Name: a novel ex vivo culture method for the embryonic mouse heart
Uploaded: 2013-05-24T13:00:00
Description: Watch this Scientific Journal Video about A Novel Ex vivo Culture Method for the Embryonic Mouse Heart at JoVE.com

我们想感谢Andrea波尔特布里的批判性阅读的手稿和美国国立卫生研究院（授＃R01HL061656）的资金支持。

1。切除胚胎心<ol><li>安乐死定时孕鼠在使用经批准的安乐死技术所需的胚胎一天。所有的实验都是在北卡罗莱纳大学教堂山分校的机构动物护理和使用委员会批准。 </li><li>涂抹适量的喷女性解剖前，用70％乙醇。打开女性的腹腔检索和切除子宫角。 </li><li>子宫角放置在培养皿中含有冷的1x磷酸盐缓冲盐水（PBS）漂洗根据需要。剖开子宫角，通过中线暴露所附的胚胎囊。 </li><li>剖开胚囊和检索胚...

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	<li>Tu, S., Chi, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+models+in+cardiac+development+and+congenital+heart+birth+defects.">Zebrafish models in cardiac development and congenital heart birth defects.</a> Differentiation. 84, 4-16 (2012).</li><li>Lavine, K. 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=Fibroblast+growth+factor+signals+regulate+a+wave+of+Hedgehog+activation+that+is+essential+for+coronary+vascular+development.">Fibroblast growth factor signals regulate a wave of Hedgehog activation that is essential for coronary vascular development.</a> Genes Dev. 20, 1651-1666 (2006).</li><li>Red-Horse, K., Ueno, H., Weissman, I. L., Krasnow, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+arteries+form+by+developmental+reprogramming+of+venous+cells.">Coronary arteries form by developmental reprogramming of venous cells.</a> Nature. 464, 549-553 (2010).</li><li>Stuckmann, I., Evans, S., Lassar, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythropoietin+and+retinoic+acid,+secreted+from+the+epicardium,+are+required+for+cardiac+myocyte+proliferation.">Erythropoietin and retinoic acid, secreted from the epicardium, are required for cardiac myocyte proliferation.</a> Dev. Biol. 255, 334-349 (2003).</li><li>Zhang, 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=The+FGF-BMP+signaling+axis+regulates+outflow+tract+valve+primordium+formation+by+promoting+cushion+neural+crest+cell+differentiation.">The FGF-BMP signaling axis regulates outflow tract valve primordium formation by promoting cushion neural crest cell differentiation.</a> Circ. Res. 107, 1209-1219 (2010).</li><li>Pillekamp, F., 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=Establishment+and+characterization+of+a+mouse+embryonic+heart+slice+preparation.">Establishment and characterization of a mouse embryonic heart slice preparation.</a> Cellular Physiology and Biochemistry: international journal of experimental cellular physiology, biochemistry, and pharmacology. 16, 127-132 (2005).</li><li>Habeler, W., Peschanski, M., Monville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+heart+slices+for+cell+transplantation+and+physiological+studies.">Organotypic heart slices for cell transplantation and physiological studies.</a> Organogenesis. 5, 62-66 (2009).</li><li>Janssen, P. M., Lehnart, S. E., Prestle, J., Hasenfuss, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+contractile+characteristics+of+human+myocardium+in+multi-day+cell+culture.">Preservation of contractile characteristics of human myocardium in multi-day cell culture.</a> J. Mol. Cell Cardiol. 31, 1419-1427 (1999).</li><li>Brandenburger, 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=Organotypic+slice+culture+from+human+adult+ventricular+myocardium.">Organotypic slice culture from human adult ventricular myocardium.</a> Cardiovasc. Res. 93, 50-59 (2012).</li><li>Weaver, M., Dunn, N. R., Hogan, B. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cardiac Development. , (2007).</li><li>Arima, S., 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=Angiogenic+morphogenesis+driven+by+dynamic+and+heterogeneous+collective+endothelial+cell+movement.">Angiogenic morphogenesis driven by dynamic and heterogeneous collective endothelial cell movement.</a> Development. 138, 4763-4776 (2011).</li></ol>

目前的培养体系带来了显着优势，为胚胎小鼠心脏研究。这种文化系统保留心肌收缩和冠状动脉丛，坏死的迹象有限，即使经过4天的文化。此外，半固体基质提供了足够的支持，以维持发展的心脏三维形态，同时允许灵活性，以合同，也培养期间举行涂珠。尽管这种支持，这个矩阵是可渗透的荧光染料，如祥发16，使荧光分析的活的心。 切片培养的方法将心部分中，包含其?...

近年来，已经成为了主要的转基因小鼠模型系统的研究开发心脏缺损。然而，其他的模型有机体，如斑马鱼，已被证明具有显着优于鼠标。三大优点的斑马鱼的外部铺设的鸡蛋，易于获得胚胎的胚胎的光学透明性，它可以轻松地可视化心脏发育;和便于应用小分子治疗调节胚胎的发育1。因此，文化技术的发展，使子宫外生长的胚胎器官将绕过，至少部分，目前研究转基因小鼠的发育过程中所经历的限制。 体外心脏培养系统已经开发了小鸡和小鼠胚胎中，使治疗用小分子，并分析如何不同心脏ferent地区沟通2-6。对于整个小鼠心脏培养，心取自胚胎的胚胎（E）12.5岁可以放置在培养基中有或无摆动2,3,5。使用这种技术，胚胎的心已被成功地培养的E13.5适合，只要3天（E10.5）3保持用摆动心培养。但是，没有研究已经报道了从旧的胚胎培养成功的心。同样，救援实验一直局限于，应用治疗剂的培养基2的全局。 切除心中，嵌入式和使用一个vibratome切片，一片文化体系，也被用于两个年轻的心，如E12.5小鼠心脏和汉堡 - 汉密尔顿36期（约在鼠标E16）小鸡心2,4,6，和老的心，如产后和成年小鼠H电子艺术和成人人心7,8。虽然胚胎分析通常使用150微米厚的部分2,4，截面厚度可以是一个伟大的为500微米，没有证据表明缺氧8。这些切片文化一直保持，只要培养两个月，与大多数片保持收缩整个这个期间9。在孤立的心肌细胞的研究相比，这些切片培养允许与它们相邻的类型的细胞的心肌细胞的共培养物，并提供一种有用的方法，用于体外分析。然而，这些文化需要更精细的设置不是简单地放置一个心培养液中（ 例如，一个vibratome切片嵌入活的心），显然是有限的，任何人分析的段内的部分的心。 鉴于上述限制，培养小鼠胚胎的心和丰富的转基因小鼠AVA研究ilable，我们开发了体外小鼠心脏文化系统类似韦弗开发体外肺癌文化的系统， 等。10我们的文化系统允许在整个胚胎小鼠心脏重塑冠状动脉循环的长期培养和可视化。此外，基质胶的使用允许心脏的地方附近的内举行的有孔玻璃珠，从而提供了一种局部治疗与治疗剂。在这些实验中，可以执行在不同发育时间点上的过程中，如冠状动脉形成比较一个给定的治疗效果。因为小分子可以通过基底膜扩散，该培养系统也可以用于培养解剖区域的心彼此靠近，以确定是否特定的细胞 - 细胞接触所必需的某些发育过程中，还是旁分泌信号从一个地区到另一个是必要的。 这种文化系统是相对简单的，和不同的切片培养系统，利用基本培养试剂和设置在大多数实验室都是现成的。总之，切除胚胎的心培养稀基底膜，它提供了一种半固体的支持。这种支持是足以维持心脏的三维形态，同时也使心脏收缩。使用这个系统，整个的心从旧的小鼠胚胎（E14.5-E16.5）可以维持在文化，长达四天。整个冠状动脉丛维持，不像切片文化，所以，任何信令发生的线索，来自不同地区的心脏仍然存在。此外，活细胞荧光染料可以渗透到基质胶允许可视化现场的心，和蛋白结合珠可以放在心脏附近，提供本地化的信号源。总之，这些优点使这种技术的理想方法研究发育过程中的胎IC小鼠心脏。

<table border="1">
<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>Timed-pregnant mice</td>
 <td></td>
 <td></td>
 <td>To be dissected at the embryonic stage of interest</td>
 </tr>
 <tr>
 <td>PBS (1x)</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Cellgro </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Sigma-Aldrich </td>
 <td>F2442 </td>
 <td></td>
 </tr>
 <tr>
 <td>Growth factor-reduced Matrigel</td>
 <td>BD Bioscience </td>
 <td>356231 </td>
 <td></td>
 </tr>
 <tr>
 <td>Syto-16</td>
 <td>Invitrogen </td>
 <td>S7578 </td>
 <td>Used as directed in 13 </td>
 </tr>
 <tr>
 <td>24-well culture plate</td>
 <td>Fisher Scientific</td>
 <td>07-200-84</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Stereoscopic microscope</td>
 <td>Nikon </td>
 <td>SMZ645</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell culture incubator</td>
 <td>Thermo </td>
 <td>3110 </td>
 <td></td>
 </tr>
</tbody>
 </table>

a novel ex vivo culture method for the embryonic mouse heart

在鼠标发展研究的胚胎在妊娠期间的交通不便阻碍了。因此，协议隔离，，培养个别器官还提供一种方法，都可视化的发展变化，并允许新的治疗策略是必不可少的。为了促进长期培养的胚胎心脏在妊娠后期，我们开发了一种半固体，稀基底膜培养协议，其中切除心脏。这基板提供了足够的支持，以维持三维结构，但有足够的灵活性允许继续收缩。简单地说，心从胚胎中切下，并冷基质胶的混合物放置在1:1稀释的生长培养基中。稀释的基质胶固化后，加入生长培养基的培养皿中。四天后解剖切除的晚期胚胎16.5天心是可行的。冠状动脉丛分析表明，该方法不扰乱冠状动脉血管发育。因此，我们提出了一种新的方法长期培养胚胎的心。

使用这种技术，心脏维持其三维形态，并且仍然是可行的，如持续收缩（ 电影1）。这些始终突出在心房心室收缩。文化，心中可以固定，无论是免疫组化或组织学检查特异性标志物的表达或结构处理。 图1A显示基地，培养24小时，固定的小鼠胚胎的心脏心室和大动脉转位，并处理免疫组化标记血管内皮细胞。这些共焦的显微照片表明，冠状动脉血管切除同等岁的胚胎?...

Watch this Scientific Journal Video about A Novel Ex vivo Culture Method for the Embryonic Mouse Heart at JoVE.com

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

在鼠标发展研究的胚胎在妊娠期间的交通不便阻碍了。为了促进长期培养的胚胎心脏在妊娠后期，我们开发了一种半固体，稀基底膜培养协议，其中切除心脏。

一种新型的<em&gt;体外</em小鼠胚胎心脏的培养方法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

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A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

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Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

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In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...