Name: the slice culture method for following development of tooth germs in explant culture
Uploaded: 2013-11-13T20:30:00
Description: Watch this Scientific Journal Video about The Slice Culture Method for Following Development of Tooth Germs In Explant Culture at JoVE.com

莎拉A. Alfaqeeh是由牙科，高等教育部，沙特阿拉伯王国的沙特类大学学院资助。

1。设置<ol><li>削解剖工具（使用润滑用矿物油一刃磨石）和灭菌前用70％乙醇喷雾和干热灭菌用于器官培养。使用12 V电源用电解2M的氢氧化钠锐化解剖针。 </li><li>准备用于胚胎器官的解剖和培养的培养基，其中包括：高级Dulbecco改良的Eagle培养基F12（DMEM F12），补充有1％的GlutaMAX和1％青霉素 - 链霉素。 </li></ol> 2。胚胎解剖<ol><li>扑杀使用调度一种方法核定由内政部或其他监管?...

<ol>
	<li>Rothova, M., Peterkova, R., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+map+of+the+dental+mesenchyme:+dynamic+development+of+the+dental+papilla+and+follicle.">Fate map of the dental mesenchyme: dynamic development of the dental papilla and follicle.</a> Developmental biology. 366, 244-254 (2012).</li><li>Ferguson, C. A., 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=Activin+is+an+essential+early+mesenchymal+signal+in+tooth+development+that+is+required+for+patterning+of+the+murine+dentition.">Activin is an essential early mesenchymal signal in tooth development that is required for patterning of the murine dentition.</a> Genes &amp; development. 12, 2636-2649 (1998).</li><li>Trowell, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+culture+of+mature+organs+in+a+synthetic+medium.">The culture of mature organs in a synthetic medium.</a> Experimental cell research. 16, 118-147 (1959).</li><li>Matalova, E., Antonarakis, G. S., Sharpe, P. T., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+of+primary+and+secondary+enamel+knots.">Cell lineage of primary and secondary enamel knots.</a> Developmental dynamics : an official publication of the American Association of Anatomists. 233, 754-759 (2005).</li><li>Mitsiadis, T. A., Tucker, A. S., De Bari, C., Cobourne, M. T., Rice, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+regulatory+relationship+between+Tbx1+and+FGF+signaling+during+tooth+morphogenesis+and+ameloblast+lineage+determination.">A regulatory relationship between Tbx1 and FGF signaling during tooth morphogenesis and ameloblast lineage determination.</a> Developmental biology. 320, 39-48 (2008).</li><li>Diep, L., Matalova, E., Mitsiadis, T. A., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+tooth+bud+mesenchyme+to+alveolar+bone.">Contribution of the tooth bud mesenchyme to alveolar bone.</a> Journal of experimental zoology. Part B, Molecular. 312B, 510-517 (2009).</li><li>Rothova, M., Feng, J., Sharpe, P. T., Peterkova, R., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+mesoderm+to+the+developing+dental+papilla.">Contribution of mesoderm to the developing dental papilla.</a> The International journal of developmental biology. 55, 59-64 (2011).</li><li>Buchtova, M., 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=Initiation+and+patterning+of+the+snake+dentition+are+dependent+on+Sonic+hedgehog+signaling.">Initiation and patterning of the snake dentition are dependent on Sonic hedgehog signaling.</a> Developmental biology. 319, 132-145 (2008).</li><li>Buchtova, M., Stembirek, J., Glocova, K., Matalova, E., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+regression+of+the+dental+lamina+underlies+the+development+of+diphyodont+dentitions.">Early regression of the dental lamina underlies the development of diphyodont dentitions.</a> Journal of dental research. 91, 491-498 (2012).</li><li>Cho, S. W., 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+primary+enamel+knot+determines+the+position+of+the+first+buccal+cusp+in+developing+mice+molars.">The primary enamel knot determines the position of the first buccal cusp in developing mice molars.</a> Differentiation; research in biological diversity. 75, 441-451 (2007).</li><li>Sakano, M., 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=Cell+dynamics+in+cervical+loop+epithelium+during+transition+from+crown+to+root:+implications+for+Hertwig's+epithelial+root+sheath+formation.">Cell dynamics in cervical loop epithelium during transition from crown to root: implications for Hertwig's epithelial root sheath formation.</a> Journal of periodontal research. , (2012).</li><li>Fisher, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+development+in+vitro+of+the+whole+and+halved+lower+molar+tooth+germ+of+the+mouse.">Morphological development in vitro of the whole and halved lower molar tooth germ of the mouse.</a> Archives of oral biology. 16, 1481-1496 (1971).</li><li>Coin, R., Schmitt, R., Lesot, H., Vonesch, J. L., Ruch, J. 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齿培养的本方法具有获得牙胚的优点是优异的，从而允许精确的谱系追踪和内上皮或间质珠放置。显影牙胚的限定的区域，因此可以专门针对。在文化牙胚的形态变化可以遵循，以及操作的效果快速评估。 的方法，然而，这只是适合于大量形成硬组织如牙质和釉质前年轻牙胚，因为这些不能准确地切碎。对于下颌骨后E15.5有必要砍前去除骨头。这具有潜在的破坏性的牙胚，这...

对于一些实验，它能够培养组织体外遵循发展是重要的。发展组织的培养提供了访问在发展的定义期间，允许操纵基因通过添加因子的培养基中，或在负载的珠子，以及通过使用转染和电穿孔1。许多实验都必须能以可视化的组织，它的增长，例如，遵循谱系标记的细胞的命运的组织形态发生是很重要的。这可以是特别有问题的组织是开发深胚胎内，这是不很明显，当组织从胚胎的块进行培养。牙齿是很好的例子，因为他们的下颌骨，上颌骨和额鼻过程中的发展。当整个下颚被培养在牙的表面上的结构可以被看作但切片固定组织后改变形态只能分析<s了&gt; 2。我们已经适应一个活切片培养技术使我们能够遵循牙胚发育和发育过程中提供接入到牙齿的不同部位。该技术培养牙胚切片在气-液界面，使用改进的Trowell方法3。这些切片培养已在直接跟随牙的形态发生，并允许不同的组件，如釉质结节与牙乳头和滤泡1,4-7的谱系追踪是非常有益的。该技术不仅限于小鼠胚胎，并已成功地用于猪的文化活切片和蛇的牙齿组织8,9。除了具有能够直观牙齿发育的好处，该切片法也具有这样的薄片组织的增加访问营养素从介质和空气从培养箱的优点。这将导致在牙胚，这符合开发在体内 ，并显示invas的改善的生长离子内皮细胞进入乳头7。相反，在整个下颌骨培养牙胚发育比那些在体内更慢和文化的中心通常是坏死的长期培养物。在切片培养，齿颚片内形成的，其与周围的显影骨和其他组织的相互作用可以被监控。在我们的方法中组织被切除，而无需在支持介质10,11嵌入和不需要任何系统的组织附着在砧板直后切碎。该方法因此无创，快速，让很多下颌骨在一个会话中切片。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>101077Y</td>
			<td>100% ethanol was diluted in distilled H2O to 70%.</td>
		</tr>
		<tr>
			<td>DMEM F12</td>
			<td>Gibco</td>
			<td>12634-010</td>
			<td>Advanced Dulbecco&#39;s Modified Eagle Medium F12</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Sigma</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>DiI (Molecular probes)</td>
			<td>Vybrant</td>
			<td>V-22885</td>
			<td>Cell-labeling solution</td>
		</tr>
		<tr>
			<td></td>
			<td>Invitrogen</td>
			<td></td>
			<td>Cell tracker CM-DiI, C-7000</td>
		</tr>
		<tr>
			<td>DiO (Molecular probes)</td>
			<td>Vybrant</td>
			<td>V22886</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Geminator 500</td>
			<td>Thomas</td>
			<td>Thomas No. 3885A20</td>
			<td>Dry-heat sterilization</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>McIlwain tissue chopper</td>
			<td>Ted Pella, Inc.</td>
			<td>10180</td>
			<td>Standard table</td>
		</tr>
		<tr>
			<td>Organ culture dish (Center-Well Organ Culture Dish)</td>
			<td>Falcon</td>
			<td>353037</td>
			<td></td>
		</tr>
		<tr>
			<td>Membranes (Cell Culture Inserts, 0.4 &#956;m pore size)</td>
			<td>BD Falcon</td>
			<td>353090</td>
			<td>PET track-etched membrane, 6-well format</td>
		</tr>
		<tr>
			<td>Metal grids (Stainless Steel - AISI 304 - Mesh)</td>
			<td>Goodfellow</td>
			<td>FE228710</td>
			<td>(Fe/Cr18/Ni10)</td>
		</tr>
		<tr>
			<td>AutoFlow Direct Heat CO2 Incubator</td>
			<td>Nuaire</td>
			<td>NU-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Picospritzer III</td>
			<td>Intracel Ltd</td>
			<td>051-0500-900 0-100 psi</td>
			<td>Single channel picospritzer III</td>
		</tr>
		<tr>
			<td>Glass capillary with filament 1 mm</td>
			<td>WPI</td>
			<td>TW100F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten wire 0.1 mm</td>
			<td>Goodfellow</td>
			<td>W005138</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten wire 0.38 mm</td>
			<td>Goodfellow</td>
			<td>W005155</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirator tubes</td>
			<td>Sigma</td>
			<td>A5177</td>
			<td>Used for mouth aspiration lineage tracers</td>
		</tr>
	</tbody>
</table>

the slice culture method for following development of tooth germs in explant culture

外植体培养，因此可以操纵开发机构在特定的时间点，是对发育生物学家的一个重要方法。对于许多器官也难以进入显影组织，以允许在体外培养过程中监测。切片培养法允许访问的组织，这样形态的动作可以遵循和特定细胞群可以有针对性地进行操作或谱系跟踪。 在本文中，我们描述切片培养的方法，它一直是在一个范围内的物种牙胚的文化非常成功。该方法提供了极好的访问牙胚，它以类似的速度，以在体内观察到的，由其他组织下巴周围发展。这允许要监视的牙齿和周围组织之间的组织的相互作用。虽然本文集中于牙胚，相同的协议可以被应用到遵循的一些发展其他器官，如唾液腺，麦氏软骨，鼻腺，舌和耳。

为了按照第一磨牙牙囊的运动，为250μm额叶切片使用上述的方法通过下颌骨服用。的牙囊是间充质包围的外釉质上皮显影齿的（OEE）的层，并且先前已被证明参与牙周6的组织的形成。下颌骨解剖在E14.5，牙齿发育的帽状期。在该切片的牙科用上皮的轮廓是清楚的，并且冷凝牙齿间充质可以被识别为周围的牙上皮细胞（ 图4A）较暗的环。的DiI注射在旁的舌侧（ 图4A）</stron...

Watch this Scientific Journal Video about The Slice Culture Method for Following Development of Tooth Germs In Explant Culture at JoVE.com

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture (Video) | JoVE

在这里，使用组织斩波我们详细的方法在下颌切片文化牙胚。这种方法允许独特的访问牙齿发育过程中，为操作和谱系跟踪，不可使用更传统的培养方法极好的机会。

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