧化铵应小心处理，应戴上手套、实验室外套，并在发动机罩中工作，以防止接触这种化学物质。

Research

JoVE Journal

Cancer Research

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

一种内在的弥漫性脑胶质瘤脑桥快速验尸细胞培养协议（DIPG）

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

科研

癌症研究

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

3维球形共培养胰腺癌细胞和成纤维细胞的制备及代谢试验

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

Using the Dot Assay to Analyze Migration of Cell Sheets

用点法分析细胞板的迁移

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

Serum and Plasma Copy Number Detection Using Real-time PCR

应用实时 PCR 技术检测血清和血浆拷贝数

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

cd8+ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

石蜡包埋三维胶质瘤神经球培养的免疫组织化学评价胶质瘤生物标志物

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

M1巨噬细胞-4T1小鼠乳腺癌细胞在共培养中噬菌体活性的时移2D成像

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

基于阻抗的癌细胞迁移和入侵的实时测量

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

测量组织肿瘤组织切片的实时药物反应

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

快速筛选工作流程，利用患者衍生胶质瘤干细胞确定GBM的潜在组合疗法

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...