The authors do not have any acknowledgements.

描述所有的程序必须按照与使用脊椎动物，其中包括由当地机构动物护理和使用委员会事先批准的准则和规定进行。 1.准备解剖仪器仪表及文化传媒<ol><li>要用于在夹层预洗所有仪器用3％的过氧化氢，然后高压釜在121℃下20分钟。过滤BGJb介质与使用0.22微米孔径大小的过滤器是在一个通风柜真空抽吸抗生素和抗真菌溶液。 </li></ol> 2.准备文化设备<ol><li>切，将支持腭器官小三角形聚碳酸酯膜过滤器，喷以70％的酒精，并允许他们干。通过将清洁和消毒等边三角形状的线栅（20毫米）在器官培养板制备中心阱器官培养的培养皿（60x15毫米）。然后填写井用1毫升的BGJB媒体文化传媒。注意:介质应足以达到网格的水平而不浸没的口味。放置膜过滤器，有光泽的一面朝下，对电网的顶部（每格3-4的膜）.Depending在实验中，处理（蛋白质，抗体或抑制剂）可以被加入到培养基</li></ol> 3.小鼠胚胎收集和准备文化<ol><li>为了获得野生型小鼠胚胎，利用定时13.5怀孕的CD-1小鼠遗传背景品系。清洁用70％乙醇的解剖区域。 注:可用于小鼠的其他菌株。我们优选CD-1小鼠，因为更大的垫料数量。 </li><li>安乐死怀孕小鼠E13.5 DPC（天性交后），用5％异氟醚颈椎脱位使用批准的协议来保证死刑。 </li><li>喷洒在腹侧的腹部表面的70％的乙醇，以避免腹部卷发棒的文书。然后腹侧打开腹腔用尖钝经营剪刀和微解剖钳定位子宫。使用镊子，解除整个子宫和削减在子宫体和子宫角的一个光操作剪刀的提示它从身体分离。 </li><li>将子宫上干净的纸巾在冰​​（根据协议指南护理和使用实验动物的）。用另一干净的纸巾，以防止污染的子宫。消毒用70％乙醇喷洒在进一步使用仪器。子宫和胚胎可以运到在冰上的实验室。 </li><li>在一个开放的层流罩，仔细地做一个小开口，利用尖钝经营剪刀和微解剖钳卵黄囊。使扩大开放，然后轻轻将其推到exteriorize从卵黄囊胚胎。 </li><li>立即胚​​胎转移至培养皿用冷的磷酸盐缓冲盐水1X（PBS）的pH为7.4。为了确保适当的阶段，前胺解剖显微镜的身体，头部大小和肢体形态下的胚胎。 不用于实验胚胎不属于在发展的权阶段:注意。 </li></ol><img alt="图2" src="/files/ftp_upload/53063/53063fig2.jpg" /> 图2.肢芽形态的前后肢被检查的数字之间织带缩进的程度。在E13.5，所有的数字凝聚突出，与数字之间的织带和远端缺口出现非常明显的。在后数字是落后了半天12上排:前肢，下排:后肢。 4.腭解剖<ol><li>在用PBS解剖显微镜下的培养皿放置一个胚胎的时间。使用小解剖剪和镊子从胚的体分开的头部。 </li><li>小心地握着鼠标颅骨高于人眼视觉用钳子（避免上颌处理）上的唇线两侧做出两个切口。然后取出下颌和舌头。通过切割在眼睛水平隔离上颌处理区，并取出大脑，眼睛，鼻中隔和毗邻的组织。 </li></ol><img alt="图3" src="/files/ftp_upload/53063/53063fig3.jpg" /> 从13.5胚胎，当腭货架具有升高的图3.腭组织收集但不进行接触，腭置于器官培养上在72小时的静态模型空气媒体接口的过滤器。（A）的水平解剖上的E13.5胚胎。（B）的蓝色恒星划分区域举行与解剖过程中钳组织。（C）查看放置在梯形滤膜解剖腭。培养皿（D）侧面的位置后，剖分口味和加入培养基。 <ol start="3"><li>将腭口朝上，取出鼻中隔和它周围的组织，保持连接到帮助引导它由前至后的主要口味。用勺子铲，并在培养皿非常仔细地转移腭（鼻朝下）将准备好的过滤器上的网格。注:调整媒体音量，允许适当的空气介质接口。 </li><li>根据体视显微镜，解剖主要腭及用镊子将腭接近对方。该腭不会熔化，如果他们不接触放置。切三角形的膜过滤器，其中腭前部的位置。梯形形状将有助于定位上腭的前部。 </li><li>单独或与两个或三个在使用BGJb培养基相同的器官培养皿培养的腭。 </li></ol> 5.培养鼠标腭<ol><li>孵育Tissu酒店在37℃下在加湿的气体混合物（5％CO 2和95％空气）72小时ES，并改变它的介质，每24小时。 </li></ol> 6.口感处理进行组织学分析<ol><li> 72小时培养用4％甲醛/磷酸缓冲盐水ö后固定的口味/ N在4℃ &#160; ℃。然后，轻轻地用移液管在培养皿用PBS洗涤组织。裹在一块实验室湿巾和地方口味的嵌入盒。 注:完成实验室湿巾口味防止在脱水过程中的损失。 </li><li>脱水组织以下70％，80％和100％的乙醇洗涤1小时，随后经常石蜡包埋过程。嵌入，将面临的托盘上腭的前部。 </li><li>由前切串行6微米的部分在冠状定向后路。一个完整的部分腭将产生350-450 secti组件。染色用苏木精和曙红（H＆E）。10分数使用先前描述的平均数融合得分，每20 个段（MFS）比例（表1）的部分。8 </li></ol>

<ol>
	<li>Nawshad, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palatal+seam+disintegration+to+die+or+not+to+die+that+is+no+longer+the+question.">Palatal seam disintegration to die or not to die that is no longer the question.</a> Developmental dynamics an official publication of the American Association of Anatomists. 237, 2643-2656 (2008).</li><li>Strong, E. B., Buckmiller, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+the+cleft+palate.">Management of the cleft palate.</a> Facial plastic surgery clinics of North America. 9, 15-25 (2001).</li><li>Witt, P. D., Marsh, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+assessing+outcome+of+surgical+repair+of+cleft+lip+and+cleft+palate.">Advances in assessing outcome of surgical repair of cleft lip and cleft palate.</a> Plastic and reconstructive surgery. 100, 1907-1917 (1997).</li><li>4miloro, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> principles of oral and maxillofafcial surgery. 209, 231-249 (1984).</li><li>Mima, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+epithelial+adhesion+molecule+CEACAM1+is+important+for+palate+formation.">Regulation of the epithelial adhesion molecule CEACAM1 is important for palate formation.</a> PloS one. 8, e61653 (2013).</li><li>Carette, M. J., Ferguson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fate+of+medial+edge+epithelial+cells+during+palatal+fusion+in+vitro+an+analysis+by+DiI+labelling+and+confocal+microscopy.">The fate of medial edge epithelial cells during palatal fusion in vitro an analysis by DiI labelling and confocal microscopy.</a> Development (Cambridge, England). 114, 379-388 (1992).</li><li>Ferguson, M. W., Honig, L. S., Slavkin, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+cultured+palatal+shelves+from+alligator+chick+and+mouse+embryos.">Differentiation of cultured palatal shelves from alligator chick and mouse embryos.</a> The Anatomical record. 209, 231-249 (1984).</li><li>San Miguel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ephrin+reverse+signaling+controls+palate+fusion+via+a+PI3+kinase+dependent+mechanism.">Ephrin reverse signaling controls palate fusion via a PI3 kinase dependent mechanism.</a> Developmental dynamics an official publication of the American Association of Anatomists. 240, 357-364 (2011).</li><li>Kang, P., Svoboda, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI+3+kinase+activity+is+required+for+epithelial+mesenchymal+transformation+during+palate+fusion.">PI 3 kinase activity is required for epithelial mesenchymal transformation during palate fusion.</a> Developmental dynamics an official publication of the American Association of Anatomists. 225, 316-322 (2002).</li><li>RD, 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> Histopathologic Technic and Practical Histochemistry. , (1965).</li><li>Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Atlas of Mouse Development. , (1992).</li><li>Taher, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+gene+expression+analysis+of+murine+limb+development.">Global gene expression analysis of murine limb development.</a> PloS one. 6, e28358 (2011).</li></ol>

The authors do not have any competing financial interests.

本文中的方案提供了从胚胎解剖腭为13.5天胚胎的方法。从小鼠胚胎腭分别在无血清的培养基中培养在95％ 的 O 2，5％CO 2的气氛下在37℃。成功的腭夹层是极其依赖于多种因素，在从安乐死的小鼠的培养完成的胚胎时在操作过程中的每个步骤。一的影响腭融合的最重要因素是启动器官培养所需要的时间;胚胎必须在13.5作为二次腭货架是水平的胚胎天，但还没有开始熔化（ 图1）...

口面裂是最主要的颅面先天缺陷。另外，当以在考虑所有可能的颅面畸形，这些是在新生儿2第二最常见的出生异常。腭裂发生在约1在每700出生在美国（美国）每年，腭裂的发生率等于475所生的子女，每月兔唇或15个孩子，每个3天裂。世界各地的孩子大约有1％生每年展品某种形式的颅面畸形学的。 腭和唇的裂需要非常昂贵和复杂的过程与谁有这种异常的患者终生影响。估计费用为每一个病人口腔裂约为$ 10万个4。与唇腭裂患者的治疗需要一个团队的医生，包括颅面外科，耳鼻喉科，遗传学家，麻醉医师，言语语言病理学家，营养学家，正牙医生，prosthodontists，心理学家，神经外科医生，和眼科医生。 在腭，次级腭产生作为副产物配对，最初垂直增长和经受舌头的鼻背上述腭搁板标高。随着海拔的配对腭增长对（在小鼠E14.5 -E15和9周人）中线。内侧缘上皮细胞（MEE），涵盖了架子尖端附着形成中线上皮煤层。 这之后是上皮至间质转变和/或凋亡，以允许充质汇合。反对MEE的附着力是一个重要事件，其​​变更导致腭裂。然而，只有少数研究调查了腭5的初始粘合性。硬腭形式由分化的间充质细胞的成骨细胞。口感可以产生谱号的畸形发展吨口味有或没有唇部的参与。 腭器官培养技术被广泛应用于很多实验室在过去30年6,7了。 在这个协议中，我们详细描述腭解剖和静态器官培养的方法。一动不动器官培养的优点是，它允许腭融合。这种技术已经被成功地用于在我们的实验室对许多聚变和信令实验8,9。然而该技术的范围广阔，可以用于每当静态器官培养系统是必需的，包括响应外源性化学药剂的评价中，在不同的途径和特定蛋白质调节的生长因子的作用。 <img alt="图1" src="/files/ftp_upload/53063/53063fig1.jpg" /> 图1.鼠标腭，小鼠腭的发展阶段。（BF）扫描 ELE二级腭ctron显微照片（SEM）的代表发展时期。红色箭头:显示腭粘附和融合的初始部分。黄箭头:点到初级和次级口味融合后，将消失之间的空间（重印从考夫曼11与许可PLOS之一）。

&#160;
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>BGJb</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Lonza Inc.</td>
			<td>17-602E</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>VWR</td>
			<td>25384-088</td>
			<td>100x15mm</td>
		</tr>
		<tr>
			<td>Center-well organ culture dish</td>
			<td>Falcon&#160;</td>
			<td>#353037</td>
			<td>60x15 mm</td>
		</tr>
		<tr>
			<td>Wire triangular grid</td>
			<td>Custom made with stainless steel wire mesh to fit the organ culture dish well.&#160;&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate filter</td>
			<td>GE&#38; Water and Process Technologies</td>
			<td>K04BP04700</td>
			<td>Black 0.4 micron, 47 mm</td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>ZEISS&#160; Stemi SR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Culture hood</td>
			<td>NUAIRE Laminar Flow</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microdissecting forceps</td>
			<td>Dumont Medical&#160;</td>
			<td></td>
			<td>#5</td>
		</tr>
		<tr>
			<td>Microdissecting scissors</td>
			<td>Kent Scientific Corporation</td>
			<td>INS14003-G</td>
			<td>Vannas Scissors, straight, 8cm long, 0.1mm tips, 5mm blades, German made</td>
		</tr>
		<tr>
			<td>Microdissecting scissors</td>
			<td>Kent Scientific Corporation</td>
			<td>INS500086</td>
			<td>Vannas Scissors, straight, 8.5cm long, 0.025mm x 0.015mm tips, 7mm blades</td>
		</tr>
		<tr>
			<td rowspan="2">Microdissecting scissors</td>
			<td rowspan="2">Kent Scientific Corporation</td>
			<td rowspan="2">INS14127-G</td>
			<td rowspan="2">Spring scissors curved curved, 10.5cm long, 8mm blades, German made</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Surgical currete (spoon spatula)</td>
			<td>Hu-friedy</td>
			<td></td>
			<td>CM 2/4</td>
		</tr>
		<tr>
			<td>Protector laboratory hood</td>
			<td>Labconco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="3">Incubator</td>
			<td rowspan="3">Thermo Scientific</td>
			<td rowspan="3"></td>
			<td>Farma</td>
		</tr>
		<tr>
			<td>Series11 water Jacket</td>
		</tr>
		<tr>
			<td>Co2 incubator</td>
		</tr>
		<tr>
			<td>PBS&#160;</td>
			<td>GIBCO</td>
			<td>10010-023&#160;</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>Fiber optic Light source</td>
			<td>Fiber-light&#160; Dolan-Jenner</td>
			<td>PL-750</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding cassette&#160;</td>
			<td>Statlab</td>
			<td>EC301</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Kim Wipes</td>
			<td rowspan="2">VWR</td>
			<td rowspan="2">470173-504</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
		</tr>
	</tbody>
</table>

method of studying palatal fusion using static organ culture

唇腭裂是最常见的所有出生缺陷。从骨髓间充质货架的次生腭形成覆盖着上皮细胞附着，形成中线上皮煤层（MES）。该理论认为MES细胞遵循上皮至间质转变（EMT），细胞凋亡和迁移，使稠腭1。在MES的完全解体是腭融合与周围间质细胞的最后一个基本阶段。我们提供味觉器官培养的方法。 在体外协议开发允许生物和分子过程的融合过程中的研究。这种技术的的应用有很多，包括评估响应外源化学试剂，监管和生长因子和特异性蛋白质的效果。腭器官培养具有许多优点，包括:在操作的开发使用体内研究认为是不可能的不同阶段。

该技术可应用于各种不同experiments.The以下研究的目的是测试尼古丁在体外对腭融合致畸作用。被用于本次实验的两个品系的小鼠，CD1和C57.Palatal组织与烟碱半硫酸盐的0.06和6mm的浓度处理。据观察，腭融合模式和时间分别在两个不同的菌株不同。在CD1小鼠，与对照组腭持续融合在时间依赖性。然而，在治疗组烟碱特别是在6毫米尼古丁浓度（图4A）延迟融合。的CD1小鼠味觉组织学?...

Watch this Scientific Journal Video about Method of Studying Palatal Fusion using Static Organ Culture at JoVE.com

Method of Studying Palatal Fusion using Static Organ Culture

腭发展研究是由腭裂，出生缺陷，它强行巨大的医疗负担，并可以留下持久的缺陷的发生率动机。我们在这里展示了一种技术，可用于研究参与腭发展和融合不同信号通路的培养腭。

使用静态器官培养研究腭融合的方法

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that ...

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Developmental Biology

8.7K 次观看.  Texas A&M University Baylor College of Dentistry. 腭发展研究是由腭裂，出生缺陷，它强行巨大的医疗负担，并可以留下持久的缺陷的发生率动机。我们在这里展示了一种技术，可用于研究参与腭发展和融合不同信号通路的培养腭。 

视频: Method of Studying Palatal Fusion using Static Organ Culture

Studying Wnt Signaling During Patterning of Conducting Airways

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis controlled by Wnt signaling is required to understand how tissues are patterned during normal development. This knowledge is also critical to determine the etiology of birth defects such as lung hypoplasia and tracheobronchomalacia. Analysis of earliest stages of development of respiratory tract imposes challenges, as the limited amount of tissue prevents the performance of standard protocols better suited for postnatal studies. In this paper, we discuss methodologies to study cell differentiation and proliferation in the respiratory tract. We describe techniques such as whole mount staining, processing of the tissue for confocal microscopy and immunofluorescence in paraffin sections applied to developing tracheal lung. We also discuss methodologies for the study of tracheal mesenchyme differentiation, in particular cartilage formation. Approaches and techniques discussed in the current paper circumvent the limitation of material while working with embryonic tissue, allowing for a better understanding of the patterning process of developing conducting airways.

The use of reporter mice coupled to whole mount and section staining, microscopy and in vivo assays facilitates the analysis of mechanisms underlying the normal patterning of the respiratory tract. Here we describe how these techniques contributed to the analysis of Wnt signaling during tracheal development.

研究Wnt信号传导过程中的航空公司图案

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis ...

科研

发育生物学

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

联体斑马鱼胚胎一代发展囊胚的手术融合

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable. Many beneficial effects of MSCs have been attributed to their ability to regulate inflammation and immunity, mainly through secretion of immunomodulatory factors such as tumor necrosis factor-stimulated gene 6 (TSG6) and prostaglandin E2 (PGE2). However, MSCs require activation to produce these factors and since the effect of MSCs is often transient, great interest has emerged to discover ways of pre-activating the cells prior to their use, thus eliminating the lag time for activation in vivo. Here we present protocols to efficiently activate or prime MSCs in three-dimensional (3D) cultures under chemically defined XF conditions and to administer these pre-activated MSCs in vivo. Specifically, we first describe methods to generate spherical MSC micro-tissues or &#39;spheroids&#39; in hanging drops using XF medium and demonstrate how the spheres and conditioned medium (CM) can be harvested for various applications. Second, we describe gene expression screens and in vitro functional assays to rapidly assess the level of MSC activation in spheroids, emphasizing the anti-inflammatory and anti-cancer potential of the cells. Third, we describe a novel method to inject intact MSC spheroids into the mouse peritoneal cavity for in vivo efficacy testing. Overall, the protocols herein overcome major challenges of obtaining pre-activated MSCs under chemically defined XF conditions and provide a flexible system to administer MSC spheroids for therapies.

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

生产和治疗间充质干行政/基质细胞（MSC）球体底漆在三维培养下无异物条件

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

器官培养和鼠标沃尔弗导管的全山免疫染色

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

脊髓培养法研究胚胎鸡组织的发展

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

使用由内皮结肠形成细胞和间质干细胞形成的共培养球体的三维血管生成测定系统

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

一种从人类胚胎干细胞中生成脑器官的静态自导方法

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

Cell-cell Fusion of Genome Edited Cell Lines for Perturbation of Cellular Structure and Function

Life is spatially partitioned within lipid membranes to allow the isolated formation of distinct molecular states inside cells and organelles. Cell fusion is the merger of two or more cells to form a single cell. Here we provide a protocol for cell fusion of two different cell types. Fused hybrid cells are enriched by flow cytometry-based sorting, followed by fluorescence microscopy of hybrid cell structure and function. Fluorescently tagged proteins generated by genome editing are imaged inside fused cells, allowing cellular structures to be identified based on fluorescence emission and referenced back to the cell type of origin. This robust and general method can be applied to different cell types or organelles of interest, to understand cellular structure and function across a range of fundamental biological questions.

The purpose of this protocol is to fuse two different cell types to create hybrid cells. Fluorescence microscopy analysis of fused cells is used to track the cell of origin of cellular organelles. This assay can be used to explore how cellular structure and function respond to perturbation by cell fusion.

基因组编辑细胞系细胞融合，用于细胞结构和功能的扰动

Life is spatially partitioned within lipid membranes to allow the isolated formation of distinct molecular states inside cells and organelles. Cell fusion ...