Name: ex utero culture of mouse embryos from pregastrulation to advanced organogenesis
Uploaded: 2021-10-19T15:00:00
Description: Watch this Scientific Journal Video about Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis at JoVE.com

这项工作由Pascal和Ilana Mantoux资助;欧洲研究理事会（ERC-CoG-2016 726497-Cellnaivety）;空乘医学研究委员会（FAMRI）;以色列癌症研究基金（ICRF）教授，BSF，Helen和Martin Kimmel干细胞研究所，Helen和Martin Kimmel创新研究奖;以色列科学基金会（ISF），密涅瓦，谢尔曼药物化学研究所，Nella和Leon Benoziyo神经系统疾病中心，David和Fela Shapell遗传疾病研究中心，Kekst家庭医学遗传学研究所，Beth Rom-Rymer博士干细胞研究基金，Edmond de Rothschild基金会，Zantker慈善基金会，Zvia Zeroni的遗产。

所有动物实验均根据魏茨曼科学研究所的动物保护指南进行，并得到相关魏茨曼研究所IACUC的批准（#01390120-1，01330120-2，33520117-2）。根据赫尔辛基兰巴姆医疗中心委员会（#RMB-0452-15）的批准，要求健康的孕妇知情同意从脐带采集血液。健康成年人被要求根据魏茨曼科学研究所赫尔辛基委员会的指导方针（#1566-3）进行知情同意。
1. 培养基准备
<ol><li>使用500mL不含?...

<ol>
	<li>New, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-embryo+culture+and+the+study+of+mammalian+embryos+during+organogenesis.">Whole-embryo culture and the study of mammalian embryos during organogenesis.</a> Biological Reviews of the Cambridge Philosophical Society. 53 (1), 81-122 (1978).</li><li>Tam, P. P., Behringer, R. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postimplantation+mouse+development:+whole+embryo+culture+and+micro-manipulation.">Postimplantation mouse development: whole embryo culture and micro-manipulation.</a> International Journal of Developmental Biology. 42 (7), 895-902 (1998).</li><li>Nicholas, J. S., Rudnick, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+rat+embryos+in+tissue+culture.">The development of rat embryos in tissue culture.</a> Proceedings of the National Academy of Sciences of the United States of America. 20 (12), 656-658 (1934).</li><li>New, D. A., Stein, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+mouse+embryos+in+vitro.">Cultivation of mouse embryos in vitro.</a> Nature. 199, 297-299 (1963).</li><li>Rivera-P&#233;rez, J. A., Jones, V., Tam, P. P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+whole+mouse+embryos+at+early+postimplantation+to+organogenesis+stages:+developmental+staging+and+methods.">Culture of whole mouse embryos at early postimplantation to organogenesis stages: developmental staging and methods.</a> Methods in Enzymology. 476, 185-203 (2010).</li><li>New, D. A. T., Coppola, P. T., Terry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+explanted+rat+embryos+in+rotating+tubes.">Culture of explanted rat embryos in rotating tubes.</a> Journal of Reproduction and Fertility. 35 (1), 135-138 (1973).</li><li>Cockroft, D. 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+comparative+and+historical+review+of+culture+methods+for+vertebrates.">A comparative and historical review of culture methods for vertebrates.</a> International Journal of Developmental Biology. 41 (12), 127-137 (1997).</li><li>Parameswaran, M., Tam, P. P. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+a+second+neural+axis+by+the+mouse+node.">Induction of a second neural axis by the mouse node.</a> Development. 120 (3), 613-620 (1994).</li><li>Aguilera-Castrejon, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+utero+mouse+embryogenesis+from+pre-gastrulation+to+late+organogenesis.">Ex utero mouse embryogenesis from pre-gastrulation to late organogenesis.</a> Nature. 593 (7857), 119-124 (2021).</li><li>Takahashi, M., Makino, S., Kikkawa, T., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+rat+serum+suitable+for+mammalian+whole+embryo+culture.">Preparation of rat serum suitable for mammalian whole embryo culture.</a> Journal of Visualized Experiments: JoVE. 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本文介绍的培养方案可以在子宫外维持适当和连续的小鼠胚胎发育长达六天，从E5.5到E11。以前，处于这些发育阶段的胚胎只能在短时间内（长达48小时）在培养物中正常发育15。将气体调节模块耦合到滚筒培养箱以精确控制氧气浓度和高压气体压力对于本文所述的正确小鼠胚胎培养至关重要。将气体压力增加到7 psi可增强氧气扩散，允许胚胎在高达21%O2 / 5%CO2的大气...

哺乳动物胚胎的宫内发育限制了对植入后发育早期阶段的研究1，2。发育中的胚胎的不可接近性阻碍了对胚胎植入子宫后发生的关键发育过程的理解，例如动物身体计划的建立，生殖层的规范或组织和器官的形成。此外，早期植入后胚胎的尺寸非常小，使得在E103之前的子宫内通过活体成像难以观察。在这些阶段无法观察和操纵活胚胎，限制了对早期植入后胚胎发生的研究，仅限于发育过程中的快照。
植入前哺乳动物胚胎体外培养的方案已建立良好，可靠且经常使用4。然而，建立能够支持哺乳动物胚胎正常生长的宫外培养系统的尝试收效甚微5。一个多世纪以来，人们已经提出了各种培养技术，主要是通过在常规静态板6，7，8或旋转瓶（滚筒培养）中培养胚胎5，9，10。事实证明，这些平台有助于扩大植入后哺乳动物发育的知识11，12，尽管对于正常胚胎存活效率低下且仅限于短期。胚胎早在培养开始后24-48 h就开始出现发育迟缓和形态异常。
本研究为建立 宫前 胚胎培养系统提供了详细说明，该系统允许在植入后发育长达六天的条件下从前胚形成阶段持续发育到高级器官发生阶段13。本文描述了通过在静态板和辊培养平台上结合培养，支持E7.5胚胎（神经板和头褶阶段）生长直至后肢形成阶段（~E11）和E5.5 / E6.5扩展培养的改进的滚轮培养方案。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m pore size filter (250 mL)</td>
			<td>JetBiofil</td>
			<td>FCA-206-250</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m pore size syringe PVDF filter</td>
			<td>Millipore</td>
			<td>SLGV033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>8-well &#181;-plates glass bottom/ibiTreat</td>
			<td>iBidi</td>
			<td>80827/80826</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle with adaptor cap for gas inlet</td>
			<td>Arad Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bungs (Hole)</td>
			<td>B.T.C. Engineering, Cullum Starr Precision Engineering</td>
			<td>BTC 06</td>
			<td>Used to seal the bottles to the drum</td>
		</tr>
		<tr>
			<td>Bungs (Solid)</td>
			<td>B.T.C. Engineering, Cullum Starr Precision Engineering</td>
			<td>BTC 07</td>
			<td>Used to seal the rotating drum</td>
		</tr>
		<tr>
			<td>Culture bottles</td>
			<td>B.T.C. Engineering, Cullum Starr Precision Engineering</td>
			<td>BTC 03/BTC 04</td>
			<td>Either Glass Bottles (Small) BTC 03 or Glass Bottles (Large) BTC 04</td>
		</tr>
		<tr>
			<td>D(+)-glucose Monohydrate</td>
			<td>J.T. Baker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond knife</td>
			<td>Fine Science Tools</td>
			<td>10100-30/45</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Pressure Gauge</td>
			<td>Shanghai Benxu Electronics Technology co. Ltd</td>
			<td>BX-DPG80</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>GIBCO</td>
			<td>11880</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Biological industries</td>
			<td>02-020-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Biological industries</td>
			<td>04-013-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas regulation module</td>
			<td>Arad Technologies</td>
			<td>HannaLab1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>GIBCO</td>
			<td>35050061</td>
			<td>glutamine</td>
		</tr>
		<tr>
			<td>Graefe forceps</td>
			<td>Fine Science Tools</td>
			<td>11052-10</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>GIBCO</td>
			<td>15630056</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgical forceps (Dumont #5, #55)</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipettes (glass)</td>
			<td>Hilgenberg</td>
			<td>3150102</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipettes (plastic)</td>
			<td>Alexred</td>
			<td>SO P12201</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Biological industries</td>
			<td>03-031-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes (60 mm and 100 mm)</td>
			<td>Falcon</td>
			<td>351007/351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision incubator system</td>
			<td>B.T.C. Engineering, Cullum Starr Precision Engineering</td>
			<td>BTC01</td>
			<td>BTC01 model with gas bubbler kit</td>
		</tr>
		<tr>
			<td>Pro-coagulant sterile test tubes (5 mL)</td>
			<td>Greiner Bio-One</td>
			<td>#456005</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat whole embryo culture serum</td>
			<td>ENVIGO Bioproducts</td>
			<td>B-4520</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscopic microscope equipped with heating plate</td>
			<td>Nikon</td>
			<td>SMZ18</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile syringes (5, 10 ml) for sera filtration</td>
			<td>Pic Solution</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14094-11</td>
			<td></td>
		</tr>
	</tbody>
</table>

ex utero culture of mouse embryos from pregastrulation to advanced organogenesis

植入后哺乳动物胚胎培养方法通常效率低下，仅限于解剖子宫后的短时间。最近已经开发了平台，用于从卵筒阶段到晚期器官发生的高度健壮和长时间的小鼠胚胎 宫外 培养。这些平台使孕前胚胎（E5.5）能够适当和忠实地发育，直到后肢形成阶段（E11）。在这些环境中，晚期胃泌尿胚胎（E7.5）在旋转瓶中生长，而从预气胚形成阶段（E5.5或E6.5）的扩展培养需要静态和旋转瓶培养的组合。此外，对O2 和CO2 浓度、气体压力、葡萄糖水平的敏感调节以及使用特定的 宫前 培养基对于胚胎的正常发育至关重要。这里提供了用于延长 宫前 小鼠胚胎培养的详细分步方案。从原肠胚形成到器官发生， 在子宫外 生长正常小鼠胚胎的能力代表了表征胚胎发育过程中不同实验扰动效应的宝贵工具。

描述的E7.5胚胎（晚期原胚期）的滚轮培养条件支持恒定和正常的胚胎生长，4个培养日后的平均效率接近75%（图2 和 表1）。胚胎发育的效率可能因不同的小鼠遗传背景而异，但始终是稳健的（图2C）。补充HBS代替HCS在 宫前 培养4天后产生约68%的效率，这取决于小鼠的遗传背景（图2D 和 表2）。 子...

Watch this Scientific Journal Video about Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis at JoVE.com

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

增强的全胚胎培养平台允许植入后小鼠胚胎的连续和稳健的 宫外 发育长达六天，从前肠胚形成阶段到高级器官发生。在该协议中，我们详细介绍了使用静态板和旋转瓶系统成功培养胚胎的标准程序。

前子宫 从前肠胚形成到晚期器官生成的小鼠胚胎培养

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. ...

这种方法对于理解哺乳动物中形成的原肠胚形成至关重要。通过允许在长期隐藏在母体子宫内的胚胎阶段对活胚胎进行实验。它允许小鼠胚胎在子宫外持续有效和适当的生长，长达6天。快速和充分的胚胎解剖和气体浓度和压力的预胃原析是成功胚胎生长的最关键步骤。要开始培养7.5天龄或更先进的小鼠胚胎，请在胚胎解剖前打开滚筒培养箱系统的旋转器和加热装置至少1小时。此外，将高压灭菌器水添加到进气瓶和出口试管中。接下来，通过按下主开关打开气体调节模块，然后使用相应的控制器将氧气和二氧化碳值设置为5%打开气体调节器并移动压力变送器中的电压开关，将气体压力设置为每平方英寸约6.5至7磅。使用数字压力表确认气体压力值。通过检查在精密培养箱内分配的充满水的试管内产生的气泡速率来监测气体流量。通过关闭或打开水瓶盖上的气流阀来设置适当的气泡速率。确保气流允许以每秒2到4个气泡的速率形成气泡，或者将气泡流量设置为气泡流出到充满水的出口管的第一个点。接下来，用2毫升培养基填充培养瓶。使用空心硅包密封瓶子，并将其插入空心旋转滚筒中进行1小时的预平衡。使用实心滚筒将旋转滚筒中的空隙密封。接下来，从安乐死的怀孕雌性小鼠身上解剖胚胎。用70%乙醇清洁腹部，用剪刀切开皮肤和腹壁。找到子宫的一端，并在卵巢和子宫之间的交叉处切开。继续沿着子宫切割直到另一端，然后转移到充满DPBS的100毫米培养皿中。在DPBS中快速清洗概念，并成对切割，以方便胚胎处理。将所有概念对移动到60毫米培养皿中的预平衡解剖培养基中，然后切入单个概念。接下来，使用一对粗镊子，通过撕裂子宫组织来去除概念的子宫壁，然后使用细小的显微外科钳，切开梨形蜕膜的尖端。将镊子插入胚胎旁边，平行于其长轴，并打开镊子将蜕膜分成两半。最后，使用细镊子，从蜕膜上抓住胚胎，将顶叶蛋黄袋从胚胎上剥离，留下完整的外蝶骨锥体附着在卵圆筒上。选择神经板或早期头部折叠阶段的胚胎，这些胚胎在外胚层中未显示损伤。将每瓶5至6个选定的胚胎转移到预平衡的玻璃培养瓶中，并将瓶子置于37摄氏度的旋转培养系统中，在5%氧气和5%二氧化碳的气氛中。为了培养预原胚和早期原肠胚和静态板，向8孔板的每个孔中加入250微升新鲜制备的宫前胚胎培养基。将板放入37摄氏度的二氧化碳培养箱内进行预平衡。如前所述，将胚胎从子宫中解剖出来后，使用微量移液管将单个胚胎转移到8孔板的每个孔中，并将板放入培养箱内，在37摄氏度下加入5%二氧化碳。在体视显微镜下对胚胎进行成像，并仅选择具有良好形成的羊膜腔且没有明显损伤且没有Reichert膜的胚胎进行培养。该协议中描述的7.5天大胚胎的滚筒培养条件支持恒定和正常的胚胎生长，4个培养天后的平均效率接近75%。胚胎发育的效率因不同的小鼠遗传背景而异，但始终保持稳健。在培养 4 天后，补充 HBS 代替 HCS 可产生约 68% 的效率。使用具有HCS和HBS的宫前胚胎培养基，以超过90%的效率正确概括6.5个胚胎和静态板的发育。从预孕胚胎开始的培养物显示出46%的正常发育效率，直到早期somite阶段，近17%的胚胎在培养6天后完成正常发育。形态发生和组织发育正常进行，直到42个索米特。此处显示了从胚胎第7.5天，6.5天和5.5日开始的培养物在胚胎中观察到的代表性发育缺陷。对外胚层细胞有轻微损伤的胚胎或保留Reichert膜的胚胎应丢弃。早期胚胎将无法正常生长或表现出严重的发育迟缓，而胚胎外胚层母细胞附着在板表面将导致进一步胚胎发育的失败。在有缺陷的胚胎中观察到的主要异常是蛋黄袋外后部区域的发育或神经褶皱生长的缺陷。在从5.5天大的胚胎发育的培养物中，经常观察到发育缺陷是存在小的，不发达的外胚层。使用这种方法，研究人员能够在发育植入后胚胎时进行各种物理或化学操作。例如，操作、细胞移植或谱系追踪。这种方法不仅为研究小鼠植入后发育铺平了道路，而且还为在来自其他物种（如人类）的胚胎中实施类似的培养方法铺平了道路。

Research

JoVE Journal

Developmental Biology

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

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

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

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

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

在对小鼠胚胎子宫内心脏番茄凝集素注射到计肾血流量

The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis ...

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

小鼠海马组织的前子宫内电和器官切片文化

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

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

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

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 ...

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

运用<em&gt;离体</em&gt;全胎儿各器官的直立滴文化的过程中鼠标研究器官发育过程

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

日本鹌鹑胚胎的卵黄囊膜主内胚上皮细胞培养

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

从小鼠胚胎植入前胚胎干细胞系的推导

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

神经冠状原脂肪衍生干细胞的分离、培养和异位诱导，来自围阴脂肪组织

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

为早期小鼠胚胎测序准备小型RNA库

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

前子宫 从前肠胚形成到晚期器官生成的小鼠胚胎培养

前子宫从前肠胚形成到晚期器官生成的小鼠胚胎培养