我们感谢肇一乘先生编辑视频录像和乐于助人的意见。我们也感谢Drs.Yuji Tsunekawa和凯驰吉崎实物录像助理。这项工作是支持KAKENHI青年科学家B和优先领域，分子由日本文部科学省的脑科学。我们承认，支持全球COE计划“全球脑科学的基础和转化研究中心”由文部科学省，日本和演化的科学和技术的核心研究（CREST）由日本科技公司从日本科学技术振兴机构（JST） 。

1。设立职业英语运动系统<ol><li>联合0.22μm的滤膜过滤，职业英语运动系统内的进口硅管 ） 。 </li><li>打开一个汽缸气体， 含 O 2（95％）和CO 2（5％）阀门。通过鼓泡瓶含有灭菌水鼓流入的气体混合物。 </li><li>调整混合气体流量50立方厘米。 </li><li>插入让出一瓶水硅管，并检查了混合气体的流量。 </li><li>在20转/分的速度旋转的鼓。 </li></ol> 2。培养基的制备<ol><li>解冻大鼠立即离心（IC）血清在37.0 ° C B）。添加D -葡萄糖（2毫克/毫升）蒸压100毫升烧杯中进入解冻血清。培养基应准备在小学文化的层面（例如，使用遮光罩）。 </li><li> 1：400稀释血清添加抗生素antimycotic（100X）的液体。 </li><li>蒸发剩余的异氟醚（由于麻醉大鼠在采血），从20分钟，在室温（RT）C）血清。 </li><li>使用0.45微米的滤膜过滤，消毒的培养基。 </li><li>在每个培养瓶中的培养基倒入3.0毫升，密封与 ​​硅塞顶部，每个瓶子，用铝箔覆盖（见文献14）D ）。 </li><li>准备三个或四个处理的培养皿中，含有台氏生理盐水发送）。 </li></ol> 3。麻醉大鼠和子宫的隔离<ol><li>深深吸入或注射麻醉药麻醉超时孕鼠。 </li><li>清洁麻醉用70％乙醇的孕鼠的腹部。 </li><li>拿起镊子的皮肤和exteriorize 与 F大剪刀切割皮肤的腹壁 ） 。 </li><li>拿起一双镊子腹壁和U形大上大剪刀腹壁纵切口胸G 级）。 </li><li>熊远肠道一双镊子的左侧和揭露​​一端连接到卵巢子宫。 </li><li>拿起一双镊子子宫底和大剪刀之间的子宫和卵巢切的位置。 </li><li>从孕鼠的子宫，取出切割子宫的另一端，直到子宫周围的脂肪。 </li><li>子宫转移到台氏培养皿中含有生理盐水。 </li><li>安乐死大鼠，取出后，子宫过度管理的麻醉剂或颈椎脱位。 </li></ol> 4。去除胚胎从子宫<ol><li>简单地说，在台氏生理盐水冲洗子宫，并转移到第二一双细镊子h 的 Petri菜）。 </li><li>在双目显微镜下，拿起一双细镊子的子宫壁和削减对面侧的子宫壁mesometrium使用眼科直剪刀的血管连接。 </li><li>眼科直剪刀的尖端插入子宫壁和蜕膜之间的空间。 antimesometrial方沿纵向切子宫壁。 </li><li>丛蜕膜在mesometrial一边单独conceptuses用细镊子一）两对从子宫。 </li><li> conceptuses转移到第三一个合适的直径灭菌玻璃吸管的Petri菜。 </li></ol> 5。大鼠胚胎的剖析<ol><li> microforceps的尖端插入蜕膜和上横切口周围两个双钳conceptus蜕膜。 </li><li>钳提示插入蜕膜，胎盘侧，使两个纵向切口顶端上的蜕膜。 </li><li>在胎盘中取出，有两个双钳抓取蜕膜。两个双钳取出剩余的蜕膜。 </li><li>拿起赖克特的膜，上水平切口，并在反胎盘侧conceptus的膜分开。 </li><li>钳的两对卵黄囊胚胎的头部附近的一个小口子，和一双眼科弯剪刀切断与卵黄囊。重要的是不损伤主要血管。 </li><li>羊膜拿起网站围绕胚胎的头两对细镊子，轻轻拔出撕破羊膜 h胚胎卵黄囊 ） 。卵黄囊，胚胎的身体应该拉到midgestation阶段，以增加胚胎的氧气供应。 </li></ol> 6。全胚胎培养<ol><li>检查胎盘，卵黄囊，心脏跳动和血液循环的条件的损害。 </li><li>意识到不伸展脐带，转让一个培养瓶用消毒​​的玻璃吸管的胚胎。台氏生理盐水培养瓶，应尽可能少。 </li><li>删除从肩鼓无孔硅插件。连接插头与一个职业英语运动系统的肩鼓垂直孔培养瓶中。要小心不要把鼓，因为在不适当的方向力肩鼓是一种非常优良的设备。瓶含有解剖胚胎被转移到一个开放的孵化器的前门文化之一应尽量避免孵化器的温度降低j） 。 </li><li>经过10小时的文化，增加混合气体的流量后24小时至75毫升和100毫升（见表1）。 </li><li>培养基应改为24小时各地转移到新的培养瓶中培养的胚胎与新鲜配制的中等。 34小时后增加的流量高达125 CC和高达150毫升后48小时（见表1） </li><li>偶尔检查计数心跳率（120-150/min即是最优的），并通过观察血液循环，培养胚胎的情况。如果需要计数的体节数判断胚胎在​​台氏生理盐水增长。在正常的发展，一个体节是在两小时内补充。 </li><li>职业英语运动完成时，停止供应气体混合物和断开的入口管和膜过滤器，以避免回冒泡瓶从入口管流。 </li></ol> 7。注释<ol class="lalpha"><li>职业英语运动系统内的温度始终保持在37.0-37.5 ° C。 </li><li> 100％大鼠IC血清是经常使用在我们的职业英语运动对大鼠和小鼠胚胎中，并在使用前保持在-20 ° C。 </li><li>我们购买IC血清，这是从与异氟醚麻醉的大鼠收集大鼠。 </li><li>一次性瓶上，也可从池本梨。瓶子和硅插头应高压灭菌。 </li><li>清扫应在室温下进行的胚胎，因为胚胎在低温或高温期间保持夹层，影响职业英语运动的后续发展。 </li><li>干热消毒清扫工具。 </li><li>应切开皮肤和腹壁，单独成一碟尽可能避免污染的头发。我们当我们改变了他们的一双新的一双大剪刀和镊子切开皮肤，分离子宫，分别。 </li><li>使用镊子，其秘诀是罚款是非常重要的解剖组织的肯定。我们用石头和油机手动提升产钳的提示。 </li><li>我们使用镊子的一侧，像刀子一样，以避免过量的压力胚胎。 </li><li>当职业英语运动系统内的温度降低反复开门，我们继续上的灯打开，以保持系统内部的温度。 </li></ol> 8。代表性的成果图1显示了解剖大鼠胚胎和体外培养的大鼠胚胎的程序。 <img src="/files/ftp_upload/2170/2170fig1.jpg" alt="图1" /> 图1。E12.5大鼠胚胎全胚胎培养。 （A）从孕鼠的子宫解剖conceptus。 （b）取消蜕膜胎盘侧。 （三）赖克特的膜去除。 （四）开幕卵黄囊。 （五）在职业英语运动开始后6小时瓶大鼠胚胎培养。 （F）的大鼠胚胎培养42小时。 D，蜕膜，R;赖克特的膜，P，胎盘; YS，卵黄囊，E，胚胎。 <table border="1"><tbody><tr><td></td><td> 0小时</td><td> 12小时</td><td> 24小时</td><td> 36小时</td><td> 48小时</td></tr><tr><td> E12.5大鼠胚胎</td><td> 95％50立方厘米</td><td> 95％，75 CC （10小时） </td><td> 95％的100毫升</td><td> 95％的125毫升（34小时） </td><td> 95％的150 CC </td></tr></tbody></table> 表1。最优氧条件。

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有成功的灭鼠职业英语运动的两个关键步骤。首先，剥离过程应该是准确的，不破坏胚胎，特别是血管。其次，程序应尽快，因为氧气和营养通过胎盘从子宫隔离后不再提供。这是对年纪较大的胚胎的关键。在E12.5大鼠职业英语运动的情况下，我们应该在30分钟内转移到培养瓶解剖胚胎。 我们已经分析了野生型和标签少量细胞用荧光染料DII或DII -标记的细胞移植在大鼠胚胎的?...

<table border="1"><tbody><tr><td> 名称 </td><td> 类型 </td><td> 公司 </td><td> 产品编号 </td><td> 评论 </td></tr><tr><td colspan="5"> 设置最多职业英语运动 </td></tr><tr><td>职业英语运动系统（10-0310） </td><td>工具</td><td>池本丽卡</td><td> 010-0310 </td><td>另外一个小的模型也可用。 </td></tr><tr><td>无孔的硅插件</td><td>工具</td><td>池本丽卡</td><td> 010-032-08 </td><td></td></tr><tr><td>混合气体气瓶</td><td>工具</td><td>日光山庄</td><td> - </td><td>含95％氧气和5％的二氧化碳。自定义的顺序。 </td></tr><tr><td>气体调节器</td><td>工具</td><td>小野制作所</td><td> WR - 11 </td><td></td></tr><tr><td> 0.22微米的过滤器Millex GS </td><td>工具</td><td> Millipore公司</td><td> SLGS033SS </td><td></td></tr><tr><td colspan="5"> 麻醉和子宫隔离 </td></tr><tr><td>大剪刀</td><td>工具</td><td> Napox </td><td> B - 7H </td><td></td></tr><tr><td>镊子</td><td>工具</td><td> Napox </td><td> A - 3 - 2 </td><td></td></tr><tr><td>处置培养皿 （90毫米× 15毫米） </td><td>工具</td><td>磐</td><td> SH90 - 15 </td><td>深型的菜是最好的解剖。 </td></tr><tr><td>异氟醚</td><td>试剂</td><td>雅培</td><td> B506 </td><td>麻醉</td></tr><tr><td>戊巴比妥钠</td><td>试剂</td><td>先灵葆雅动物保健</td><td> - </td><td>麻醉</td></tr><tr><td>台氏生理盐水</td><td>试剂</td><td> - </td><td> - </td><td>根据文献的协议。 2。保存在4 ° C。 </td></tr><tr><td>定时妊娠的SD大鼠</td><td>动物</td><td>查尔斯河实验室日本</td><td> - </td><td></td></tr><tr><td colspan="5"> 培养基的制备</td></tr><tr><td>文化玻璃瓶</td><td>工具</td><td>池本丽卡</td><td> 010-032-05 </td><td></td></tr><tr><td>处置培养瓶</td><td>工具</td><td>池本丽卡</td><td> 010-032-06 </td><td></td></tr><tr><td>硅孔插头</td><td>工具</td><td>池本丽卡</td><td> 010-032-07 </td><td></td></tr><tr><td>铝箔</td><td>工具</td><td>任何公司</td><td> - </td><td></td></tr><tr><td>灭菌袋</td><td>工具</td><td> Hogy </td><td> HM - 26 </td><td>对于培养瓶</td></tr><tr><td>灭菌袋</td><td>工具</td><td> Hogy </td><td> HM - 14A </td><td>硅插头</td></tr><tr><td> 0.45微米的过滤器Millex医管局</td><td>工具</td><td> Millipore公司</td><td> SLHA033SS </td><td></td></tr><tr><td> 50毫升锥形管</td><td>工具</td><td>碧迪</td><td> 352070 </td><td></td></tr><tr><td> 100毫升烧杯中</td><td>工具</td><td>磐</td><td> TE - 32 </td><td></td></tr><tr><td>大鼠集成电路血清</td><td>试剂</td><td>查尔斯河实验室日本</td><td> - </td><td>请参阅：邦彦森崎先生， 电话：+81-（0）45-474-9336传真：+81-（0）45-474-9340 </td></tr><tr><td> D（+）-葡萄糖</td><td>试剂</td><td>瓦科</td><td> 041-00595 </td><td></td></tr><tr><td>抗生素antimyotic液体</td><td>试剂</td><td> Gibco公司</td><td> 15240 </td><td></td></tr><tr><td colspan="5"> 解剖胚胎 </td></tr><tr><td>眼科直剪刀</td><td>工具</td><td> Napox </td><td> MB50 - 7 </td><td>切割子宫壁</td></tr><tr><td>眼科弯剪刀</td><td>工具</td><td> Napox </td><td> MB54 - 2 </td><td>切割卵黄囊</td></tr><tr><td>钳＃5 </td&gt; <td>工具</td><td>活力</td><td> T6715 </td><td></td></tr><tr><td>钳＃5F </td><td>工具</td><td>雷吉娜</td><td> T6819 </td><td></td></tr><tr><td>玻璃吸管</td><td>工具</td><td> - </td><td> - </td><td>由手使用巴斯德吸管。 </td></tr><tr><td>灭菌袋</td><td>工具</td><td> Hogy </td><td> HM - 4 </td><td>对于玻璃移液器</td></tr><tr><td>双目显微镜</td><td>工具</td><td>徕卡</td><td> Mz7s </td><td></td></tr><tr><td>光</td><td>工具</td><td>徕卡</td><td>华彩150XD </td><td></td></tr><tr><td>用油石</td><td>工具</td><td>内田洋子</td><td> 833-2000 </td><td>钳锐化</td></tr><tr><td>机械油</td><td>工具</td><td>内田洋子</td><td> 835-0000 </td><td>钳锐化</td></tr></tbody></table>

the method of rodent whole embryo culture using the rotator-type bottle culture system

全胚胎培养（WEC）技术已在1950年开发新和他的同事，和发育生物学的1应用。虽然哺乳动物胚胎的发展和增长是严重依赖对胎盘功能，职业英语运动技术使我们能够在胚胎一天（五）6.5 E12.5相应midgestation阶段，在有限的时期在文化大鼠和小鼠胚胎体外条件鼠标或在大鼠2，3，4 E8.5 - E14.5。职业英语运动，我们可以直接目标所需的领域使用细的玻璃毛细管的胚胎，因为胚胎可以在显微镜下操作。因此，灭鼠职业英语运动是非常有用的技术时，我们要研究动态postimplanted哺乳动物胚胎的发育过程。到今天为止，几种类型的职业英语运动的系统已经开发1。其中，肩，瓶型文化体系是最流行 ​​和最适合长期midgestation，即胚胎的文化，后E9.5和E11.5的小鼠和大鼠， 分别为1 。在这个视频协议，我们展示了我们的标准程序后E12.5使用了原来的旋转系统，这是由新的Cockroft 5，6设计精致模型大鼠职业英语运动，并引入职业英语运动技术的研究在哺乳动物发育的各种应用生物学。

Watch this Scientific Journal Video about The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System at JoVE.com

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

全胚胎培养技术，使我们文化的大鼠和小鼠胚胎<em&gt;体外</em&gt;在midgestation阶段对应有限的时期条件。在这个视频协议，我们将演示后，用肩瓶型文化体系E12.5大鼠全胚胎培养我们的标准程序。

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 ...

the-method-rodent-whole-embryo-culture-using-rotator-type-bottle

Research

JoVE Journal

Biology

23.6K 次观看.  Tohoku University Graduate School of Medicine. 全胚胎培养技术，使我们文化的大鼠和小鼠胚胎体外在midgestation阶段对应有限的时期条件。在这个视频协议，我们将演示后，用肩瓶型文化体系E12.5大鼠全胚胎培养我们的标准程序。

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

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

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

Method for Culture of Early Chick Embryos ex vivo New Culture

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

文化早期鸡胚体外（新文化）的方法

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

科研

生物学

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

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

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

小鼠胚胎的龙文化，一个系统来评价分枝的分子机制

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

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 ...

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 ...

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

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

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 ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

大鼠血清适用于哺乳动物全胚胎培养的制备

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

胞外酸性 pH 培养体系的建立

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

使用三维共培养系统NICO-1评估卵巢癌干细胞样细胞的血管遗传特性

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera frugiperda (Sf9) cells with Sf9 cells infected with baculovirus (BV)-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap in serum-free suspension culture. Cells are cultured in a flask in an orbital shaker or Wave bioreactor. To release the AAV particles, producer cells are lysed in buffer containing detergent, which is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the cell lysate using AVB Sepharose column chromatography, which binds AAV particles. Bound particles are washed with PBS to remove contaminants and eluted from the resin using sodium citrate buffer at pH 3.0. The acidic eluate is neutralized with alkaline Tris-HCl buffer (pH 8.0), diluted with phosphate-buffered saline (PBS), and further concentrated with tangential flow filtration (TFF). The protocol describes small-scale pre-clinical vector production compatible with scale-up to large-scale clinical-grade AAV manufacturing for human gene therapy applications.

Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System

In this protocol, AAV2 vector is produced by co-culturing Spodoptera frugiperda (Sf9) insect cells with baculovirus (BV)-AAV2-green fluorescent protein (GFP) or therapeutic gene and BV-AAV2-rep-cap infected Sf9 cells in suspension culture. AAV particles are released from the cells using detergent, clarified, purified by affinity column chromatography, and concentrated by tangential flow filtration.