Name: a 3d spheroid model for glioblastoma
Uploaded: 2020-04-09T13:00:00
Description: Watch this Scientific Journal Video about A 3D Spheroid Model for Glioblastoma at JoVE.com

这项工作得到了Transcan 2017，ARC 2017，癌症联盟（吉龙德和查伦特-海洋委员会）的支持。约里斯·古扬是图卢兹大学医院（CHU图卢兹）奖学金的获得者。

所有患者均获得书面同意（根据当地道德委员会的规定，从挪威卑尔根的豪克兰医院获得）。我们的协议遵循我们机构人类研究伦理委员会的准则。
1. 生成均匀大小的肿瘤球体
注：干细胞在神经基础培养中培养，补充B27补充剂、肝素、FGF-2、青霉素和链霉素，如前文第12条所述。这些细胞在培养中自发地形成球体。
<ol><li>用5 mL磷酸盐?...

<ol>
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(150), e60122 (2019).</li><li>Boye, 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=The+role+of+CXCR3/LRP1+cross-talk+in+the+invasion+of+primary+brain+tumors.">The role of CXCR3/LRP1 cross-talk in the invasion of primary brain tumors.</a> Nature Communications. 8, 1571 (2017).</li><li>Dejeans, N., 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=Autocrine+control+of+glioma+cells+adhesion+and+migration+through+IRE1alpha-mediated+cleavage+of+SPARC+mRNA.">Autocrine control of glioma cells adhesion and migration through IRE1alpha-mediated cleavage of SPARC mRNA.</a> Journal of Cell Science. 125, 4278-4287 (2012).</li><li>Golembieski, W. A., Ge, S., Nelson, K., Mikkelsen, T., Rempel, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+SPARC+expression+promotes+U87+glioblastoma+invasion+in+vitro.">Increased SPARC expression promotes U87 glioblastoma invasion in vitro.</a> International Journal of Developmental Neuroscience. 17, 463-472 (1999).</li><li>Daubon, T., 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=Deciphering+the+complex+role+of+thrombospondin-1+in+glioblastoma+development.">Deciphering the complex role of thrombospondin-1 in glioblastoma development.</a> Nature Communications. 10, 1146 (2019).</li><li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tube+travel:+the+role+of+proteases+in+individual+and+collective+cancer+cell+invasion.">Tube travel: the role of proteases in individual and collective cancer cell invasion.</a> Cancer Research. 68, 7247-7249 (2008).</li><li>Das, A., Monteiro, M., Barai, A., Kumar, S., Sen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MMP+proteolytic+activity+regulates+cancer+invasiveness+by+modulating+integrins.">MMP proteolytic activity regulates cancer invasiveness by modulating integrins.</a> Scientific Reports. 7, 14219 (2017).</li><li>Schaeffer, D., Somarelli, J. A., Hanna, G., Palmer, G. M., Garcia-Blanco, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+migration+and+invasion+uncoupled:+increased+migration+is+not+an+inexorable+consequence+of+epithelial-to-mesenchymal+transition.">Cellular migration and invasion uncoupled: increased migration is not an inexorable consequence of epithelial-to-mesenchymal transition.</a> Molecular and Cellular Biology. 34, 3486-3499 (2014).</li><li>de Gooijer, M. 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肿瘤球体测定非常适合研究肿瘤特征，包括增殖、入侵和迁移，以及细胞死亡和药物反应。癌细胞侵入形成侵入性微肿瘤的3D基质，如图4B，C所示。在入侵过程中，基质金属蛋白酶（MMP）消化肿瘤细胞13周围的基质，MMP抑制剂（例如GM6001或Rebimastat）可能损害细胞入侵，但不能迁移14。迁移和入侵涉及重叠但独立的分子事件<sup cl...

在大多数使用原细胞系或商业上可用的细胞系的研究中，在塑料表面作为单层培养物生长的细胞上进行检测。在 2D 中管理单元格培养表示缺点，因为它不模仿体内 3D 细胞环境。在2D培养物中，整个细胞表面与介质直接接触，改变细胞生长，改变药物可用性。此外，非生理塑料表面触发细胞分化1。为克服这些困难，我们开发了三维文化模型。它们具有模仿肿瘤2的多细胞架构和异质性的优点，因此可被视为实体肿瘤3的更相关的模型。球类复杂的形态有助于更好地评价药物渗透和抗药性4。球体中的肿瘤异质性影响氧和营养的扩散，以及对药理剂的反应（图1A）。当球体尺寸达到300μm时，氧气的扩散会改变，在球体中心引起缺氧环境（图1A，C）。代谢物也较少穿透细胞层和补偿代谢反应发生5。当球形的直径增加时，可以观察到坏死核，进一步模仿许多固体癌症中发现的特征，包括恶性脑癌胶质细胞瘤（GBM）6。
文献77、88报道了胶质细胞瘤的几种2D或3D入侵测定。二维测定主要用于研究在薄矩阵层或博伊登腔室测定9中水平平面上的入侵。三维检测用经典的胶质细胞系10来描述3D球形培养。更复杂的变异表现为对抗培养中肿瘤球类对脑器官的入侵11。然而，开发一种易于利用和可重复的检测对于任何实验室都仍然很重要。我们开发了一种协议，从患者样本中产生胶质细胞瘤干细胞样细胞。这些检测的量化易于管理，只需要开放访问的在线软件。简单地说，肿瘤片被切成小块，酶消化。从消化中提取的单细胞在神经基础培养基中培养。4-7天后，球形结构自发形成。在小鼠模型中植入颅内后，它们形成肿瘤，表现出一个被伪细胞包围的坏死核12。这与 GBM 患者的特征非常相似。
在本文中，我们描述了我们根据确定数量的细胞生产球体以确保可重复性的协议。两种补充基质可用于此目的：母质和胶原蛋白 I 型，Matrigel 富含生长因子，并模仿细胞连接和迁移所需的哺乳动物基膜。另一方面，胶原蛋白I型，一种基质的结构元素，是最常见的纤维外细胞基质，用于细胞入侵测定。在这里，我们通过执行迁移和增殖测定来说明我们的 GBM 球形模型。分析不仅在固定的时间点进行，而且通过实时成像监测球体扩张和细胞运动。此外，还进行了电子显微镜的可视化形态细节。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 well round-bottom plate</td>
			<td>Falcon</td>
			<td>08-772-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Gibco</td>
			<td>A11105-01</td>
			<td>Stored at 4 &#176;C, sphere dissociation enzyme</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>12587</td>
			<td>Stored at -20 &#176;C, defrost before use</td>
		</tr>
		<tr>
			<td>Basic Fibroblast Growth Factor</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td>Stored at -20 &#176;C, defrost before use</td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS 10X</td>
			<td>Pan Biotech</td>
			<td>P04-53-500</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Fiji software</td>
			<td>ImageJ</td>
			<td></td>
			<td>Used to analyze pictures</td>
		</tr>
		<tr>
			<td>Flask 75 cm2</td>
			<td>Falcon</td>
			<td>10497302</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Stored at -20 &#176;C, diluted to a final concentration of 0.2 mg/mL in cold NBM</td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td>Sigma</td>
			<td>M0512</td>
			<td>Diluted in NBM for a 2% final concentration</td>
		</tr>
		<tr>
			<td>NBM</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Penicillin - Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Trypan blue 0.4%</td>
			<td>ThermoFisher</td>
			<td>T10282</td>
			<td>Used to cell counting</td>
		</tr>
		<tr>
			<td>Type I Collagen</td>
			<td>Corning</td>
			<td>354236</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
	</tbody>
</table>

a 3d spheroid model for glioblastoma

二维（2D）细胞培养物在体内肿瘤生长不令人满意地模仿。因此，开发了三维（3D）培养球形模型。这些模型在神经肿瘤学领域可能特别重要。事实上，脑肿瘤有侵入健康大脑环境的倾向。我们在这里描述了一个理想的3D胶质母细胞瘤球体为基础的测定，我们开发研究肿瘤入侵。我们提供所有技术细节和分析工具，以成功执行此测定。

如协议部分所述，编制了球类，并就迁移、入侵、扩散和显微镜进行了观测。为了测量球形结构不同区域的缺氧，用车盒性水合酶IX染色来确定缺氧活性（图1A-C）。在球形中心观察到更多的CAIX阳性细胞（图1A-C）。位于球状核中的缺氧细胞往往比周围的细胞更甘油。线粒体可以成像，以便进一步分析，?...

Watch this Scientific Journal Video about A 3D Spheroid Model for Glioblastoma at JoVE.com

A 3D Spheroid Model for Glioblastoma

在这里，我们描述了一种容易使用的胶质细胞瘤入侵测定。这种测定适用于胶质母细胞瘤干细胞。还介绍了斐济宏，用于轻松量化入侵、移徙和扩散。

胶质母细胞瘤的3D球形模型

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

二维细胞培养不能充分模仿体内肿瘤的生长。为了改进，3D 程序已经开发使用球形。我们的3D胶质母细胞瘤球类检测特别适合研究脑肿瘤进入健康脑肿瘤环境。三维培养模型可用于概括肿瘤的多细胞结构、异质性和代谢状态，从而对药物效应进行更严格的评估。确保不要触摸井底或完全去除上清液，将凝胶留在冰上，并迅速将细胞添加到凝胶中。要生成大小均匀的肿瘤球体，首先用5毫升PBS清洗感兴趣的肿瘤细胞培养，用0.5至1毫升分离酶治疗细胞。在37摄氏度下5分钟后，停止使用4至4.5毫升PBS的反应，将分离的细胞转移到含有10毫升完整生长介质的15毫升圆锥管中。计数后，每8毫升完全生长介质的1倍10至6细胞重新悬浮细胞，辅以2%甲基纤维素浓度的两毫升，将悬浮液转移到无菌系统容器中。然后将100微升的细胞加入96井圆形底板的每个井中，并将该细胞放入37摄氏度5%的二氧化碳培养箱中，为期三至四天。进行入侵测定，当肿瘤细胞形成大小均匀的球类时，将每个细胞结构转移到500微升管中，用200微升PBS洗涤球体一次。第二次洗涤后，小心地将球体转移到100微升新鲜准备的胶原蛋白基质中，并将每个球体悬浮液添加到平底96井板的每个井的中心。在37摄氏度下孵育30分钟后，在每一井的凝胶顶部加入100微升完全生长介质。一定要把所有的球体放在每一个井的中心，以最好的成像肿瘤细胞入侵。为了进行迁移测定，当肿瘤细胞形成大小均匀的球状体时，在37摄氏度下，在生长介质中涂上每毫升Matrigel0.2毫克的六井板，30分钟。在孵育结束时，取出马特里格尔，并添加两毫升的完全生长介质到每一个井。将球体从50微升体积的圆形底板转移到六孔板的单个孔中，并放入细胞培养箱24小时。第二天，在37摄氏度下，用每毫升10毫微克的球状物染色30分钟。在入侵或迁移分析后进行图像采集时，请将板放在配备摄像机的 Brightfield 共合一显微镜的舞台上，并在适当的实验期间获取图像。要以半自动方式分析图像，请将图像导入斐济，然后单击插件、宏、交互式解释器。复制并粘贴适应的紫色循环。保留紫色句子，并根据需要添加感兴趣的绿色句子。然后，要测量每个球体的面积，请单击"分析和测量"。为了测量球形结构不同区域的缺氧，可以使用碳氢化物IX染色来确定缺氧活性。在这个具有代表性的分析中，在球体中心观察到更多的碳氢化物IX阳性细胞。位于球状核中的低氧细胞往往比核心周围的细胞更糖解。线粒体可以通过电子显微镜进行成像，以作进一步分析。球体直径与组成 3D 细胞结构的细胞密度直接相关，斐济宏可用于量化每个球体内的细胞的增殖、入侵或迁移，或每个球体内的细胞迁移。球体核心的增加反映了细胞增殖的刺激。线粒体呼吸链的复杂 I 抑制后，线粒体中绝大多数 ATP 生产在 72 小时后减少增殖 20%。氯化氢处理可增强胶原蛋白 I 型入侵在 24 小时内，但与控制未经处理的球类相比，球类的迁移面积减少了 1.5 倍。根据实时成像评估，球体表现出高内部动力学并快速移动。球体的大小应相对均匀，应注意将球体操纵到井的中心，以获得最佳成像效果。该方法还可用于研究肿瘤细胞增殖、迁移和死亡，以及细胞外基质、细胞受体或细胞内蛋白质的表达。球体也可以封装，并在去除胶囊时研究细胞表面张力增加的影响。

Research

JoVE Journal

Cancer Research

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

共培养化验，以研究胶质母细胞瘤入侵的巨噬细胞和小胶质细胞刺激

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

科研

癌症研究

PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

PET 与 MRI 引导下微器对大鼠胶质瘤模型的照射

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

基于球形的头颈部鳞状细胞癌3D 细胞培养模型的治疗试验

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

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 ...

A 3D Organotypic Melanoma Spheroid Skin Model

3D 脊髓黑色素瘤球形皮肤模型

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

体外3D 球形培养模型小鼠前列腺叶的鉴定、组织学特征及解剖

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

A Spheroid Killing Assay by CAR T Cells

一种用汽车 t 细胞进行的切杀检测

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

评估癌细胞3D球形培养中的细胞活力和死亡

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

3D球形模型作为癌症相关纤维细胞分化和入侵体内研究的更多生理系统

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

通过高分辨率共聚焦显微镜对3D肿瘤球体入侵的影响表征

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and ...