The work was funded by the University of Zurich. The authors would like to thank Estrela Neto and Dr. Meriem Lamghari for helping in establishing the co-culture conditions.

所有的小鼠根据瑞士动物福利法和符合州兽医办公室，苏黎世的规定维护和处理。 1.准备材料解剖，文化传媒，微流体装置<ol><li>高压釜显微解剖镊子和剪刀（121℃，灭菌时间20分钟），并把它们存储在无菌容器中。 </li><li>通过将它们在1M HCl中24小时，在定轨摇床上，在37℃消毒盖玻片（24毫米×24毫米）。用无菌，蒸馏水用乙醇99％冲洗三次，2 O和三次。干燥然后将盖玻片，在37℃或在无菌流罩。最后，高压灭菌或暴露的盖玻片于UV光（30分钟）以完成杀菌。盖玻片然后可被储存在70％乙醇。 </li><li>使用无菌镊子小心地从包中取出的AXIS轴突隔离设备，并将其放置在无菌培养皿。 </LI&gt; <li>使用无菌活检穿孔（直径:仅1mm）中创建的培养腔室对应每采样1孔进行培养（ 图1）。 注意:不要冲太靠近微槽，因为他们可能会被施加的压力而损坏。 </li><li>通过佛光普照他们70％乙醇消毒AXIS轴突隔离设备。干那么Axon公司AXIS分离设备和盖玻片完全在无菌流罩。等待至少3小时的，然后再继续。 注:不完全的干燥，将导致在微流体器件的装配不良。 </li><li>放置每个盖玻片到35毫米的培养皿或成孔的6孔平板内。 </li><li>将AXIS轴突隔离装置上盖玻片，并轻轻按下但坚定地用钳子用弯曲的两端，以使隔离装置和玻璃盖玻片之间的完全粘连。 </li><li>在每个培养室，吸移管150微升聚-D-赖氨酸（0.1mg / ml的无菌蒸馏水的H 2O）。放置在真空下，微流体装置进行5分钟，以便从培养腔室除去所有的空气。 </li><li>如果空气仍然可以在腔室，再吸移管内看到了聚-D-赖氨酸溶液进入腔室。 </li><li>孵育用聚-D-赖氨酸O / N的装置，在37℃。 </li><li>用无菌蒸馏水H 2 O洗室三个时间</li><li>填用150微升的层粘连蛋白的工作溶液（Sigma-Aldrich公司，5微克/毫升，在PBS或无血清培养基）腔室，并孵育​​O / N在37℃。 </li><li>制备50ml培养基的三叉神经节培养物37，组成如下:48毫升Neurobasal培养基，加入1ml的B-27，100U / ml青霉素/链霉素，2mM的L-谷氨酰胺，5纳克/毫升神经生长因子（NGF） ，下午12时25阿糖胞苷。 </li><li>制备50ml培养基的牙胚培养37，组成如下:40毫升的DMEM-F12，将10毫升胎牛血清（FBS，终浓度:20％），100U / ml青霉素/链霉素，2mM的L-谷氨酰胺，150微克/毫升抗坏血酸。 </li></ol> 2.小​​鼠胚胎生成和解剖<ol><li>根据阴道塞确定胚胎的年龄（阴道塞:0.5的发展，E0.5胚胎天），并通过形态学标准确认。对于这个协议，我们一般使用E14.5-E17.5小鼠胚胎。 </li><li>清洁清扫面积和70％乙醇的立体镜。 </li><li>通过颈椎脱位牺牲孕妈妈。鼠标与第一和第二手指的颈部阻挡到电网，和拉带尾决定。 </li><li>解剖周围皮肤下腹部，并用剪刀打开腹部。定位子宫:怀孕期间的这种后期，子宫大量填充腹腔。 </li><li>解剖出子宫，并装在填充用PBS在冰上的管中。当在冰上，可将组织放置几个小时。根据机构指引抛弃母亲的尸体</li> &lt;LI&gt;解剖出胚胎从子宫并从他们胚外组织释放他们。放置在PBS中的胚胎在冰上。 </li><li>用剪刀斩首的胚胎，并使用显微解剖剪（ 图2A）从头部的其余部分分开的下颚。除去恰恰下颌，而不损坏三叉神经节;因为后者被定位在靠近下颌，其意外损坏是可能的。保留下颚和头部在冷PBS中的其余部分，在冰上。 </li><li>解剖TG，搭头，并将其放置到一个夹层玻璃培养皿，以前充满冷PBS。使用镊子，消除皮肤和颅骨。然后取出端脑和小脑通过将端脑和电梯下面镊子;端脑和小脑将翻转起来，把裸露的头骨底部。 </li><li>本地化的三叉神经节（在图2B中示出）。使用镊子TG的从三叉神经分开。消除使用解剖针为刀三叉预测的残余。将解剖的TG在培养皿中填充有冷PBS，并保持他们在冰上。 </li><li>解剖胚胎的牙齿，把下颚，以前从头骨分离，到夹层玻璃培养皿，充满冷PBS。用解剖针的刀，去除舌头和周围的下颚皮肤。通过沿颚的中线切割分开的左和右半爪。的牙胚很容易看到， 如图1C所示 。采用分离解剖针牙胚和去除多余的非牙齿组织。将解剖牙胚在培养皿中填充有冷PBS，并保持他们在冰上。 </li></ol> 3.微流控共培养<ol><li>解剖后，从微流体装置去除粘连。填写室与200微升respecti的已经媒体。 </li><li>用钳子，转移轻轻解剖TG和牙胚成通过冲压（ 图1D）形成的孔。确保牙胚不浮动，他们下沉，直到他们联系盖玻片。 </li><li>培养在37℃培养箱的样品中，5％的CO 2。 </li><li>改变培养基每48小时。不要空室完全，也不要直接将吸取文化室。的腔室将导致内室空气气泡的形成完全排空;直接移液到腔室会导致轴突损伤。为了避免这些问题，取出指向孔的外侧的吸移管的介质;类似地，吸液管上位于相对于腔室的孔中的侧面的新鲜培养基中。 </li><li>在培养期间，共培养物可以很容易地通过时间推移显微镜成像。共培养物可以保持超过10天。 </li><li>培养后，牙周D，吹打150微升PBS为每室一井，并让PBS流过腔三次洗室。 </li><li>除去PBS中并通过吸取150微升多聚甲醛的4％（在PBS中）在每室一个阱固定的样本;在室温下孵育15分钟。 </li><li>在3.6中所述，用PBS洗两次室。 </li><li>进行进一步的分析。 </li></ol>

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D., Bianco, F., Matteoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacology+on+microfluidics:+multimodal+analysis+for+studying+celll-cell+interaction.">Pharmacology on microfluidics: multimodal analysis for studying celll-cell interaction.</a> Current opinion in pharmacology. 13 (5), 821-828 (2013).</li><li>Neto, E., Alves, C. J., 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=Sensory+neurons+and+osteoblasts:+close+partners+in+a+microfluidic+environment.">Sensory neurons and osteoblasts: close partners in a microfluidic environment.</a> Integrative Biology. , (2014).</li><li>Pagella, P., Neto, E., Jim&#233;nez-Rojo, L., Lamghari, M., Mitsiadis, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+co-culture+systems+for+studying+tooth+innervation.">Microfluidics co-culture systems for studying tooth innervation.</a> Frontiers in physiology. 5 (August), (2014).</li><li>Connor, R., Tessier-Lavigne, M. <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+maxillary+factor,+a+maxillary+process-derived+chemoattractant+for+developing+trigeminal+sensory+axons.">Identification of maxillary factor, a maxillary process-derived chemoattractant for developing trigeminal sensory axons.</a> Neuron. 24, 165-178 (1999).</li></ol>

The authors declare that they have no competing financial interests.

上一页牙神经支配的体外研究都是基于传统的联合培养三叉神经节和牙齿的组织或细胞26,28,29的。这些研究进行主要调查这些细胞或组织上的感觉轴突38上的吸引力的影响。虽然引进外地显著的进步，几个技术问题被提出。牙胚开始培养后37几天就变质。基于这些观察，生长的神经元和牙齿在相同的培养条件影响涉及这两个组织之间的串扰的分子的任何最终分析。需要最佳培养条件以保持三叉神经节和牙齿组织的生理分子谱。 微流体系统已用于迄今共培养的神经元和多种细胞类型中优化的媒体34,36。该微流体系统可以代表更多的信心充分的体内的情况下，其中，神经细胞体，轴突终端和靶组织通常暴露于不同的细胞和分子的微环境。最近，微流体装置已被用于共培养整个背根神经节和成骨细胞36。我们最近表明，三叉神经节和牙齿能够存活很长一段时间时，共培养在微流体装置37。此外，我们证明了来自不同发育阶段的牙齿在这些体外条件对三叉神经支配相同的排斥或吸引力的影响，他们体内 37展出维护。 描述的方案需要实践和手巧，特别是用于对三叉神经节和牙胚的显微解剖。三叉神经解剖时容易撕裂。因此，我们建议使用解剖针，而不是剥离力ps的，为了去除周围的神经节的组织。同样地，应特别注意，同时解剖牙胚，以避免损坏牙齿上皮而从周围间质组织中分离它服用。此外，在共培养的第一天，特别应注意在处理培养箱，因为过度的振动会影响三叉神经节粘附到培养盖玻片。在共培养期间，媒体应该被删除，通过吸取入井，相反在培养腔室的一侧加入。拟任或移液介质中的文化室接近会导致轴突损伤。 描述的协议可以被修改来研究几种胚胎的器官和胚胎或产后组织和细胞类型的神经支配。微流体系统可以代表一个合适的平台，允许更长的时间培养对相互作用的神经元和不断增长的发球的研究日。而且，从牙科室中的神经元的分离允许的特定蛋白定位和定量33,37的影响的分析。阻断性抗体或重组蛋白质，也可以加入到微流体装置的分开的隔室，用于分析其对神经元和牙齿组织的效果。例如，治疗三叉神经节神经生长因子的支持他们的生存，并允许神经元生长。然而，在这项工作中神经生长因子是从牙胚隔室，其中，齿源性信号是唯一负责神经元吸引或排斥不存在。同样，其他的重组分子，抗体或药物可用于操纵系统在控制的条件。 提出的协议的主要限制驻留在商用微流体装置的相对高的成本和基本上2D-建立共培养体系。事实上，虽然整个器官可以是培养D，两个机构和轴突生长在玻璃盖玻片2D贴壁培养条件;该系统的定制将需要以获得神经支配的立体，逼真的表示。 最后，微流体装置是最佳的用于调查神经在显影或再生机构的作用，并允许在最佳培养条件下的神经元和靶组织之间的相互作用的研究。

支配起着器官和组织1,2的发育，稳态和再生的关键作用。此外，支配参与干细胞增殖，动员和分化3的调节- 5。实际上，实现了在颜面部复杂的组织最近的研究已经表明，唾液的发育和再生腺体6,7-期间副交感神经是必要的上皮的祖细胞的功能。类似地，已经证明，神经支配是必要的味蕾8的开发和维护- 11。因此，为了分析如牙神经支配在其他重要的口面部的器官和组织的发展却忽视的作用是很重要的。 尽管成年齿的丰富的神经支配的，相反于主体，开发板的所有其它器官和组织平齿开始在最早产后阶段进行支配。牙齿作为发展的口腔外胚层和中颅神经嵴源性间充质顺序和相互交流的结果。这些相互作用产生的上皮衍生成釉细胞和间充质来源的牙本质细胞，它们负责牙釉质和牙本质，分别为12的形成。从颈上神经节三叉神经节和交感神经感觉神经支配牙齿的成人13 - 15。在胚胎发生期间，神经纤维从向显影牙胚三叉神经节项目发出并逐步包围他们，但他们不渗透到牙乳头间质13。神经纤维进入牙髓间充质在与成牙本质细胞分化和牙本质基质沉积16关联更先进的发展阶段。牙髓的神经支配是并发症eted不久后在口腔中13牙齿萌出。先前的研究已经表明，各种脑信号和神经营养蛋白牙胚16期间参与神经支配的调节- 19。早先的研究已经清楚地表明，是支配牙齿形成鱼20的先决条件。最近的研究显示，在小鼠门齿该牙齿间充质干细胞的体内平衡是由感觉神经经由音猬（SHH）21的分泌调节。然而，神经支配在齿起始，发展和再生中的作用仍然是在哺乳动物中22高度争议- 24。 体内研究的大量已经在多种动物模型13,25,26的发展和修复过程提供了有关牙齿组织的神经支配的模式的重要信息。然而，大多数这些APPRO疼痛不是最佳突出神经纤维和靶器官和组织之间的相互作用的分子基础。共培养物构成一个有价值的方法进行调查，并操纵一个受控和隔离的环境26的神经纤维和牙齿之间的相互作用- 29。与此同时，共培养受到各种技术调整。例如，神经和特定牙组织（ 例如，牙髓，牙囊，牙上皮）通常需要不同的培养基中，以保证组织存活的时间30长时间- 32。 在过去的几十年中，使用相同的培养基中常规共培养物已进行很短的时间（ 例如 ，2天），以调查对感觉神经纤维27显影口腔和牙齿组织的吸引力或排斥力的影响- 29。然而，延长培养期必须调查神经支配牙齿形态发生和细胞分化的影响，并研究神经纤维靶器官内分枝的动态。因此，非连续的共培养将是更适合于神经，牙齿组织相互作用的研究执行。 微流体系统允许共培养的神经元和不同类型的细胞在它们的合适的培养基。在这些器件中，牙组织和神经元被分离在不同的隔室，同时允许从神经细胞体通过微通道朝向含有它们的靶组织33中的隔室的轴突的生长。微流体装置已经被用于研究癌症和新生血管35的神经元和胶质细胞34,35之间的相互作用，以及细胞与细胞的相互作用。此外，这些系统已经被用于研究DORS之间的相互作用人根神经节和成骨细胞36。 我们最近证明，三叉神经节（TG）和牙齿能够存活很长一段时间时，共培养在微流体装置37。此外，我们已经表明，来自不同发育阶段的牙齿在这些体外条件对三叉神经支配同一斥力或引力作用，它们显示出在体内 37保持。该协议提供了有关一个简单，功能强大且灵活的方式，共同培养神经节/神经与靶组织，并研究在一个可控和隔离的环境互动等特定分子的角色的信息。

<table><tbody><tr><td>AXIS Axon Isolation Devices</td><td>Millipore</td><td>AX15010-TC</td><td>Microchannels of different lenght are available</td></tr><tr><td>Laminin</td><td>Sigma Aldrich</td><td>L2020</td><td></td></tr><tr><td>Neurobasal</td><td>Gibco</td><td>21103-049</td><td></td></tr><tr><td>B27</td><td>Gibco</td><td>17504</td><td></td></tr><tr><td>Recombinant Mouse beta-NGF</td><td>R&amp;amp;D Systems</td><td>1156-NG-100</td><td>Human and Rat beta-NGF (R&amp;amp;D Systems) are equivalent</td></tr><tr><td>DMEM-F12</td><td>Gibco</td><td>11320-033</td><td></td></tr></tbody></table>

analysis of developing tooth germ innervation using microfluidic co-culture devices

支配起着器官和组织的发育，稳态和再生的关键作用。然而，这些现象背后的机制尚未很好地理解爱好。特别是，在神经支配牙齿发育和再生的作用被忽略。 几个在体内的研究已经在多种动物模型的发展和修复过程提供了有关牙齿组织的神经支配的模式的重要信息。然而，大多数这些方法不是最优的，突出的神经纤维和靶器官和组织之间的相互作用的分子基础。 共培养物构成一个有价值的方法进行调查，并操纵一个受控和隔离的环境神经纤维和齿之间的相互作用。在过去的几十年中，使用相同的培养基中常规共培养物已进行很短的时间（ 例如 ，2天）研究制定对感觉神经纤维口腔和牙齿组织的吸引力和排斥力的影响。但是，延长培养期需要调查支配牙齿形态发生和细胞分化的影响。 微流体系统允许共培养的神经元和不同类型的细胞在它们的合适的培养基。我们最近证明，三叉神经节（TG）和牙齿能够存活的很长一段时间时，共培养在微流体装置，并且它们在这些条件下保持它们显示在体内同一神经支配模式。 在此基础上，我们将描述如何分离和共培养显影三叉神经节和牙胚在微流体的共培养system.This协议描述了一个简单而灵活的方式来共培养神经节/神经和靶组织，并研究的作用在对照这种相互作用的特定分子olled和孤立的环境。

这些结果表明，分离的三叉神经节用的微流体装置的一个隔室生长，此外，所分离的牙胚的发展是持续的时间在微流体装置的另一个室长。不同的培养基中使用了两个室，两个室之间的微槽允许扩展从朝显影牙胚三叉神经节轴突的。 图3表示神经丝通过免疫荧光37可视化，在鼠标的共培养胚胎三叉神经节和小鼠胚胎门齿中所描述的微流体的共培养系统。鼠标门牙发展体内期间不支配;一致，从三叉神经节轴突由显影牙胚中培养高达10天废除图3A示出了轴突室在此概述<html>共培养体系。 图3B和3C示出内轴突腔和微园的神经突的渐进倍率。牙胚（或任何共培养的器官或组织）能够从微流体装置可以容易地除去，并分别如分析，处理以用于组织学染色或用于基因表达分析。 <img alt="图1" src="/files/ftp_upload/53114/53114fig1.jpg" /> 图1.浏览安装微流体的共培养装置（A）的从上方的; （二）部分横向的文化室（CC）红（伊红）突出显示和蓝色（甲苯胺蓝）;微槽（毫克）可以被看作是在培养室之间的白线。神经节和共培养器官/组织是通过在设备中的冲压孔（PH）放置到适当的培养室中。媒体添加和删除经由所述孔（MW）。<html> "https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53114/53114fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/53114/53114fig2.jpg" /> 图2.（A）小鼠胚胎头部侧面观（发展E14.5胚胎天）。黑线表示下颚应，以保持两者三叉神经节和牙胚的完整性进行切割。 （B）小鼠胚胎头（E14.5）去除下颌，头骨和端脑后。黑色箭头指示三叉神经节（TG）。 （C）的小鼠胚胎（E14.5）下颌切除舌后。黑色箭头表示不同的牙胚的本地化（公司:中切牙; FM:第一恒磨牙）。 （D）的微流体的共培养装置的示意图。p_upload / 53114 / 53114fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53114/53114fig3.jpg" /> 图3.共培养三叉神经节，并从野生型E15.5小鼠胚胎切牙牙胚。轴突室（ 一 ）概述（神经丝，DAPI）。比例尺:300微米。 （ 二）高放大倍率的微槽和突起成长为轴突室（神经丝，DAPI;荧光灯和明图像叠加）。比例尺:150微米。 （C）高倍显示轴突的微槽（神经丝）内的增长。比例尺:75微米<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53114/53114fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices at JoVE.com

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

微流控采用共培养开发设备牙胚支配分析

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

analysis-developing-tooth-germ-innervation-using-microfluidic-co

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8.2K 次观看.  University of Zurich. Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

视频: Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

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 ...

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神经科学

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

Pediatric high-grade gliomas (pHGG) represent childhood and adolescent brain cancers that carry a rapid dismal prognosis. Since there is a need to overcome the resistance to current treatments and find a new way of cure, modeling the disease as close as possible in an in vitro setting to test new drugs and therapeutic procedures is highly demanding. Studying their fundamental pathobiological processes, including glutamatergic neuron hyperexcitability, will be a real advance in understanding interactions between the environmental brain and pHGG cells. Therefore, to recreate neurons/pHGG cell interactions, this work shows the development of a functional in vitro model co-culturing human-induced Pluripotent Stem (hiPS)-derived cortical glutamatergic neurons pHGG cells into compartmentalized microfluidic devices and a process to record their electrophysiological modifications. The first step was to differentiate and characterize human glutamatergic neurons. Secondly, the cells were cultured in microfluidic devices with pHGG derived cell lines. Brain microenvironment and neuronal activity were then included in this model to analyze the electrical impact of pHGG cells on these micro-environmental neurons. Electrophysiological recordings are coupled using multielectrode arrays (MEA) to these microfluidic devices to mimic physiological conditions and to record the electrical activity of the entire neural network. A significant increase in neuron excitability was underlined in the presence of tumor cells.

Recent works uncover the neuronal impact on high-grade pediatric glioma (pHGG) cells and their reciprocal interactions. The present work shows the development of an in vitro model co-culturing pHGG cells and glutamatergic neurons and recorded their electrophysiological interactions to mimic those interactivities.

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 ...

癌症研究

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

神经元共培养与单细胞精度的制备

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

研究对牙髓寄生虫信号反应的神经质生长的共同培养方法

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

发育生物学

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The continuously growing mouse incisor provides an excellent model to study tissue-specific stem cells. The epithelial tissue-specific stem cells of the incisor are located at the proximal end of the tooth in a niche called the cervical loop. They provide a continuous influx of cells to counterbalance the constant abrasion of the self-sharpening tip of the tooth. Presented here is a detailed protocol for the isolation and culture of the proximal end of the mouse incisor that houses stem cells and their niche. This is a modified Trowell-type organ culture protocol that enables in vitro culture of tissue pieces (explants), as well as the thick tissue slices at the liquid/air interface on a filter supported by a metal grid. The organ culture protocol described here enables tissue manipulations not feasible in vivo, and when combined with the use of a fluorescent reporter(s), it provides a platform for the identification and tracking of discrete cell populations in live tissues over time, including stem cells. Various regulatory molecules and pharmacological compounds can be tested in this system for their effect on stem cells and their niches. This ultimately provides a valuable tool to study stem cell regulation and maintenance.

The Trowell-type organ culture method has been used to unravel complex signaling networks that govern tooth development and, more recently, for studying regulation involved in stem cells of the continuously growing mouse incisor. Fluorescent-reporter animal models and live-imaging methods facilitate in-depth analyses of dental stem cells and their specific niche microenvironment.

使用Trowell型器官培养研究牙科干细胞的调节

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

切片培养法继牙胚在植文化的发展

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

生物学

A Gut-on-a-Chip Model to Study the Gut Microbiome-Nervous System Axis

The human body is colonized by at least the same number of microbial cells as it is composed of human cells, and most of these microorganisms are located in the gut. Though the interplay between the gut microbiome and the host has been extensively studied, how the gut microbiome interacts with the enteric nervous system remains largely unknown. To date, a physiologically representative in vitro model to study gut microbiome-nervous system interactions does not exist. 
To fill this gap, we further developed the human-microbial crosstalk (HuMiX) gut-on-chip model by introducing induced pluripotent stem cell-derived enteric neurons into the device. The resulting model, 'neuroHuMiX', allows for the co-culture of bacterial, epithelial, and neuronal cells across microfluidic channels, separated by semi-permeable membranes. Despite separation of the different cell types, the cells can communicate with each other through soluble factors, simultaneously providing an opportunity to study each cell type separately. This setup allows for first insights into how the gut microbiome affects the enteric neuronal cells. This is a critical first step in studying and understanding the human gut microbiome-nervous system axis.

neuroHuMiX is an advanced gut-on-a-chip model to study the interactions of bacterial, epithelial, and neuronal cells under proximal and representative co-culture conditions. This model allows unravelling of the molecular mechanisms underlying the communication between the gut microbiome and the nervous system.

用于研究肠道微生物组-神经系统轴的肠道芯片模型

The human body is colonized by at least the same number of microbial cells as it is composed of human cells, and most of these microorganisms are located ...

Establishing a Co-Culture of Dental Pulp Cells and Trigeminal Neurons for Cross-Communication

Source: Barkley, C. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/60809">A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals.</a>&#160;J. Vis. Exp. (2020).
This video showcases a technique for establishing a co-culture of trigeminal neurons and primary dental pulp cells using a transwell filter. In this physically separated condition, mesenchymal cells obtained from dental pulp tissue secrete paracrine signaling molecules that traverse through the pores, promoting the differentiation and migration of neurons towards the mesenchymal cells establishing a co-culture system.

建立牙髓细胞和三叉神经神经元的共培养以进行交叉通讯

1. Plate Preparation
NOTE: Be sure the plate lid is on during all incubation and rinsing steps outside of the sterile tissue culture hood to prevent ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Co-culture and Interaction Studies

Generating Neuromuscular Junctions Using a Microfluidic Device

Source: Stoklund Dittlau, K. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/62959">Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices.</a>&#160;J. Vis. Exp. (2021)
The video demonstrates the formation of neuromuscular junctions using a microfluidic device. Neural precursor cells and muscle progenitor cells are seeded into different wells within the device and left to mature into motor neurons and myotubes, respectively. The creation of a volume and chemical gradient leads to the development of active neuromuscular junctions.

使用微流体设备生成神经肌肉接头

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
All reagents and equipment used in ...

Advanced Neuronal Models

Analyzing Tooth Germ and Trigeminal Ganglia Interactions Using a Microfluidic Co-culture System

Source: Pagella, P., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/53114">Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices.</a> J. Vis. Exp. (2015).
This video demonstrates the utilization of a microfluidic device to study the interactions between tooth germ pulp and trigeminal ganglia. It showcases the placement of tissues inside the microfluidic device and provides a detailed visualization of trigeminal neurite growth toward the tooth germ through the device&#39;s microgrooves. Additionally, it highlights the role of tooth-derived molecular factors in promoting or inhibiting neurite extension into the tooth germ pulp, which is crucial for ensuring efficient postnatal tooth innervation.