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

1。设置<ol><li>削解剖工具（使用润滑用矿物油一刃磨石）和灭菌前用70％乙醇喷雾和干热灭菌用于器官培养。使用12 V电源用电解2M的氢氧化钠锐化解剖针。 </li><li>准备用于胚胎器官的解剖和培养的培养基，其中包括:高级Dulbecco改良的Eagle培养基F12（DMEM F12），补充有1％的GlutaMAX和1％青霉素 - 链霉素。 </li></ol> 2。胚胎解剖<ol><li>扑杀使用调度一种方法核定由内政部或其他监管机构定时交配怀孕的女性。剖析了周围的小腹皮肤，切用剪刀打开腹部。找出两个子宫角，它们共同连接在较低的中线。 </li><li>解剖出子宫，并装在填充有在冰上介质的管。磨片N于冰上，可将组织放置数小时。 </li><li>取出子宫并放置在陪替氏培养皿中的培养基显微镜下。在可能的胚胎解剖应在层流罩下进行，以减少污染。 </li><li>解剖出来自子宫的胚胎，并从他们的胚外组织释放他们。 </li><li>使用钨针杀头的胚胎和磁头转移到介质的新鲜菜。 </li><li>通过将针头插入口腔隔离从磁头的下颌骨和切断通过朝向头部的背面（ 图1A和1B）。分离的下颌骨，可暂时储存在冰上介质，同时等待斩波。 </li><li>对于整个下颌骨片阶段E11.5-E15.5是理想的牙胚文化。 E15.5后下颌骨发育骨太硬切碎。牙片仍然可以提出，但骨（齿骨形成和牙槽骨）必须首先被删除砍9前清扫。使用钨针和镊子小心取出骨头。 </li></ol> 3。胚胎切片<ol><li>使用标准表组织斩波器（ 图1C）准备下颌骨组织切片。用新鲜叶片后约200下颌骨，并确保它在于安装盘平齐时，它下降。 </li><li>清洁安装光盘并在使用前用70％乙醇刀片。护理必须使用本机时，应考虑，因为刀片非常锋利。 </li><li>根据试样的年龄之间200-400微米的切割距离设定斩波，并且取决于整个牙胚是否是必需的。设置最大的剑锋。 </li><li>用吸管或镊子转移到下颌骨的塑料安装光盘。安排在光盘制作确认方向仍然是清晰的下颌骨。显影舌可被用作用于确定方位的有用的地标。对于臼齿使用自动对焦rontal /横向取向（参见斩波图1B中的平面），而对于门牙用的矢状切片，通过下颌骨，以反映这些牙齿的不同取向，因为他们成长（参见砍在图2A的平面）。 </li><li>从各地使用滤纸以稍干上安装盘下颌骨组织去除多余的介质。 </li><li>立即干燥后，将光盘上的斩波器的圆形不锈钢工作台。打开机器的电源，然后按启动。该表由左到右，而在同一时间的斩波臂承载叶片升高和下降高达200转/分钟进行自动遍历。 </li><li>关闭机器，一旦组织已经完全切片。确保刀片臂在上升位置，取出光盘。不要将手指下的刀片手臂或切碎时，让机器自行。 </li><li>拿安装盘，立即加入培养基吨Ø为了光盘上的切片，以防止过度干燥。小心地使用钨针，然后吸移管插入介质的小培养皿中（ 图2A）从盘的底部撞出切片组织中。 </li><li>用针管分离的切片，然后选择包含在立体显微镜下（ 图1D，1E，和2B）感兴趣的组织的组织切片。将所选切片新鲜培养皿中等。 </li></ol> 4。切片文化<ol><li>通过将大约2毫升灭菌水至外井，以防止脱水，并通过在盘的中心定位的金属网格准备中央孔器官培养皿中准备培养物（ 图3A和3B）。 </li><li>切割片大约4×1.5cm的从不锈钢网片准备的金属网格。用打孔器，使在中心的孔和弯曲的侧面，以形成交错末端。电网应该适合平板横贯中部不错，躺在平行的菜（ 图3A和3B）的底部。高压灭菌电网使用前消毒。 </li><li>切下一块的透明聚对苯二甲酸乙酯（PET）膜（孔径0.4微米）大到足以覆盖在网格中的孔。将切膜进入与所选择的切片的菜。 </li><li>将切片在膜的顶部，然后小心地将膜出来的介质中，用切片展平在中心。 </li><li>将过滤器上的网格在圆形孔的中心，并加入大约1毫升培养物中至井，直至膜的水平。 </li><li>为操作添加的蛋白质或小分子抑制剂的培养基（例如8）。 </li><li>拍摄片的形态在培养的第0天的纪录。 </li><li>将菜在5％CO2的潮湿气氛<sUB&gt; 2，在37℃的培养箱中培养。 </li><li>拍摄的培养，定期为平均7天。改变培养基每48-72小时。对于摄影从下方（ 图2C-E）查看切片用光源。 </li></ol> 5。谱系追踪如果谱系追踪是必需的，亲脂性染料，如DiI荧光或DIO可以在步骤4中，切片培养之前被注入牙胚。 <ol><li>使用针拔出器拉玻璃毛细管针。 </li><li>打破使用镊子将针的尖端和下入染料溶液放置小费。离开了几分钟，而染料通过毛细管作用填充小费。 </li><li>将填充针插入支架，并通过管道连接到注射器或口吸气。 </li><li>放置在培养基中接触片由于被标记的区域中的针的尖端。适用于空气压力使少量的染料出针的的ð到组织中。 </li><li>卸下针头和检查下荧光标记。 </li><li>成功标记切片可以转移到过滤器，如步骤4中所述培养。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

齿培养的本方法具有获得牙胚的优点是优异的，从而允许精确的谱系追踪和内上皮或间质珠放置。显影牙胚的限定的区域，因此可以专门针对。在文化牙胚的形态变化可以遵循，以及操作的效果快速评估。 的方法，然而，这只是适合于大量形成硬组织如牙质和釉质前年轻牙胚，因为这些不能准确地切碎。对于下颌骨后E15.5有必要砍前去除骨头。这具有潜在的破坏性的牙胚，这...

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

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

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

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

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

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

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

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

切片培养法继牙胚在植文化的发展

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

the-slice-culture-method-for-following-development-tooth-germs

Research

JoVE Journal

Biology

14.0K 次观看.  King's College London. 在这里，使用组织斩波我们详细的方法在下颌切片文化牙胚。这种方法允许独特的访问牙齿发育过程中，为操作和谱系跟踪，不可使用更传统的培养方法极好的机会。 

视频: The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

器官型E18大鼠脑切片文化

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

科研

生物学

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.  

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

基因枪子弹的制备和神经元的基因枪转染切片文化

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

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 ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

文化和维护人类胚胎干细胞

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

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 ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

鼠类全胚胎培养方法，使用的肩型瓶培养系统

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

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 ...

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 ...

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.