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>

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	<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）。

<table><tbody><tr><td>BGJ&lt;sub&gt;b&lt;/sub&gt;</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>100 x15 mm</td></tr><tr><td>Center-well organ culture dish</td><td>Falcon&amp;nbsp;</td><td>#353037</td><td>60 x15 mm</td></tr><tr><td>Wire triangular grid</td><td>Custom made with stainless steel wire mesh to fit the organ culture dish well.&amp;nbsp;&amp;nbsp;</td><td></td><td></td></tr><tr><td>Polycarbonate filter</td><td>GE&amp;amp; Water and Process Technologies</td><td>K04BP04700</td><td>Black 0.4 micron, 47 mm</td></tr><tr><td>Stereo microscope</td><td>ZEISS&amp;nbsp; 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&amp;nbsp;</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, 8 cm long, 0.1mm tips, 5 mm blades, German made</td></tr><tr><td>Microdissecting scissors</td><td>Kent Scientific Corporation</td><td>INS500086</td><td>Vannas Scissors, straight, 8.5 cm long, 0.025 mm x 0.015 mm tips, 7 mm blades</td></tr><tr><td>Microdissecting scissors</td><td>Kent Scientific Corporation</td><td>INS14127-G</td><td>Spring scissors curved curved, 10.5 cm long, 8 mm blades, German made</td></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>Incubator</td><td>Thermo Scientific</td><td></td><td>Farma</td></tr><tr><td>Series11 water Jacket</td><td></td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td></td><td></td><td></td></tr><tr><td>PBS&amp;nbsp;</td><td>GIBCO</td><td>10010-023&amp;nbsp;</td><td>1X</td></tr><tr><td>Fiber optic Light source</td><td>Fiber-light&amp;nbsp; Dolan-Jenner</td><td>PL-750</td><td></td></tr><tr><td>Embedding cassette&amp;nbsp;</td><td>Statlab</td><td>EC301</td><td></td></tr><tr><td>Kim Wipes</td><td>VWR</td><td>470173-504</td><td></td></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

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

咽弓的体外培养研究心脏和肌肉祖细胞和自己的优势

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

科研

发育生物学

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

腭扩展口腔生物膜分析，荧光原位杂交和激光共聚焦显微镜

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

医学

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

人力蕈状味觉​​乳头细胞的分离和培养

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

神经科学

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 ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

原代培养及质粒电穿孔尔蒂小鼠器官。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

生物学

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

We present a protocol for isolation and culture of primary mouse embryonic palatal mesenchymal cells for time-lapse imaging of two-dimensional (2D) growth and wound-repair assays. We also provide the methodology for analysis of the time-lapse imaging data to determine cell-stream formation and directional motility.

原代小鼠胚胎腭间充质细胞的分离和延时成像，以分析集体运动属性

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and ...

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The continuously growing mouse incisor provides an excellent model to study tissue-specific stem cells. The epithelial tissue-specific stem cells of the incisor are located at the proximal end of the tooth in a niche called the cervical loop. They provide a continuous influx of cells to counterbalance the constant abrasion of the self-sharpening tip of the tooth. Presented here is a detailed protocol for the isolation and culture of the proximal end of the mouse incisor that houses stem cells and their niche. This is a modified Trowell-type organ culture protocol that enables in vitro culture of tissue pieces (explants), as well as the thick tissue slices at the liquid/air interface on a filter supported by a metal grid. The organ culture protocol described here enables tissue manipulations not feasible in vivo, and when combined with the use of a fluorescent reporter(s), it provides a platform for the identification and tracking of discrete cell populations in live tissues over time, including stem cells. Various regulatory molecules and pharmacological compounds can be tested in this system for their effect on stem cells and their niches. This ultimately provides a valuable tool to study stem cell regulation and maintenance.

The Trowell-type organ culture method has been used to unravel complex signaling networks that govern tooth development and, more recently, for studying regulation involved in stem cells of the continuously growing mouse incisor. Fluorescent-reporter animal models and live-imaging methods facilitate in-depth analyses of dental stem cells and their specific niche microenvironment.

使用Trowell型器官培养研究牙科干细胞的调节

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The ...

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

真菌形态、环形和味觉味蕾的全镶嵌染色、可视化和分析

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

人鼻上皮类器官的培养和成像

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

从成人小鼠腭中分离和培养原代口腔角质形成细胞

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

使用静态器官培养研究腭融合的方法

摘要

探索更多视频

咽弓的体外培养研究心脏和肌肉祖细胞和自己的优势

腭扩展口腔生物膜分析，荧光原位杂交和激光共聚焦显微镜

人力蕈状味觉乳头细胞的分离和培养

小鼠第二腭融合的实时成像

原代培养及质粒电穿孔尔蒂小鼠器官。

原代小鼠胚胎腭间充质细胞的分离和延时成像，以分析集体运动属性

使用Trowell型器官培养研究牙科干细胞的调节

真菌形态、环形和味觉味蕾的全镶嵌染色、可视化和分析

人鼻上皮类器官的培养和成像

从成人小鼠腭中分离和培养原代口腔角质形成细胞

使用静态器官培养研究腭融合的方法

摘要

探索更多视频

咽弓的体外培养研究心脏和肌肉祖细胞和自己的优势

腭扩展口腔生物膜分析，荧光原位杂交和激光共聚焦显微镜

人力蕈状味觉​​乳头细胞的分离和培养

小鼠第二腭融合的实时成像

原代培养及质粒电穿孔尔蒂小鼠器官。

原代小鼠胚胎腭间充质细胞的分离和延时成像，以分析集体运动属性

使用Trowell型器官培养研究牙科干细胞的调节

真菌形态、环形和味觉味蕾的全镶嵌染色、可视化和分析

人鼻上皮类器官的培养和成像

从成人小鼠腭中分离和培养原代口腔角质形成细胞

人力蕈状味觉乳头细胞的分离和培养