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>剖开胚囊和检索胚胎。将胚胎在第二个培养皿用冷PBS。返回培养皿子宫角冰。 </li><li>杀头胚胎提高访问胸壁。如果基因分型是必要的，删除和保存的尾巴在Eppendorf管置于冰上。 </li><li>与胚胎在它的后面，切开胸壁心脏和肺的可视化。根据在舞台上，胸壁可能是透明的。 </li><li>使用镊子小心地抬起心脏和削减以下的船只。然后，切成以上的大血管，释放心。如果有必要，去除多余的组织。 </li><li>将心的24孔板中，充满冷PBS上的菜放在冰。 </li><li>重复上述步骤，直到心中所有的胚胎已被切除。 </li></ol> 2。文化集<ol><li>在层流罩中，冷的基底膜，然后选择所需的培养基中（ 如 10％胎牛血清（FBS）的Dulbecco改进的Eagle培养基（DMEM））按1:1的比例相结合。不要预暖的培养基。 </li><li>保持稀的基底膜上的冰，移液器500-1000微升的稀释液入井的24孔培养皿中，也保持在冰上。 </li><li>在引擎盖，仔细挑选，并把切除心脏在一个良好的含基质胶的培养皿中，培养皿中，同时保持在冰上。 </li><li>如果嵌入的心的取向是至关重要的：在倒置显微镜和周围的空间，用70％乙醇适量的喷。在引擎盖，放置在一个良好的含基底膜培养板切除的心脏，同时仍然在冰上。然后，将板在显微镜和快速定位切除心脏的基底膜开始凝固。 </li><li>培养皿放置在潮湿的37℃培养箱（5％CO 2），并允许基质胶固化约30分钟。 </li><li>加入1 ml预热的培养基以及包含心。 </li><li>心中可以留在培养至少四天。建议改变培养基中每两天。 </li></ol> 3。固定和可视化<ol><li>如果需要，添加了荧光活细胞染料（ 如祥发-16）的可视化实时心脏。摄影会仅限于对心脏的一侧，基于嵌入的位置，在该位置上是可见的。 </li><li>如果将进行培养后分析（ 如整装成像或切片），去掉培养基更换用冷的4％的甲醛。烹调放置在冰上30分钟。 </li><li>寒冷的甲醛和冰促进基底膜的溶解后，更换新鲜的甲醛固定液（基底膜）和修复心中一夜之间在4°C。 </li><li>洗心中的缓冲液（ 如 PBS或Tris），并存储在4°C，直到进一步的分析。 </li></ol>

<ol>
	<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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmp4+and+Fgf10+play+opposing+roles+during+lung+bud+morphogenesis.">Bmp4 and Fgf10 play opposing roles during lung bud morphogenesis.</a> Development. , 127-2695 (2000).</li><li>Viragh, S., Challice, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+the+epicardium+and+the+embryonic+myocardial+circulation+in+the+mouse.">The origin of the epicardium and the embryonic myocardial circulation in the mouse.</a> Anat. Rec. 201, 157-168 (1981).</li><li>Kirby, M. 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>

作者宣称，他们有没有竞争经济利益。

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 ...

a-novel-ex-vivo-culture-method-for-the-embryonic-mouse-heart

Research

JoVE Journal

Biology

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

13.2K 次观看.  University of North Carolina at Chapel Hill. 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。

视频: A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

科研

生物学

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

在子宫内和前体内的基因表达在小鼠视网膜神经节细胞的电穿孔

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

小鼠胚胎的龙文化，一个系统来评价分枝的分子机制

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food.
Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out.
To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area.
We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.

We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.

文化与偏振刺激人体肠道黏膜植的新方法

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

胚胎心室血管内皮细胞的分离

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

鼠胚体外发育无血清全胚胎培养系统

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

小鼠胚胎皮肤和Live成像黑素细胞迁移的体外培养

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

从发展胚胎小鼠心脏瓣膜区蛋白分离

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

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

在c-fos蛋白免疫组织学检测：一个有用​​的工具，中央通路在特定的生理反应所涉及的标记<em&gt;在体内</em&gt;和<em&gt;离体</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...