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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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?...

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 border="0" cellpadding="0" cellspacing="0">
	<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&#38;D Systems</td>
			<td>1156-NG-100</td>
			<td>Human and Rat beta-NGF (R&#38;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</em...

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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 ...

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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.