Name: organotypic culture method to study the development of embryonic chicken tissues
Uploaded: 2018-08-25T12:30:00.000+00:00
Duration: 5 min 12 s
Description: Watch this Scientific Journal Video about Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues at JoVE.com

作者要感谢格雷戈里 Haller (圣文森特大学) 在发展《议定书》方面的初步工作。作者还要感谢尼古拉斯. 琼斯 (圣文森特大学) 的技术专长和协助, 拍摄和制作手稿的视听部分。丹尼尔安德鲁斯得到了来自 MSVU 和加拿大自然科学和工程研究委员会 (NSERC) 的资助,通过了一项本科生研究奖。塔玛拉. Odendaal 是 NSERC 发现补助金的支持。

注: 对于胚胎阶段, 利用汉堡包和哈密尔顿12 (HH) 分期表。
1. 胚胎孵化
<ol><li>在37°c 1 °c 和40% 湿度的无菌、温度控制的孵化器中孵化受精的鸡卵。</li>
	<li>每天把鸡蛋 1x, 让它们孵化到 HH34 (受精后8天)。</li>
</ol>2. 材料的制备和灭菌
<ol><li>12胚, 高压釜2升蒸馏水, 1 升0.85% 鸡盐水, 12 玻璃吸管, 1 盒纸组织, 24 2.5 厘米 x 2.5 厘米的半多孔滤纸的正方形, 和 24 2.5 厘米 x 2.5 厘米正方形的钢丝网, 边缘弯曲向下。要消毒的材料, 蒸压他们在121摄氏度至少15分钟。 注: 材料的热处理可以提前完成。</li>
	<li>通过擦拭两侧、货架和门, 用70% 乙醇消毒孵化器。在孵化箱内放置2塑料容器, 其中含有蒸压蒸馏水, 以保持湿度。Prewarm 培养孵化器到37°c 1 °c 和 ~ 40% 湿气。确保孵化器完全黑暗;如果门是透明的, 用锡纸盖住它。</li>
	<li>Prewarm 100 毫升的营养培养基和1毫升的青霉素放置在培养皿中。</li>
	<li>用纸巾盖住工作区, 用70% 乙醇喷洒在工作台上消毒。消毒2对镊子, 解剖剪刀, 剃刀刀片, 和一个塑料勺子与70% 乙醇的类似方式。</li>
	<li>放置12无菌100毫米培养皿和24无菌35毫米培养皿在板凳上使用。确保餐具在使用前保持在无菌袋中。</li>
</ol>3. 胚胎准备
注意: 从这一点起, 戴上防护防尘面罩, 避免污染文化。细菌或真菌感染将破坏文化, 并有风险迅速蔓延到所有文化的孵化器。
<ol><li>打开鸡蛋, 将胚胎移植到含有0.85% 只生理盐水的100毫米培养皿中。根据汉堡和汉密尔顿分期指南12中描述的解剖特征, 分期胚胎, 并确认胚胎在 HH34。</li>
	<li>切开颈部, 然后用消毒剃刀刀片在中线对开胚胎的头部。使用无菌钳, 取出大脑。保持喙完好。</li>
</ol>4. 文化设置
<ol><li>放置 2 35 毫米培养皿内100毫米培养皿。</li>
	<li>在每35毫米培养皿内放置一个钢丝网。</li>
	<li>使用镊子, 转移1的一分为二头到一块2.5 厘米 x 2.5 厘米半孔滤纸与眼睛朝上。确保组织是安全的, 并不会滑倒轻微倾斜。</li>
	<li>使用镊子, 小心地将眼睛组织和滤纸放在钢丝网的顶部, 在35毫米培养皿中的 1, 形成一个凸起的阶段与眼睛组织在顶部。</li>
	<li>重复步骤4.3 和4.4 与另一半头。</li>
	<li>使用无菌的玻璃吸管, 小心地添加营养介质到每35毫米培养皿, 直到它达到过滤纸的水平。不要将半头浸入营养培养基中。</li>
	<li>在每个培养皿中加入50µL 1万毫升青霉素-链霉素到营养培养基中。</li>
	<li>将一张纸巾折叠成一个小正方形, 放入100毫米培养皿内。用蒸压蒸馏水滋润它。</li>
	<li>将培养皿放入无菌, 深色孵化器, 在37°c 1 °c 和40% 湿度4天。</li>
	<li>对每个胚胎重复步骤3和4。 注: 需要的胚胎数量将取决于具体的实验;在这里, 我们描述了12胚胎的协议。</li>
</ol>5. 文化维护
<ol><li>每天一次, 顶部的塑料容器在孵化器与新鲜, 蒸压蒸馏水。</li>
	<li>每日检查所有的细菌或真菌感染的文化。已解体或介质更改颜色的区域性被感染。丢弃所有受感染的区域性。</li>
</ol>6. 固定
<ol><li>培养后, 从孵化器中取出所有菜肴。</li>
	<li>使用一对镊子, 轻轻地从滤纸上取出眼睛, 注意不要撕掉纸巾。</li>
	<li>将4% 多聚甲醛中的眼部组织固定在4摄氏度或10% 中性缓冲的福尔马林中过夜, 室温下在1x 磷酸盐缓冲盐水中。</li>
	<li>在1x 磷酸盐缓冲盐水中储存4摄氏度的组织。</li>
</ol>

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作者没有什么可透露的。

本协议利用已建立的组织培养技术, 从体外培养8 (HH34) 的胚胎4天, 实现鸡眼的生长。这种网格培养方法最初是由 Trowell15描述的。我们优化了一个协议, 从托比和霍尔的1991研究16利用半孔膜与网格, 研究诱导信号之间的分离组织层的胚胎鸡眼15。用这种方法, 蟑螂培养了14天的老鸡股骨6。这种方法也证明了有效的培养小鼠?...

胚胎鸡肉 (鸡鸡) 是生物学领域中常用的一种优良的模型生物。其潜伏期约为21天, 许多卵可以同时孵化, 使实验快速、高效。也许最重要的是 , 胚胎也很容易控 , 从而能够对关键的发育过程以及驱动这些过程的基因和蛋白质进行广泛的研究。
胚胎鸡眼是一个复杂的器官,通过与许多其他身体系统相似的不同组织的相互作用而发展。这种方法使研究这些组织的发展, 特别是在发展的先进阶段。例如, 多层视网膜可能对那些研究神经系统发育的人特别感兴趣。其他的方法, 使研究其他眼睛组织, 如角膜, vitreal 体, 晶状体, 巩膜, 和眼睑是有益的研究人员。鸡胚眼还含有一系列扁平骨, 即巩膜听骨, 可作为研究脊椎动物1膜内骨诱导和骨化的模型。
目前, 有许多方法用于研究胚胎发育。Microinjections 抑制性抗体或其他抑制分子2,3, 手术植入微球浸泡在抑制剂4, 和电穿孔5是所有的方法, 可用于 downregulate 基因或者是胚胎中感兴趣的蛋白质。类似的方法用于 upregulate 蛋白质。这些方法不是没有限制的。例如, 当使用化学物质来改变胚胎发育时, 必须对胚胎的致命影响进行评估, 这就限制了使用上述方法在足够低的剂量下对应用地点进行本地化, 以确保胚胎。
体外组织培养已被广泛应用于各种生物体中, 用于研究发展, 可用于绕过上述一些限制。例如, 股骨6、羽毛芽7、8和四肢9的鸡都用组织培养方法进行了研究, 如老鼠10的睾丸和植物的根和茎11。这些方法给予科学家高度的控制组织发展, 例如有能力波动的温度和改变养分的可用性。从整个胚胎中分离出的组织也使得它更不易受到化学物质的致命影响, 从而使得在全球范围内进行更高浓度的操纵研究。体外培养的另一个显著优点是保存组织的细胞环境;组织的排列保持相对不变, 从而可以研究不同组织类型9之间的相互作用。因此,在体外培养打开门的其他实验方法没有在体内或蛋模型。
目前, 使用化学物质研究胚胎鸡眼的发展尤其具有挑战性。许多胚外膜覆盖胚胎, 使其难以应用微球或化学物质;胚胎在卵子中也非常活跃, 因为它变老了, 使已经很难的方法复杂化了。该协议能够方便地接触眼睛及其周围组织, 消除这些障碍, 同时也为检查眼睛内的发育过程提供了新的机会。本协议的建立是为了研究在胚胎眼中的巩膜听骨的诱导。

<table><tbody><tr><td>35 mm cell culture petri dishes</td><td>Corning</td><td>353001</td><td>easy grip tissue culture dish, polystyrene, non-pyrogenic</td></tr><tr><td>100 mm cell culture petri dishes</td><td>Corning</td><td>353003</td><td>tissue culture dish, polystyrene, non-pyrogenic</td></tr><tr><td>paper tissue</td><td>Kimtech</td><td>34155</td><td>Kimtech Science Brand Task Wipers, 280 per carton</td></tr><tr><td>wire mesh</td><td>n/a</td><td>n/a</td><td>stainless steel wire mesh (grid size 0.7 mm)</td></tr><tr><td>disposable glass pipettes</td><td>VWR</td><td>14673-010</td><td>Borosilicate glass disposable 5 3/4&quot;</td></tr><tr><td>nutrient medium</td><td>Gibco</td><td>12591-038</td><td>Fitton-Jackson Modification, [+] L-glutamine with phenol red (BGJB)</td></tr><tr><td>penicillin-streptomycin</td><td>Sigma-Aldrich</td><td>P4458</td><td>10000 units/mL penicillin streptomycin solution stabilised</td></tr><tr><td>filter paper</td><td>Whatman</td><td>1454 090</td><td>semi-porous filter paper 90mm</td></tr><tr><td>fertilized chicken eggs</td><td>Dalhousie University Agricultural College</td><td>n/a</td><td>can be obtained from local farms</td></tr><tr><td>sodium chloride (NaCl)</td><td>EMD</td><td>SX0420-3</td><td>sodium chloride crystals, reagent grade</td></tr><tr><td>1 L glass bottle</td><td>VWR</td><td>89000-240</td><td>1 L pyrex autoclavable glass bottle</td></tr><tr><td>ethanol</td><td>Fisher Scientific</td><td>BP82011</td><td>70% molecular biology grade</td></tr><tr><td>tupperware containers</td><td>n/a</td><td>n/a</td><td>store-bought and sterilized with EtOH</td></tr><tr><td>disposable razor blades</td><td>VWR</td><td>55411-050</td><td>single edge industrial razor blades (surgical carbon steel)</td></tr><tr><td>plastic spoons</td><td>n/a</td><td>n/a</td><td>store-bought and sterilized with EtOH</td></tr><tr><td>dust mask</td><td>3M</td><td>n/a</td><td>3M 8500 Comfort Mask</td></tr><tr><td>paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>paraformaldehyde, reagent grade, crystalline</td></tr><tr><td>neutral-buffered formalin</td><td>Fisher Scientific</td><td>72210</td><td>10% neutral buffered formalin</td></tr><tr><td>phosphate buffered saline (PBS)</td><td>n/a</td><td>n/a</td><td>10X phosphate buffered saline pH 7.4 (137mM NaCl, 2.5mM KCl, 4.3mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 1.4mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td></tr><tr><td>15 ml falcon tubes</td><td>VWR</td><td>21008-216</td><td>presterilized centrifuge tubes</td></tr><tr><td>forceps</td><td>FST</td><td>n/a</td><td>fine forceps</td></tr><tr><td>chick saline&amp;nbsp;</td><td>n/a</td><td>n/a&amp;nbsp;</td><td>0.85% NaCl</td></tr><tr><td>tinfoil</td><td>n/a</td><td>n/a</td><td>store-bought</td></tr><tr><td>paper towel</td><td>n/a</td><td>n/a</td><td>store-bought</td></tr></tbody></table>

organotypic culture method to study the development of embryonic chicken tissues

胚胎鸡通常被用作脊椎动物发育的可靠模型有机体。它的可达性和短潜伏期使其成为实验的理想选择。目前, 对鸡胚中这些发育途径的研究是通过在局部部位应用抑制剂和药物, 在低浓度下使用多种方法进行的。体外培养是一种技术, 它能够研究与宿主生物体分离的组织, 同时绕过与整个胚胎工作时存在的许多物理限制, 如胚胎的易感性高剂量的潜在致命化学物质。在这里, 我们提出了一个脊髓培养的方法, 以培养胚胎鸡半头的体外, 这为检查发展过程的新的机会, 超越目前建立的办法。

采用这种方法, 可以在体外培养8天的胚胎鸡眼 (HH34), 4 天。四天的蛋发展对应于 HH38。
这种培养方法支持发展的羽毛芽周围的眼睛和眼睑 (图 1B)。这些羽毛芽不存在于蛋在 HH34 之前的培养 (图 1A), 表明羽毛芽发育是在培养期间开始, 因为它是在蛋<sup class="xre...

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Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

脊髓培养法研究胚胎鸡组织的发展

在这里, 我们提出了一个脊髓培养协议, 培育胚胎鸡器官体外.利用这种方法, 可以对胚胎鸡组织的发育进行研究, 同时保持对培养环境的高度控制。

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

organotypic-culture-method-to-study-development-embryonic-chicken

Research

JoVE Journal

Developmental Biology

5.9K 次观看.  Mount Saint Vincent University. 在这里, 我们提出了一个脊髓培养协议, 培育胚胎鸡器官体外.利用这种方法, 可以对胚胎鸡组织的发育进行研究, 同时保持对培养环境的高度控制。

视频: Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

优化<em&gt;前OVO</em&gt;鸡胚培养，以发展的高级阶段

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

科研

发育生物学

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

水下滤纸三明治长期<em&gt;防爆大毛</em&gt;早期鸡胚时间推移成像

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

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 ...

生物学

Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the formation of quail-chicken chimeras and the use of the chorioallantoic membrane (CAM) to sustain the development of ectopic tissues date back to the last century. Nowadays, the combination of these classical techniques with recent in vitro methodologies offers novel prospects to further explore organ formation.
Here we describe a two-step approach to study early- and late-stages of organogenesis. Briefly, the embryonic region containing the presumptive territory of the organ is isolated from quail embryos and grown in vitro in an organotypic system (up to 48 h). Cultured tissues are subsequently grafted onto the CAM of a chicken embryo. After 10 days of in ovo development, fully formed organs are obtained from grafted tissues. This method also allows the modulation of signaling pathways by the regular administration of pharmacological agents and tissue genetic manipulation throughout in vitro and in ovo developmental steps. Additionally, developing tissues can be collected at any time-window to analyze their gene-expression profile (using quantitative PCR (qPCR), microarrays, etc.) and morphology (assessed with conventional histology and immunochemistry).
The described experimental procedure can be used as a tool to follow organ formation outside the avian embryo, from the early stages of organogenesis to fully formed and functional organs.

This article provides an updated approach to the classical quail-chicken chimera system to study organ formation, by combining novel in vitro and in ovo experimental procedures.

鸟类模型中器官形成早期和晚期的两步探讨: 胸腺和甲状旁腺器官发生范式

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

鸡胚胎脊髓片培养协议

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

神经科学

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed contributions to unveiling key processes in developmental biology.
Herein is described an optimized method to isolate embryonic tissues from quail and chickens by microsurgery and enzymatic digestion while preserving its biological properties. After isolation, tissues from both species are associated in an in vitro organotypic assay for 48 h. Quail and chicken tissues can be discriminated by distinct nuclear features and molecular markers allowing the study of the cellular cross-talk between heterospecific association of tissues. This approach is, therefore, a useful tool for studying complex tissue interactions in developmental processes with highly dynamic spatial modifications, such as those occurring during pharyngeal morphogenesis and the formation of the foregut endoderm-derived organs. This experimental approach was first developed to study the epithelial-mesenchymal interactions during early-stages of thymus formation. In this, the endoderm-derived prospective thymic rudiment and mesoderm-derived mesenchyme, were isolated from quail and chicken embryos, respectively.
The capacity of the associated tissues to generate organs can be further tested by grafting them onto the chorioallantoic membrane (CAM) of a chicken embryo. The CAM provides nutrients and allows gas exchanges to the explanted tissues. After 10 days of in ovo development, the chimeric organs can be analyzed in the harvested explants by conventional morphological methods. This procedure also allows studying tissue-specific contributions during organ formation, from its initial development (in vitro development) to the final stages of organogenesis (in ovo development).
Finally, the improved isolation method also provides three-dimensionally (3D) preserved embryonic tissues, that can also be used for high-resolution topographical analysis of tissue-specific gene-expression patterns.

This article provides a method to isolate pure embryonic tissues from quail and chicken embryos that can be combined to form ex vivo chimeric organs.

胸腺标本分离和鸡刺激器官的形成

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

一个 EX - OVO鸡胚胎培养系统适用于成像和显微外科的应用

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

The soaked bead assay involves targeted delivery of test reagent at any developmental time point to study the regulation of cell differentiation and morphogenesis. A detailed protocol, applicable to any experimental animal model, for preparing three different types of soaked beads and implanting these in the interdigit of a chicken embryo is presented.

使用浸泡珠测定分析鸡胚胎发生过程中的细胞分化，形态发生和图案化

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of ...

禽胚胎皮肤外植体培养方案的全面回顾

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin explant cultures to examine different aspects of skin development are described. First, ex vivo organ cultures and manipulations offer researchers opportunities to observe and study the development of feather buds directly. Skin explant culture can grow for 7 days enabling direct analysis of cellular behavior and 4D imaging at intervals during this growth period. This also allows for physical and molecular manipulations of culture conditions to visualize tissue response. For example, growth factor-coated beads can be applied locally to induce changes in feather patterning in a limited area. Alternatively, viral transduction can be delivered globally in the culture media to up or downregulate gene expression. Second, the skin recombination protocol allows researchers to investigate tissue interactions between the epidermis and mesenchyme that are derived from different skin regions, different life stages, or different species. This affords an opportunity to test the time window in which the epithelium is competent to respond to signals and its ability to form different skin appendages in response to signals from different mesenchymal sources. Third, skin reconstitution using dissociated dermal cells overlaid with intact epithelium resets skin development and enables the study of the initial processes of periodic patterning. This approach also enhances our ability to manipulate gene expression among the dissociated cells before creating the reconstituted skin explant. This paper provides the three culture protocols and exemplary experiments to demonstrate their utility.

作者聚焦:深入了解当前的实验性禽皮外植体方法

Here we describe protocols for three types of avian embryonic skin explant cultures that can be used to examine tissue interactions, 4D imaging timelapse movie (3D plus time), global or local perturbation of molecular function, and systems biology characterization.

使用禽皮肤外植体研究组织模式和器官发生

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin ...

脊髓培养法研究胚胎鸡组织的发展

摘要

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优化

水下滤纸三明治长期

文化早期鸡胚体外（新文化）的方法

鸟类模型中器官形成早期和晚期的两步探讨: 胸腺和甲状旁腺器官发生范式

鸡胚胎脊髓片培养协议

胸腺标本分离和鸡刺激器官的形成

一个 EX - OVO鸡胚胎培养系统适用于成像和显微外科的应用

使用浸泡珠测定分析鸡胚胎发生过程中的细胞分化，形态发生和图案化

使用禽皮肤外植体研究组织模式和器官发生

使用禽皮肤外植体研究组织模式和器官发生