Name: co-culture of glutamatergic neurons and pediatric high-grade glioma cells into microfluidic devices to assess electrical interactions
Uploaded: 2021-11-17T13:00:00
Description: Watch this Scientific Journal Video about Co-culture of Glutamatergic Neurons and Pediatric High-Grade Glioma Cells Into Microfluidic Devices to Assess Electrical Interactions at JoVE.com

这项工作得到了Satt Conectus计划，斯特拉斯堡大学基金会，«J'ai demandé la lune»，«Une roulade pour Charline»，«LifePink»，«Franck，Rayon de Soleil»和«Semeurs d'Etoile»协会的资助。我们感谢受HGGs影响的儿童和家庭对这项研究的贡献和支持。

对于该协议，与使用人类材料相关的认证编号为DC-2020-4203。
1. 微流控装置的制造、制备和处理
<ol><li>使用传统的光刻技术制造SU-8模具18。 注：为此目的，设计了两个光刻掩模，以在硅晶圆衬底上构建两层光刻胶结构和一层薄的SU-8 2005光刻胶层（3.2μm高，6±1μm宽），该层在SU-8 2100（200μm高，1 mm宽， 和13毫米长）（见 补充图S1）。</li>
	<li>通?...

<ol>
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这项工作描述了一种准确的功能 体外 模型，以评估微流体装置中人hiPS衍生的皮质谷氨酸能神经元与脑肿瘤细胞之间的相互作用。本方案的关键步骤之一是谷氨酸能神经元中的hiPS分化，这通过Nestin和Sox2免疫荧光染色的减少以及mGluR2和vGLUT1染色的同时出现得到了证实。然而，很少有神经祖细胞留下来，因为只有一半的谷氨酸能细胞表达mGluR2，这意味着异质性神经元细胞群。总而言之，这些?...

小儿高级别胶质瘤 （pHGG） 表现出扩展的基因型和表型多样性，具体取决于患者年龄、肿瘤解剖学位置和扩展以及分子驱动因素1。它们是侵袭性脑肿瘤，目前可用的治疗方案控制不佳，是儿童和青少年脑癌相关死亡的主要原因2。因此，超过80%的患者在诊断后2年内复发，他们的中位生存期为9-15个月，这取决于大脑位置和驱动突变。缺乏治愈性治疗是实验室研究的主要动力，并突出了对新的创新治疗方法的迫切需要。为此，开发了患者来源的细胞系（PDCL），希望在二维（2D）系和/或三维（3D）神经球中提供pHGG多样性3 。然而，这些患者来源 的体外 细胞培养物并不能模拟所有脑变量情况。这些模型不考虑pHGG中通常描述的宏观和微观神经解剖环境。
通常，年幼儿童的pHGG主要发生在脑桥和丘脑区域，而青少年和年轻人的HGG集中在皮质区域，特别是在额颞叶1。这些跨儿科年龄的位置特异性似乎涉及导致胶质瘤发生的不同环境以及肿瘤细胞和特定神经元活动之间错综复杂的网络4，5，6。虽然机制尚未确定，但pHGG主要从神经前体细胞沿着星形胶质细胞和少突胶质细胞谱系的分化轨迹发展而来。虽然这些神经胶质谱系的作用长期以来一直局限于对神经元的简单结构支持，但现在已经明确确定，它们完全整合到神经回路中，并表现出复杂的双向神经胶质 - 神经元相互作用，能够重组大脑的结构区域并重塑神经元回路4，7，8.此外，越来越多的证据表明，中枢神经系统（CNS）在脑癌的发生和进展中起着至关重要的作用。最近的工作集中在神经元活动上，这似乎通过分泌的生长因子和直接的电化学突触通讯来驱动神经胶质恶性肿瘤的生长和有丝分裂6，9。反过来，高级别胶质瘤细胞似乎随着谷氨酸能神经元活动的增加而影响神经元功能，并调节它们在结构和电上集成的电路的运行9。因此，使用患者衍生的模型和控制神经元作用的新型神经科学工具的研究表明，神经元活动对胶质瘤的位置，生长和进展具有电路特异性影响。参与神经胶质瘤的这些神经元投射中的大多数是谷氨酸能的，并通过谷氨酸分泌物进行交流。通常描述特定的谷氨酸能生物标志物，如 mGluR2 或 vGlut1/26。
有趣的是，尽管其分子异质性，但儿童和成人高级别胶质瘤对谷氨酸能神经元活动和其他分泌因子（如神经木质素-3或BDNF（脑源性神经营养因子））表现出典型的增殖反应4，6，10，11，12，13.在皮质区域，儿童和成人HGGs可以通过增加谷氨酸分泌诱导神经元过度兴奋，并抑制GABA中间神经元，导致与癫痫网络活动相关的胶质瘤14，15。最重要的是，神经回路可以通过神经胶质瘤来重塑，这些神经胶质瘤推动特定的神经任务，例如语言，并且可以征用额外的有组织的神经元活动9。
基于这一原理，必须充分阐明对胶质瘤细胞和神经元之间双向通信的理解，并将其与 体外 pHGG方法的早期阶段相结合。这种创新的建模对于理解和测量药物测试期间神经元电活动的影响以及预测pHGG对大脑回路的反应至关重要。神经科学工具的最新发展，如微流体装置和pHGG研究工作，是开发新建模方法的床，现在能够将大脑微环境整合到体外 pHGG模型中3，16，17，18，19。结合使用多电极阵列（MEA）的电生理记录，微流体器件20，21，22 提供了模拟生理条件的可能性，同时记录整个神经网络的电活动并在多种条件下提取网络连接参数。该器件23，24 首先允许将细胞精确地沉积在直接在MEA上的腔室中。该技术能够控制MEA上的细胞接种密度和均匀性，并精细控制培养基交换，这是人类神经祖细胞直接分化成设备的关键步骤。此外，本沉积室可以在不同的时间点接种多个细胞。
因此，本研究旨在开发一种功能 性体外 模型，将人类多能干（hiPS）衍生的皮质谷氨酸能神经元和pHGG衍生的细胞共培养成微流体装置，并记录它们的电活动以评估两个细胞群之间的电相互作用。首先，在培养的不同阶段[第4天（D4）作为hiPS细胞，第21天（D21）和第23天（D23），作为谷氨酸能成熟神经元]的微流体装置中获得hiPS衍生的皮质谷氨酸能神经元并表征。对于共培养的第二步，使用了两种pHGG模型：商业化的儿科UW479系和从患者肿瘤（BT35）3引发的pHGG细胞，带有H3.3 K27M驱动突变。最后，我们在pHGG细胞接种前在D21和共培养48小时后在D23处对谷氨酸能细胞进行了电生理学记录。并在同一微流体装置中。谷氨酸能神经元与pHGG细胞之间的相互作用的特征在于记录的电生理活性显着增加。

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			<td >256MEA100/30iR-ITO-w/o</td>
			<td >MCS</td>
			<td >256MEA100/30iR-ITO-w/o</td>
			<td ></td>
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		<tr >
			<td >40 &#181;m probe for Scepter counter&#160;</td>
			<td >Dutscher</td>
			<td >53750</td>
			<td ></td>
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		<tr >
			<td >60 &#181;m probe for Scepter counter&#160;</td>
			<td >Dutscher</td>
			<td >51999</td>
			<td ></td>
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			<td >Accutase</td>
			<td >Sigma</td>
			<td >A6964</td>
			<td ></td>
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			<td >Ala -Gln (GlutaMAX)</td>
			<td >Sigma</td>
			<td >G8541</td>
			<td ></td>
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			<td >Axel Observer 7 Microscope</td>
			<td >Zeiss</td>
			<td >431007-9904-000</td>
			<td ></td>
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			<td >Cell culture flask with cap with filter membrane 70 mL Falcon&#174;</td>
			<td >Dutscher</td>
			<td >353109</td>
			<td ></td>
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			<td >Class II Biological Safety Cabinet</td>
			<td >Thermo Scientific</td>
			<td >HERASafe type KS12</td>
			<td ></td>
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			<td >Colibri 7 LED</td>
			<td >Zeiss</td>
			<td >4230529710-000</td>
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			<td >
			<div>Cortical Glutamatergic Neurons</div>
			</td>
			<td >BrainXell</td>
			<td >BX-0300</td>
			<td ></td>
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		<tr >
			<td >DMEM/F-12 (1:1) GlutaMAX</td>
			<td >Gibco</td>
			<td >31331-028</td>
			<td ></td>
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			<td >DMEM/F12 Medium</td>
			<td >Sigma</td>
			<td >D8437</td>
			<td ></td>
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		<tr >
			<td >DPBS 1X</td>
			<td >Dutscher</td>
			<td >L0615-500</td>
			<td ></td>
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			<td >EasYFlaskTM&#160;cell culture flasks 75cm3</td>
			<td >Nunc</td>
			<td >156499</td>
			<td ></td>
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			<td >Foetal Bovine Serum (FBS)</td>
			<td >Dutscher</td>
			<td >500105</td>
			<td ></td>
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			<td >GDNF</td>
			<td >Peprotech</td>
			<td >450-10</td>
			<td ></td>
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			<td >Geltrex</td>
			<td >Life Technologies</td>
			<td >A1413201</td>
			<td ></td>
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			<td >Human BDNF</td>
			<td >Peprotech</td>
			<td >450-02</td>
			<td ></td>
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			<td >Incubator</td>
			<td >Memmert</td>
			<td >IC0150med</td>
			<td ></td>
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			<td >MCS InterFace Boarder</td>
			<td >MCS</td>
			<td >181205-MEA2100-11240</td>
			<td ></td>
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		<tr >
			<td >MEA2100</td>
			<td >MCS</td>
			<td >181205-MEA2100-11240</td>
			<td ></td>
		</tr>
		<tr >
			<td >Micropipette P10</td>
			<td >Sartorius</td>
			<td >LH-729020</td>
			<td ></td>
		</tr>
		<tr >
			<td >Micropipette P100</td>
			<td >Sartorius</td>
			<td >LH-729050</td>
			<td ></td>
		</tr>
		<tr >
			<td >Micropipette P1000</td>
			<td >Sartorius</td>
			<td >LH-729070</td>
			<td ></td>
		</tr>
		<tr >
			<td >Micropipette P200</td>
			<td >Sartorius</td>
			<td >LH-729060</td>
			<td ></td>
		</tr>
		<tr >
			<td >Microtube Eppendorf 1,5 ml Safe-Lock</td>
			<td >Dutscher</td>
			<td >33290</td>
			<td ></td>
		</tr>
		<tr >
			<td >MultiChannel Experimenter</td>
			<td >MCS</td>
			<td >-</td>
			<td ></td>
		</tr>
		<tr >
			<td >N2 Supplement-A</td>
			<td >StemCell</td>
			<td >7152</td>
			<td ></td>
		</tr>
		<tr >
			<td >Neurobasal Medium</td>
			<td >Life Technologies</td>
			<td >21103049</td>
			<td ></td>
		</tr>
		<tr >
			<td >Neurocult SM1 neuronal supplement</td>
			<td >StemCell</td>
			<td >5711</td>
			<td ></td>
		</tr>
		<tr >
			<td >Non filter tip 0.1 - 10 &#181;l ClearLine&#174; sterile in removable-lid rack&#160;</td>
			<td >Dutscher</td>
			<td >030570ACL</td>
			<td ></td>
		</tr>
		<tr >
			<td >Non filter tip 1 - 200 &#181;l ClearLine&#174; sterile in removable-lid rack&#160;</td>
			<td >Dutscher</td>
			<td >032260CL</td>
			<td ></td>
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		<tr >
			<td >Non filter tip 50 - 1250 &#181;l ClearLine&#174; sterile in removable-lid rack</td>
			<td >Dutscher</td>
			<td >134760CL</td>
			<td ></td>
		</tr>
		<tr >
			<td >Non-essential amino acids (NEAA) without L-glutamine</td>
			<td >Dutscher</td>
			<td >X0557-100</td>
			<td ></td>
		</tr>
		<tr >
			<td >Pipeteur Pipet-Aid XP Gravity</td>
			<td >Drummond</td>
			<td >4000202/4038202</td>
			<td ></td>
		</tr>
		<tr >
			<td >Pipette for cell culture 10 mL Falcon&#174;&#160;</td>
			<td >Dutscher</td>
			<td >357551</td>
			<td ></td>
		</tr>
		<tr >
			<td >Pipette for cell culture 5 mL Falcon&#174;&#160;</td>
			<td >Dutscher</td>
			<td >357543</td>
			<td ></td>
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		<tr >
			<td >Plaque chauffante (CultureTemp)</td>
			<td >Belart</td>
			<td >370151000</td>
			<td ></td>
		</tr>
		<tr >
			<td >Poly-D-Lysine</td>
			<td >Sigma</td>
			<td >P6407</td>
			<td ></td>
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		<tr >
			<td >Primovert microscope</td>
			<td >Zeiss</td>
			<td >415510-1100-000</td>
			<td ></td>
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		<tr >
			<td >Scepter (Handheld Automated Cell Counter)</td>
			<td >Millipore</td>
			<td >PHCC00000</td>
			<td ></td>
		</tr>
		<tr >
			<td >TGF-&#946;1</td>
			<td >Peprotech</td>
			<td >100-21C</td>
			<td ></td>
		</tr>
		<tr >
			<td >Tube with conical bottom 15 mL (bulk) Falcon&#174;&#160;</td>
			<td >Dutscher</td>
			<td >352096</td>
			<td ></td>
		</tr>
		<tr >
			<td >Tube with conical bottom 50 mL (bulk) Falcon&#174;&#160;</td>
			<td >Dutscher</td>
			<td >352070</td>
			<td ></td>
		</tr>
	</tbody>
</table>

co-culture of glutamatergic neurons and pediatric high-grade glioma cells into microfluidic devices to assess electrical interactions

儿科高级别胶质瘤（pHGG）代表儿童和青少年脑癌，预后迅速恶化。由于需要克服对当前治疗的耐药性并找到一种新的治疗方法，因此 在体外 环境中尽可能接近地对疾病进行建模以测试新药物和治疗程序要求很高。研究它们的基本病理生物学过程，包括谷氨酸能神经元的过度兴奋性，将是理解环境大脑和pHGG细胞之间相互作用的真正进步。因此，为了重建神经元/pHGG细胞相互作用，这项工作展示了一种功能 性体外 模型的发展，该模型将人类诱导的多能干（hiPS）衍生的皮质谷氨酸能神经元pHGG细胞转化为区室化的微流体装置以及记录其电生理修饰的过程。第一步是区分和表征人类谷氨酸能神经元。其次，将细胞培养在具有pHGG衍生细胞系的微流体装置中。然后将大脑微环境和神经元活动纳入该模型，以分析pHGG细胞对这些微环境神经元的电影响。电生理记录使用多电极阵列（MEA）耦合到这些微流体装置，以模拟生理条件并记录整个神经网络的电活动。在肿瘤细胞存在的情况下，神经元兴奋性的显着增加被强调。

在研究谷氨酸能神经元和胶质瘤细胞之间的电相互作用之前，对hiPS衍生的皮质谷氨酸能神经元进行了表征，以验证在微流体装置中培养它们的可行性（图1A）。使用Nestin，Sox2，mGlurR2（代谢性谷氨酸受体2）和vGLUT1免疫染色（ 如图1A（2-7）所示）评估其表征。由于巢蛋白是神经祖细胞存活、更新和丝裂原刺激增殖所需的中?...

Watch this Scientific Journal Video about Co-culture of Glutamatergic Neurons and Pediatric High-Grade Glioma Cells Into Microfluidic Devices to Assess Electrical Interactions at JoVE.com

Co-culture of Glutamatergic Neurons and Pediatric High-Grade Glioma Cells Into Microfluidic Devices to Assess Electrical Interactions

最近的工作揭示了神经元对高级别小儿胶质瘤（pHGG）细胞的影响及其相互相互作用。目前的工作显示了一种 体外 模型的发展，该模型共培养pHGG细胞和谷氨酸能神经元，并记录了它们的电生理相互作用以模仿这些相互作用。

将谷氨酸能神经元和儿科高级别胶质瘤细胞共培养到微流体装置中以评估电相互作用

Pediatric high-grade gliomas (pHGG) represent childhood and adolescent brain cancers that carry a rapid dismal prognosis. Since there is a need to ...

恶性胶质瘤细胞与其周围神经元之间的相互作用越来越受到关注。在这里，我们展示了一种体外模型的发展，该模型共培养衍生的皮质谷氨酸能神经元和肿瘤细胞。用于共培养的微流体装置与多电极阵列耦合，允许研究不同的细胞类型，并在培养物的不同时间点进行电活动记录。该方法对于功能和机制研究以及分析阻断儿科高级别胶质瘤细胞迁移和与神经元相互作用的药理药物的作用特别有用。为了开始培养商业化的人IPS衍生的皮质谷氨酸能神经元，通过移液器抽吸清微流体装置的入口和出口储库，只让装置通道充满培养基。通过在培养基中每毫升悬浮液添加10微升650万人IPS细胞来播种人IPS细胞，并让设备在引擎盖下放置15分钟以允许细胞附着。15分钟后，用50微升的第四天培养基填充入口和出口储液槽，然后将设备转移到培养箱中，将细胞保持在37摄氏度和5%二氧化碳23天。如文中所述，每三到四天更换一次介质。要使用标准相差显微镜对细胞进行计数并评估活力，请在第4天，第21天和第23天拍摄显微镜照片。在DMEM和F-12 GlutaMAX中培养UW479和BT35细胞系，并在细胞培养瓶中补充10%胎牛血清。在整个实验过程中，在37摄氏度的受控环境中，在正常氧条件下保持细胞培养。观察每个细胞系以达到80%的汇合度。在谷氨酸能神经元培养后的第21天，通过胰蛋白酶消化UW479和BT35细胞开始共培养。将胰蛋白酶消化的UW479和BT35细胞接种在每个专用微流体装置中成熟贴壁谷氨酸能神经元的顶部。在37摄氏度和5%二氧化碳的受控环境中，用谷氨酸能神经元D11和更高培养基保持共培养物两天。使用图像分析软件分析的显微图片对儿科高级别胶质瘤细胞进行计数，以评估其活力并计算细胞百分比。将MFD放在记录设备中，并使用市售软件在第21天对谷氨酸能神经元进行电生理记录。在接种儿科高级别胶质瘤细胞之前，对作为与共培养平行培养的对照培养的分化谷氨酸能神经元进行第二次电生理记录。在共培养两天后的第23天进行另一次电生理记录。使用巢蛋白、SOX2、代谢性谷氨酸受体II和VGLUT1免疫染色对人IPS衍生的皮质谷氨酸能神经元进行了表征和评估。巢蛋白是一种中间丝蛋白，它在未分化的中枢神经系统细胞中表达。SOX2也在CNS中神经上皮的未分化细胞中表达。巢蛋白和SOX2阳性细胞百分比从第4天下降到第21天，证实了谷氨酸能神经元的分化状态。微观图像揭示了谷氨酸能神经元在微流体装置上的逐渐分布。当将BT35和UW479加入培养物中时，观察到第21天胶质瘤细胞接种后存在漂浮细胞，其逐渐消失直到第23天并在装置中粘附。BT35和UW479的活细胞百分比具有可比性，并暗示患者来源的细胞系可以得到适当使用。培养第23天的尖峰检测和光栅图显示，在添加儿科高级别胶质瘤细胞后，电活动增加。为了监测神经网络活动的同步性，记录了BT35或谷氨酸能神经元的瞬时放电速率。第23天，单独培养和共培养的谷氨酸能神经元之间的电活动差异显著。进一步的方法论发展可以包括网络的功能评估与突发分析和网络的特殊时间互相关。现在，研究人员有了一个有前途的工具来探索儿科高级别胶质瘤肿瘤系和高PSC神经元的相互作用和药理学靶向。可以使用几种肿瘤线和几种神经元类型。

Research

JoVE Journal

Cancer Research

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

将斑马鱼小儿脑肿瘤移植入免疫能力的宿主，用于长期研究肿瘤细胞行为和药物反应

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

科研

癌症研究

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

测定急性髓系白血病可测残留病和白血病干细胞的骨髓标本和过程综合协议

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

骨髓间充质干细胞在浆组织中的分离及与癌细胞联合培养研究其相互作用

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

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 ...

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

利用卡尔瓦里亚共同培养和教条法测量骨重塑和再造肿瘤-骨微环境

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength ...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

胶质母细胞瘤干细胞在模式神经元上共同培养，以研究迁移和细胞相互作用

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

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 ...

Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma

小儿弥漫性中线胶质瘤的活-3D细胞免疫细胞化学测定

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using ...

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

高级别浆液性卵巢癌患者来源类器官的生成和培养

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the ...

Quantifying Telomere Length Using Non-Radioactive Chemiluminescence Detection

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Optimization of Performance Parameters of the TAGGG Telomere Length Assay (Video) | JoVE 

TAGGG端粒长度测定性能参数优化

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...