Name: a neuronal and astrocyte co-culture assay for high content analysis of neurotoxicity
Uploaded: 2009-05-05T00:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity at JoVE.com

作者想感谢博士。里克瑞安Umesh制作帕特尔，杰夫蒂尔，马修许，汉吉尔卡Mistry在发展这些项目和协议的支持和Michele Hatler。

细胞制备： <ol><li>直到〜70％至80％融合（细胞检测，文化生长介质中的星形胶质细胞神经​​元/播种前，除非细胞从解冻或隔离直接播种）。 </li><li>通过方法适合于细胞类型的兴趣分离细胞培养瓶/板。如果有必要，大衣检测井盘与D -聚赖氨酸或细胞外基质蛋白，增强细胞的粘附。联合培养的星形胶质细胞和神经元可能同时或顺序号种子。对于连续的种子队，电镀的第一层作为“基础”的星形胶质细胞是建议，由神经元的电镀和分化，如有必要。细胞类型，随后的培养时间，感兴趣的参数进行适当调整细胞密度等，根据文化，年龄，细胞来源和播种密度，小学文化的差异很大，GFAP表达或突起生长增殖率 - 是很重要的表征和优化您的电池系统，提供为您的实验模型中大多数生物的相关性，以及提供有效的成像，分割和使用的HCS（见图1）分析。加入细胞板（种子细胞可能在90μL的媒体，以促进毒素治疗，在下面的步骤3中所述）后，使板在室温下在水平表面上坐15-30分钟，使即使是细胞分布。在此期间，孵育细胞生长介质中至少24小时（37 ° C / 5％的CO 2），然后切换到分化培养条件下（例如，低血清/ NGF对PC12细胞），适当的。继续文化，直到细胞达到所需的合流或分化水平，改变在适当的时间间隔为细胞类型的媒体。 </li><li>细胞治疗（控制化合物，测试化合物等）可在任何时候，这种文化期间，利息的处理时间，当然适当。丙烯酰胺，双氧水和K - 252A是作为神经毒性控制化合物。提供了充足的试剂是重复的12点的剂量反应曲线（包括一个 生署2 O或DMSO控制范围内设定的剂量反应）所有五个96孔板。该化合物是提供250X的浓度（K - 252A，假设为1μm的最大处理）或10X（丙烯酰胺和过氧化氢，假设分别为100毫米和10毫米，最大的治疗）。推荐的治疗准备工作涉及半的日志（1：√10）连续稀释250X化合物在DMSO，复方稀释液稀释10X（10X控制卫生署2的化合物可能会连续稀释后，直接在无菌的dh 2 O或复方稀释液）。每次治疗10μL，然后可能被加入到90μL，每口井已经存在的文化传媒，作最后的1X浓度（0.4％DMSO或10％DH 2 O ）。样本数据是在37 ° C之前，以固定为24或96小时复方治疗。 </li></ol>细胞固定和免疫荧光染色： 注：染色时间约2.5小时后固定。不要让井干染色步骤之间。试剂的愿望和豁免应进行低流速，以减少由于流体剪切任何细胞的损失 。 所有建议“是指每以及”卷单井的96孔微孔板。所有“建议每96孔板”卷包括液体处理量损失超过25％。所有的染色步骤是在室温（RT） 。所有缓冲区和抗体的解决方案是稳定的，在室温下至少24小时。 <ol start="4"><li>在文化时期的结束，预温HCS固定的解决方案（2X）室温（RT）或37 ° C，如果需要的话（12 mL/96-well板）。在化学通风柜，加入100μL/孔直接向文化传媒，并允许在室温下30分钟的修复。删除固定液/毒素包含媒体，并在符合危险废物的规定处置（见MSDS）。如果立即着手染色，冲洗两次，每孔200μL的HCS免疫缓冲区。另外，如果板沾在稍后的时间，冲洗两次与200μL洗涤缓冲液，然后离开井和存储板，紧紧密封在4 º C，直到染色第二次冲洗量。 </li><li>如果固定样本已储存在4℃染色前，200μL的HCS免疫缓冲区冲洗两次，然后进行染色协议。 </li><li>准备工作βIII兔抗微管蛋白/鼠标抗GFAP HCS（6 mL/96-well板）主要抗体的解决方案如下：每个解冻的抗体加入60微升至5.88毫升的HCS免疫缓冲区。拌匀。删除以前的免疫缓冲液冲洗。加入50μL小学抗体溶液到每口井和1小时在室温下孵育。 </li><li>删除第一抗体解决方案。用200μLHCS免疫缓冲区冲洗三次。 </li><li>准备工作的HCS中抗体/赫斯特的HCS核染色（6 mL/96-well板）的解决方案如下：每个解冻二级抗体加入30μL和30μL解冻赫斯特的HCS核染色5.91毫升的HCS免疫缓冲区。拌匀，由轻保护的解决方案。删除以前的HCS免疫缓冲区冲洗。加入50μL的HCS中抗体/赫斯特的HCS核染色溶液，孵育1小时，在避光RT。 </li><li>删除的HCS中学抗体/赫斯特HCS核染色解决方案。用200μLHCS免疫缓冲液冲洗两次。 </li><li>删除以前的HCS免疫缓冲区冲洗。用200μL的HCS洗涤缓冲液冲洗两次，留下第二次冲洗量在井。 </li><li>立即密封板和形象，或存放在4℃板避光，直到成像准备。 </li></ol>图像采集和分析： <ol start="12"><li>染色板的成像和分析，可能是各种可用的HCS平台，包括细胞分析仪（通用电气医疗集团），ArrayScan（ThermoFisher科学）或歌剧（Perkin Elmer公司）后执行。在表1提供的成像和分析的一些指导方针： </li></ol><table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td style="background:#000000; color:#FFFFFF;" colspan="4"> HCS222图像采集准则 </td></tr><tr><td> 检测试剂 </td><td> 物镜 </td><td> 激发滤光片范围[峰/带宽（NM）] </td><td> 排放过滤范围[峰/带宽（nm）] </td></tr><tr><td>赫斯特的HCS核染色</td><td> 20X </td><td>四十○分之三百六十○ </td><td>四十零分之四百六十○（或五十分之五百三十五如有必要） </td></tr><tr><td> HCS二抗，FITC标记的驴抗兔IgG </td><td> 20X </td><td>四十零分之四百八</td><td>五十零分之五百三十五</td></tr><tr><td> HCS二抗，Cy3标记的驴抗小鼠IgG </td><td> 20X </td><td>五十零分之五百三十五</td><td>五十○分之六百</td></tr></tbody></table> <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td style="background:#000000; color:#FFFFFF;" colspan="4" width="545"> HCS222图像分析准则 </td></tr><tr><td> 晶胞参数 </td><td> 检测 </td><td> 分割/测量 </td><td> 理由 </td></tr><tr><td>细胞数，核特性</td><td>赫斯特的HCS核染色</td><td>核区（460 nm发射通道）。计算原子核的数目。 DNA含量（核武器强度）或核领域的分析，也有可能。 </td><td>使用细胞数，核特性，以确定细胞的损失，毒性表型等，可以“过滤器”βIII-微管蛋白或GFAP表达获得单独的神经元和星形胶质细胞计数/刻画（强烈推荐）的原子核。 </td></tr><tr><td> βIII-微管蛋白的表达，突起生长</td><td> HCS二抗，FITC标记的</td><td>胞内区（535 nm发射通道）。 FITC标记的信号可能是用来区分突起的神经元细胞体（例如，通过最小/平均细胞的身体部位，最小/最大突起的长度和宽度）。确定参数，如总长度突起，突起计数/细胞等</td><td>突起生长的测量可在神经细胞分化过程中或化学性损伤，疾病状态等调制可以“过滤”βIII-微管蛋白的表达，获得不同的神经元的特性（强烈推荐）的胞体。 </td></tr><tr><td> GFAP的表达，星形胶质细胞区 </td><td> HCS二抗，Cy3标记的共轭 </td><td>胞内区（600 nm发射通道）。 Cy3标记的信号可以用来定义星形胶质细胞的细胞质分割。确定参数，如平均细胞质的信号强度，细胞面积，等</td><td> GFAP的表达和星形胶质细胞面积的星形胶质细胞的发展，或作为一种化学性损伤，疾病状态的结果可能会在调制，等“过滤器”与GFAP的表达，获得独特的星形胶质细胞的特性（强烈推荐）的细胞体。 </td></tr></tbody></table> 表1图像采集和分析指南- HCS222βIII-Tubulin/GFAP（联合培养）含量代表性的成果： <img src="/files/ftp_upload/1173/figure31173.png" alt="图3" /> 图1。 βIII-微管蛋白和GFAP immunofluorescence的混合大鼠神经细胞培养。 镀前几天神经播种在文化，星形胶质细胞所产生的原发性大鼠海马神经元或大鼠PC12细胞的原代大鼠海马星形胶质细胞共培养。主神经元培养一个额外的11天，而PC12s培养分化条件下（低血清/ NGF）增加2天。根据HCS222检测协议进行处理，固定和免疫细胞。细胞在细胞分析仪1000（3.3），20X（主神经元）或10X（PC12）物镜成像上的GE和分析细胞分析仪1000工作站（3.4）轴突生长和多目标分析算法中使用了GE顶板。赫斯特的HCS核染色（蓝色），βIII-微管蛋白（绿色）和GFAP（红色）荧光的融合图像。单独分析βIII-微管蛋白的荧光通道，允许突起生长分割（中间面板：概述蓝（初级神经元的胞 ​​体）或黄色（PC12），绿色（主神经元）或淡蓝色（PC12 ）的突起）。 GFAP的渠道分析允许星形胶质细胞的分割（ 底部面板：细胞核，细胞质蓝色概述绿色）。 GFAP的原代大鼠海马神经元/星形胶质细胞的共同文化的分割演示的能力，区分核/胞体，GFAP（+），（绿色概括）和GFAP（ - ）（红色），使不同的细胞计数神经元和星形胶质细胞混合培养设置。 <img src="/files/ftp_upload/1173/figure41173.png" alt="图4" /> <img src="/files/ftp_upload/1173/figure4.51173.png" alt="图4第2部分" /> 图2。海马神经元/星形胶质细胞共同培养有毒强调的原代大鼠的剂量反应。 原代大鼠海马星形胶质细胞接种于96孔板（六）在生长介质和培养6天。原代大鼠海马神经元（直接从解冻），然后上方的镀前的星形胶质细胞，接种于培养基中培养11天。细胞经连续稀释的丙烯酰胺或过氧化氢的文化在过去24小时（最大浓度= 100mm和10mm的分别）。根据HCS222检测协议进行处理，固定和免疫细胞。细胞成像上的GE细胞分析仪1000（3.3），20X（10场/孔）和分析（核/胞浆/突起分割）使用GE的细胞分析仪1000工作站（3.4）轴突生长和多目标分析算法。所列数据是平均± SEM，N = 3。多个参数（突起生长，GFAP的强度，星形胶质细胞面积和神经元或星形胶质细胞计数）为剂量依赖性的发展趋势进行了调查。

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M., Gass&#243;, S., V&#237;lchez, D., Rodr&#237;guez-Farr&#233;, E., Sanfeliu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotoxic+effects+of+trimethyltin+and+triethyltin+on+human+fetal+neuron+and+astrocyte+cultures:+a+comparative+study+with+rat+neuronal+cultures+and+human+cell+lines.">Neurotoxic effects of trimethyltin and triethyltin on human fetal neuron and astrocyte cultures: a comparative study with rat neuronal cultures and human cell lines.</a> Toxicol Lett. 152, 35-46 (2004).</li></ol>

作者Millipore公司的所有员工。

迄今为止， 在体外实验旨在研究使用混合培养的神经元和星形胶质细胞神经毒性风险评估中已有限。这是广为接受的神经胶质细胞中不可分割的神经元提供了空间和代谢支持，并在调节神经功能，包括神经细胞迁移，分化和突触活动的发挥至关重要的作用。胶质细胞的星形胶质细胞也释放细胞因子和其他可溶性因子，这是许多毒素的神经诱导周围神经组织的不良反应，以及促进双方的宽容能力。展示的神经胶质细胞相互作用的深入，合作文化的研究，星形胶质细胞已证明，以保护神经元免受氧化应激的毒性，而星形胶质细胞的功能，如抗氧化防御和细胞能量水平的维护，减值已被证明看房影响神经元的存活。最近的研究表明，星形胶质细胞在体外培养的神经毒性测试系统的使用可能被证明更切合人体中枢神经系统的结构和功能的神经细胞，单独。尽管越来越多的神经元，星形胶质细胞相互作用的生理意义的认识，以前一直难以执行这些相互作​​用的定量分析，在完整的细胞。高通量筛选的到来，使发展的强大的新的分析方法来解决这个问题。 在这项研究中，我们已经表明，HCA的可能是由多个神经元和星形胶质细胞在一个单一的检测表型的定量分析。在初级神经元，我们已经完成了的神经元的数量，神经轴突计数和突起的长度，如神经毒性标志的定量测量。在星形胶质细胞，反应性胶质化是一个已知的神经毒性的生物标志物的特点，GFAP的表达，星形胶质细胞的数量和星形胶质细胞形态的改变。我们已经证明，这些端点的每个人都可以很容易地通过HCA的评估。这些研究支持神经特异性的生物标志物HCA，可在体外试验中使用的电池的一部分，迅速 ​​屏幕为神经中毒的端点化学品的大量的概念。 Millipore公司的HCS222为神经元和星形胶质细胞共培养高含量分析试剂盒是由高品质的检测试剂和协议，同时在多种神经毒性的细胞模型的分析βIII-微管蛋白和胶质纤维酸性蛋白（GFAP）。高度验证，该试剂盒提供的试剂，允许用户规范检测，减少检测，检测变异，再现产生一个高信号背景比的图像。

<table><tbody><tr><td>Rabbit Anti-&amp;#946;III-Tubulin HCS Primary Antibody, 100X</td><td></td><td>Part No. CS201672</td><td>1 vial containing 300 &amp;#x03BC;L&lt;br/&gt;Millipore HCS222 Kit Component</td></tr><tr><td>HCS Secondary Antibody (donkey anti-rabbit IgG, FITC conjugate), 200X</td><td></td><td>Part No. CS201649</td><td>1 vial containing 150 &amp;#x03BC;L&lt;br/&gt;Millipore HCS222 Kit Component</td></tr><tr><td>Mouse Anti-GFAP HCS Primary Antibody, 100X</td><td></td><td>Part No. CS201671</td><td>1 vial containing 300 &amp;#x03BC;L&lt;br/&gt;Millipore HCS222 Kit Component</td></tr><tr><td>HCS Secondary Antibody (donkey anti-mouse IgG, Cy3 conjugate), 200X</td><td></td><td>Part No. CS200437</td><td>1 vial containing 150 &amp;#x03BC;L&lt;br/&gt;Millipore HCS222 Kit Component</td></tr><tr><td>H&amp;#8211;chst HCS Nuclear Stain, 200X</td><td></td><td>Part No. CS200438</td><td>1 vial containing 150 &amp;#x03BC;L&lt;br/&gt;Millipore HCS222 Kit Component</td></tr><tr><td>HCS Fixation Solution with Phenol Red, 2X</td><td></td><td>Part No. CS200434</td><td>1 bottle containing 100 mL&lt;br/&gt;Millipore HCS222 Kit Component</td></tr><tr><td>HCS Immunofluorescence Buffer, 1X</td><td></td><td>Part No. CS200435</td><td>1 bottle containing 1000 mL&lt;br/&gt;Millipore HCS222 Kit Component</td></tr><tr><td>HCS Wash Buffer, 1X</td><td></td><td>Part No. 2007643</td><td>1 bottle containing 500 mL&lt;br/&gt;Millipore HCS222 Kit Components</td></tr><tr><td>Acrylamide (Control Compound), 10X</td><td></td><td>Part No. CS201679</td><td>1 vial containing 500 &amp;#x03BC;L of 1M acrylamide in dH&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt;Millipore HCS222 Kit Component</td></tr><tr><td>Hydrogen Peroxide (Control Compound), 10X</td><td></td><td>Part No. CS201730</td><td>1 vial containing 500 &amp;#x03BC;L of 100 mM hydrogen peroxide in dH&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt;Millipore HCS222 Kit Components</td></tr><tr><td>K-252a (Control Compound), 250X</td><td></td><td>Part No. CS201741</td><td>1 vial containing 100 &amp;#x03BC;L of 250 &amp;#x03BC;M K-252a in DMSO&lt;br/&gt;Millipore HCS222 Kit Component</td></tr><tr><td>DMSO for Compound Serial Dilution</td><td></td><td>Part No. CS200441</td><td>1 bottle containing 10 mL&lt;br/&gt;Millipore HCS222 Kit Component</td></tr><tr><td>Compound Dilution Buffer</td><td></td><td>Part No. CS200442</td><td>1 bottle containing 25 mL&lt;br/&gt;Millipore HCS222 Kit Component</td></tr><tr><td>Plate Sealers</td><td></td><td>Part No. CS200443</td><td>10 each&lt;br/&gt;Millipore HCS222 Kit Components</td></tr><tr><td>tissue culture-treated black/clear bottom microplates </td><td></td><td></td><td></td></tr><tr><td>HCS imaging/analysis system</td><td></td><td></td><td></td></tr></tbody></table>

a neuronal and astrocyte co-culture assay for high content analysis of neurotoxicity

高含量的分析（HCA）的分析相结合细胞自动成像和强大的图像分析算法和检测试剂，允许在一个单一的检测多个细胞表型的测量。在这项研究中，我们利用HCA的发展为神经毒性的新的分析。神经毒性评估的药物安全性评价的重要组成部分，以及作为一个环保工作的重要重点。此外，神经也是一个广为接受的的<em&gt;在体外</em&gt;标记的神经退行性疾病如阿尔茨海默氏症和帕金森氏病的发展。最近据报道，HCA的神经元的筛查中的应用。 HCA的检测标签βIII-微管蛋白的神经细胞，可以提供高通量，非主观的神经元的数量，神经轴突计数和突起的长度，所有这些都表明神经毒性作用，如参数的定量测量。然而，星形胶质细胞的作用仍然未开发的，在这些车型。星形胶质细胞具有不可或缺的作用，在维护中枢神经系统（CNS）的动态平衡，当他们在应对有毒物质或疾病状态激活的神经保护作用和neurodegradation关联。 GFAP的是一种中间丝蛋白，主要表达在中枢神经系统的星形胶质细胞。星形胶质细胞的活化（胶质细胞增生），GFAP的表达上调，通常伴有星形胶质细胞增生和肥大。这种反应性胶质化的过程中已经提出的神经系统造成损害的早期标志。 GFAP的定量分析的传统方法是通过免疫分析。这种方法无法提供信息，对细胞定位，形态和细胞数量是有限的。我们确定，HCA可以用来克服这些限制，并同时测量多个与胶质细胞增生相关的功能 - GFAP表达的变化，星形胶质细胞肥大，和星形胶质细胞的增殖 - 在一个单一的检测。在共同的文化研究，星形胶质细胞已被证明，以防止有毒侮辱的几种类型的神经元和批判性的影响神经元的存活。最近的研究表明，在使用的星形胶质细胞<em&gt;在体外</em&gt;神经毒性测试系统可能证明比单独的神经细胞对人体的中枢神经系统的结构和功能。因此，我们已经开发出神经元和星形胶质细胞共培养HCA的检测，包括协议和验证，目标具体分析βIII-微管蛋白和胶质纤维酸性蛋白（GFAP）检测试剂。此实验可同时分析神经轴突生长，神经胶质增生，神经元和星形胶质细胞的形态和在多种细胞模型的神经元和星形胶质细胞的发展，一种新型的，非主观的，高通量检测神经毒性评估。该试剂盒拥有增强检测神经毒性和神经科学的研究和药物发现提高生产力的巨大潜力。

Watch this Scientific Journal Video about A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity at JoVE.com

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

本文介绍了一种新的协议和试剂，为敏感的神经毒性作用的化合物和治疗联合培养的神经元和星形胶质细胞使用高含量的分析测量设计。结果表明，高含量的分析，代表着一个令人兴奋的新技术，神经毒性评估。

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

a-neuronal-astrocyte-co-culture-assay-for-high-content-analysis

Research

JoVE Journal

Biology

25.1K 次观看.  Millipore Inc.  日本語 

视频: A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is very poor 1. The failure of axon regeneration is a combined result of several factors including the hostile glial cell environment, inhibitory myelin related molecules and decreased intrinsic neuron regenerative capacity 2. Astrocytes are the most predominant glial cell type in central nervous system and play important role in axon functions under physiology and pathology conditions 3. Contrast to the homologous oligodendrocytes, astrocytes are a heterogeneous cell population composed by different astrocyte subpopulations with diverse morphologies and gene expression 4. The functional significance of this heterogeneity, such as their influences on axon growth, is largely unknown.
To study the glial cell, especially the function of astrocyte heterogeneity in neuron behavior, we established a new method by co-culturing high purified dorsal root ganglia neurons with glial cells obtained from the rat cortex. By this technique, we were able to directly compare neuron adhesion and axon growth on different astrocytes subpopulations under the same condition.
In this report, we give the detailed protocol of this method for astrocytes isolation and culture, dorsal root ganglia neurons isolation and purification, and the co-culture of DRG neurons with astrocytes. This method could also be extended to other brain regions to study cellular or regional specific interaction between neurons and glial cells.

In this protocol, we described a new method to study the influence of glial cell heterogeneity on axon growth with an in vitro co-culture system. Rat cortical glial cells were cultured to confluence and cocultured with highly purified rat dorsal root ganglia neurons. Different glial cell influence on neurons adhesion and axon growth was compared directly in the same culture. This method provides a new way to directly study the glial cell heterogeneity influence on neuron adhesion and axon growth.

研究胶质细胞轴突的生长异质性的影响，使用一个新的共培养法

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is ...

科研

神经科学

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

神经元对神经胶质细胞在微流控文化互动平台（MCP）的神经元轴突和神经胶质细胞共培养系统的影像学分析

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

一个多室中枢神经系统的神经胶质细胞共培养的微流体平台

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

生物学

A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides reliable analysis of neuronal morphology together with quantitative measurements of modifications on neurite length and synaptic protein localization and abundance upon treatment with small molecule compounds. In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment can be performed to assess, distinguish and rank commercial chemical compounds based on their potential developmental neurotoxicity effect.
Even though cell lines are nowadays widely used in compound screening assays in neuroscience, they often differ genetically and phenotypically from their tissue origin. Primary cells, on the other hand, maintain important markers and functions observed in vivo. Therefore, due to the translation potential and physiological relevance that these cells could offer neurite outgrowth assay and neurotoxicity assessment can considerably benefit from using human neural progenitor cells (hNPCs) as the primary human cell model.
The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology.&#34;

The presented protocol describes a method for a neurite outgrowth assay and neurotoxicity assessment of small molecule compounds.

神经石生长分析与神经毒性评估与人类神经原生细胞衍生神经元

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides ...

发育生物学

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

间接神经元的星形胶质细胞共培养试验:一个<em&gt;体外</em&gt;设置 - 为神经元的神经胶质细胞相互作用的详细调查

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

研究神经-胶质相互作用在缺血中的作用的细胞培养模型

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

A Scalable Method to Study Neuronal Survival in Primary Neuronal Culture with Single-cell and Real-Time Resolution

Neuronal loss is at the core of many neuropathologies, including stroke, Alzheimer's disease, and Parkinson's disease. Different methods were developed to study the process of neuronal survival upon cytotoxic stress. Most methods are based on biochemical approaches that do not allow single-cell resolution or involve complex and costly methodologies. Presented here is a versatile, inexpensive, and effective experimental paradigm to study neuronal survival. This method takes advantage of sparse fluorescent labeling of the neurons followed by live imaging and automated quantification. To this aim, the neurons are electroporated to express fluorescent markers and co-cultured with non-electroporated neurons to easily regulate cell density and increase survival. 
Sparse labeling by electroporation allows a simple and robust automated quantification. In addition, fluorescent labeling can be combined with the co-expression of a gene of interest to study specific molecular pathways. Here, we present a model of stroke as a neurotoxic model, namely, the oxygen-glucose deprivation (OGD) assay, which was performed in an affordable and robust homemade hypoxic chamber. Finally, two different workflows are described using IN Cell Analyzer 2200 or the open-source ImageJ for image analysis for semi-automatic data processing. This workflow can be easily adapted to different experimental models of toxicity and scaled up for high-throughput screening. In conclusion, the described protocol provides an approachable, affordable, and effective in vitro model of neurotoxicity, which can be suitable for testing the roles of specific genes and pathways in live imaging and for high-throughput drug screening.

Here, we present a simple, potent, and versatile methodology to investigate neuronal survival upon cytotoxic stress in primary cortical neurons with cellular resolution in real time or in fixed material.

Neuronal loss is at the core of many neuropathologies, including stroke, Alzheimer's disease, and Parkinson's disease. Different methods were developed to ...

 用于 CNS 相互作用研究的原代小胶质细胞和神经元共培养方案

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting&#160;reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

作者聚焦:用于研究疾病条件下小胶质细胞-神经元相互作用的体外共培养模型

This protocol describes a microglia-neuronal co-culture established from primary neuronal cells isolated from mouse embryos at embryonic days 15-16 and primary microglia generated from the brains of neonatal mice at post-natal days 1-2.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

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