We thank Mr. Hajime Ichijo for video recording and helpful advice concerning editing the video. We also thank the Osumi lab members for animal care. This work is supported by a Grant-in-Aid for Scientific Research on Innovative Area, Neural Diversity and Neocortical Organization, from MEXT of Japan (to N. O.). T. K. was supported by a Research Fellowship of JSPS for Young Scientists.

注：動物実験は、実験動物の管理と使用に関する健康ガイドの国立研究所に従って行った。東北大学医学部の動物実験委員会は、ここに記載の実験手順を承認した。 1。麻酔および開腹<ol style="margin-left: 40px;"><li>採血のために、特定の病原体を含まない雄のSDラットを使用しています。水を提供しながら、少なくとも18時間、ラットを絶食。血液を収集するための年齢（550〜650グラム）の6-8ヶ月で退職した男性のブリーダーラットを使用してください。 </li><li> 10分間、2.0〜3.5 L /分で、麻酔を導入するために、麻酔装置に接続ボックスに、吸入麻酔薬であるイソフルラン2.5から4.0パーセントを流れる。麻酔を導入するボックスにラットを転送します。 <ol><li>ピンセットで足に刺激に対する応答の低下を確認することによって、麻酔を確認してください。ラットのWIの鼻をカバー深く採血時にラットを麻酔するための麻酔器に接続されたマスクを番目。濃度および流量は、それぞれ、2.5から4.0パーセントと2.0〜3.5 L /分に維持される。 注：この試薬 ​​展示イソフルラン22で麻酔したラットから得られた血清中で培養した胚に比べ胚発生の遅れで麻酔したラットから採取される血清中の胚が培養されたため、ハロタン吸入麻酔のために使用すべきではありません。 </li></ol></li><li>毛皮の汚染を避けるために、麻酔したラットの腹部に70％エタノールを注ぐことによって皮膚を消毒する。ピンセットで下部領域で消毒し、皮膚や腹壁をピックアップし、内臓を露出させ、大きなハサミで胸部領域に向けて、同時に、これらの層を切った。ピンセットと腹腔の外腸を反映している。 </li><li>と後肢に向けて皮膚や腹壁をカット大鋏は、さらに後方腹部を露出させる。ピンセットと腹腔の外に余分な脂肪を反映している。 </li><li>鉗子の2ペアを使用して正中線で内臓脂肪をピックアップし、腹部大動脈を露出させ、平行方向に慎重に脂肪を分割します。簡単に腹部大動脈の分岐部を識別するために、縦方向に内臓脂肪を分割するには、この手順を繰り返します。加えて、大動脈は拍動性であり、この特性を利用して、隣接する大静脈と区別することができる。 </li><li>腹部大動脈の周りの脂肪や鉗子の2ペアを慎重に分岐をピックアップし、腹部大動脈に脂肪を分離する。 </li></ol> 2。採血<ol style="margin-left: 40px;"><li>下方向に斜面で、大動脈の分岐部に直接頭蓋20ミリリットルの注射器に接続されている21のG針の先端を慎重に挿入します。 1で注射器を保持する手、そして雄ラットからの血液の15ミリリットルを収集するために、ゆっくりとシリンジを引き出します。 </li><li>注射器から針を外し、2氷冷10-mlの滅菌試験管（ 図1A）に血液を注ぐ。血栓の形成を遅らせるために、遠心分離まで氷上にチューブを保つ。一度に複数のチューブを遠心分離することにより、遠心分離時間を短縮。 </li><li>開胸して心臓や断頭を切断することによって、麻酔したラットを安楽死させる。 </li></ol> 3。ラットの血清の準備<ol style="margin-left: 40px;"><li>上下の層（ 図1C）に血液を分離するために1,200×gで、室温（RT）で5分間採取した血液を遠心分離する。 </li><li>フィブリンクロットを修正する1.5〜2時間、4℃でチューブを保つ。 </li><li>湾曲したピンセットを使用して、上層のフィブリン塊をピックアップし、血清を分離するために、フィブリン塊を絞る。すると、DOが直面湾曲したピンセットでフィブリン塊を押す2層（ 図1D）との界面近傍wnwards。 </li><li>さらに、フィブリン塊を分離するためにステップ3.3を繰り返します。 </li><li> 1200 XGおよびRT（ 図1E）で5分間試験管を遠心分離する。 </li><li>慎重に層流気流キャビネットに滅菌ピペットを用いて、15​​ミリリットルの滅菌チューブに血清を移す。 </li><li>残りの赤血球を除去するために1,200×gで、室温で5分間収集ラット血清を遠心分離する。 </li><li>プールチューブの底に残留赤血球（ 図1F）による汚染を避けるために、滅菌ピペットを用いて注意深く新しい15ミリリットル滅菌チューブに血清。 </li><li>補体系を不活性化するために30分間56℃の水浴中で血清をインキュベートする。 </li><li>層流気流キャビネットにRTにラット血清のチューブを冷却します。血清は、個々の滅菌チューブに10ミリリットルに等分しなければならない。使用します（ 図1H）まで-20℃でチューブを保管してください。 </li></ol>

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No conflicts of interest are declared.

WECの実験における再現性のある結果は、高品質のICラット血清、正確胚解剖技法3を使用することに依存している。空腹時には、血液中のグルコース濃度を標準化する必要があるため、採血前に絶食期間を短くしないでください。ホルモンレベルが発情周期に係る雌の動物に変化するため、雌ラット血清の調製のために使用すべきではない、およびエストラジオールホルモンのこのよ?...

モデル動物種々の分子および細胞レベルでの発達のメカニズムを調べるために発生生物学において使用される。例えば、両生類や鳥類は、広くこれらの胚が母親の外側を開発するための胚を直接操作するのに適している古典的なモデル動物として使用されている。これらの動物とは対照的に、哺乳類の胚は母親の子宮で成長し、後の段階での成長は、子宮の機能に決定的に依存している。したがって、そのような直接早い段階でのマウスおよびラット由来のもののような哺乳動物の胚を操作するために、典型的には困難である。 1960年代には、デニスの新連続酸素供給や熱制御1とWECの装置を用いて哺乳動物全胚培養（WEC）の手法を確立した。 WECでは、マウスおよびラットの胚は、生体外 （ すなわち、子宮の外側）に成長することができます。 WECの技術は、多くの場合、様々なchemicを添加することによって奇形学において使用されたがらは、培養培地中に化合物が、この技術はさらに、哺乳動物2-4の固有の発達のメカニズムを調べるために、種々の発生生物学研究に使用されている。例えば、WECは、蛍光色素5、細胞移植6、および7リポフェクションおよびエレクトロ8-13を介して遺伝子導入を用いて、野生型及び変異胚におけるこのような細胞標識のような他の技術と組み合わされる。 近年、 子宮操作の後の段階でげっ歯類の胚における発生過程を分析するために使用されており、エレクトロポレーション技術14-16と組み合わされている。しかしながら、これらの技術は、初期の段階で胚にDNA溶液の正確な局所注射を達成する困難さに起因し、妊娠中期の胚の着床後の操作には適していない。初期胚への超音波ガイド下細胞移植およびウイルスベクターの注入が（ すなわち、子宮内でマウスE8.5-E-9.5）は、以前に17,18に報告されている、優れたスキルが高い成功率でこれらの実験を行うために必要とされる。したがって、高いアクセシビリティエクスビボでWECは、マウスおよびラット胚の操作に対して利点を有する。 雄性ラットから調製し、直ちに遠心分離した（IC）ラット血清は、多くの場合、WEC媒体に使用される。胚（ すなわち、ラット又はマウスE10.0においてE8.0より以前）着床後の段階で培養すると、合成培地およびICラット血清の混合物はしばしばWEC 19のための媒体として使用される。全く、現在入手可能な代替メディアが許さないので、妊娠中期での培養胚（ すなわち 、E8.0-12.5マウス胚またはラット胚におけるE10.0-E14.5）で、100％の血清を培地として使用する必要があります胚は、2日以上、インビトロで正常に成長する。 高品質のラット血清の調製物であるWECの実験で再現性を達成するための重要なステップ。遅延遠心分離に比べて、ICは、赤血球の大部分が既にフィブリン塊から分離されているので、血清はフィブリン塊を圧搾することによって収集されるときに溶血を低減するという利点を有する。溶血性ラット血清、ラットおよびマウス胚の正常な成長を支えることができないように、即時の遠心分離を用いて血清の調製は、遅延された遠心分離を用いて調製することが好ましい。我々のプロトコルは、他のプロトコルに比べて2つの手順が含まれている18〜20（ すなわち 、前最初の遠心分離に氷上で採取した血液を貯蔵し、最初の遠心分離の後、4℃で2時間採取した血液サンプルを維持する）。前者のステップは、血栓の形成を遅らせることができ、後者のステップは、簡単にスクイズのため、フィブリン塊の凝固を促進する。そのため、我々のプロトコルは、初心者で使用することができます。しかし、採血し、血清を再生する単にプロトコルブックを参照することにより正確に抽出が19-21は非常に困難である。このビデオの記事では、我々は雄ラット、遠心分離により血清の抽出の腹部大動脈から採血を含み、最適なICのラット血清の調製のための私達の標準的なプロトコルを示しています。

<table><tbody><tr><td>Isoflurane</td><td>Abbott</td><td>B506</td><td>For rat anesthesia.</td></tr><tr><td>Large scissors</td><td>Napox</td><td>B-7H</td><td>Stelized scissors for cutting the skin and muscle of rats.&amp;nbsp;</td></tr><tr><td>Curved forceps</td><td>Napox</td><td>A-3-2</td><td>Stelized forceps for picking up the skin of rats and squeezing the fibrin clot.</td></tr><tr><td>Sprague-Dawley (SD) rat</td><td>Charles Rivers Laboratories</td><td></td><td>Retired male rats from colony in the lab or purchased retired male rats.</td></tr><tr><td>Syringe (20 ml)</td><td>TERUMO</td><td>SS-29ESZ</td><td></td></tr><tr><td>Needle (21 G x 5/8&quot;)</td><td>TERUMO</td><td>NN-2116R</td><td></td></tr><tr><td>Sterile test (spitz) tube (10 ml)</td><td>ASIAKIZAI</td><td>1101C000B-10</td><td>For collection of boold</td></tr><tr><td>Sterile disposable pipette</td><td>Eiken Chemical</td><td>CD2000 No.4</td><td></td></tr><tr><td>Sterilie disposable tube (15 ml)&amp;nbsp;</td><td>Falcon</td><td>2196</td><td></td></tr></tbody></table>

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.

図1は、血液細胞、血清を分離するための記載された手順の代表的な結果を示している。我々は、一般的に引退した雄性ラット（ 図1A）からの血液の15ミリリットルを得る。遠心分離によって、採取した血液、血清、矢印で示されているフィブリン塊、および血液細胞（ 図1C）を含む下層を含有する上層とに分離することができる。重要なことには、溶血...

Watch this Scientific Journal Video about Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture at JoVE.com

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

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.

哺乳類の全胚培養に適したラット血清の調製

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

preparation-of-rat-serum-suitable-for-mammalian-whole-embryo-culture

Research

JoVE Journal

Biology

13.8K ビュー.  Jichi Medical University. 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.

ビデオ: Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

共培養系における神経結合の形成を評価するための光遺伝学的アプローチ

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

発生生物学

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

無血清単層培養中のマウス胚性幹細胞の神経分化

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

早期着床後のマウス胚への細胞やDNAの正確​​ローカライズ転送する方法

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as multiple sclerosis and transverse myelitis. Myelination is required to not only facilitate rapid impulse propagation within the central nervous system, but also to provide trophic support to underlying axons. Oligodendrocyte progenitor cells (OPCs) can be studied in vitro to help identify factors that may promote or inhibit oligodendrocyte differentiation. To date, many of the methods available to evaluate this process have either required large numbers of cells, thus limiting the number of conditions that can be investigated at any one time, or labor-intensive methods of quantification. Herein, we describe a protocol for the isolation of large numbers of highly pure OPCs together with a fast and reliable method to determine oligodendrogenesis from multiple conditions simultaneously. OPCs are isolated from P5-P7 neonatal rat cortices and grown in vitro for three days prior to differentiation. Four days after differentiation, oligodendrogenesis is evaluated using a dual-infrared fluorescence-scanning assay to determine expression of the myelin protein.

Oligodendrocytes are the myelinating cells of the central nervous system. This protocol describes a method for the isolation and culture of their precursors, oligodendrocyte progenitor cells, from rat cortices, as well as a fast and reliable quantitative method to evaluate oligodendrogenesis in vitro in response to experimental factors.

デュアル赤外線蛍光スキャンを用いてラットオリゴデンドロサイト前駆細胞培養およびOligodendrogenesisの定量化の準備

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

マウス胚性造血内皮細胞の単離

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

ラット胚神経細胞の分離と文化：クイックプロトコル

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

神経科学

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

ニューロンとグリアの開発研究ラットの脳の集計文化の調製

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

生物学

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

An enhanced platform for whole-embryo culture allows continuous and robust ex utero development of postimplantation mouse embryos for up to six days, from pregastrulation stages until advanced organogenesis. In this protocol, we detail the standard procedure for successful embryo culture using static plates and rotating bottle systems.

元ウテロ マウス胚の採虫前から高度器官形成までの培養

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

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.

腱板断型ボトルの培養システムを使用してげっ歯類全胚培養の方法

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

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.

無血清全胚培養系で、マウス胚発生

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

哺乳類の全胚培養に適したラット血清の調製

要約

さらに動画を探す

この動画の章

共培養系における神経結合の形成を評価するための光遺伝学的アプローチ

無血清単層培養中のマウス胚性幹細胞の神経分化

早期着床後のマウス胚への細胞やDNAの正確ローカライズ転送する方法

デュアル赤外線蛍光スキャンを用いてラットオリゴデンドロサイト前駆細胞培養およびOligodendrogenesisの定量化の準備

マウス胚性造血内皮細胞の単離

ラット胚神経細胞の分離と文化：クイックプロトコル

ニューロンとグリアの開発研究ラットの脳の集計文化の調製

元ウテロマウス胚の採虫前から高度器官形成までの培養

腱板断型ボトルの培養システムを使用してげっ歯類全胚培養の方法

無血清全胚培養系で、マウス胚発生

哺乳類の全胚培養に適したラット血清の調製

要約

さらに動画を探す

この動画の章

共培養系における神経結合の形成を評価するための光遺伝学的アプローチ

無血清単層培養中のマウス胚性幹細胞の神経分化

早期着床後のマウス胚への細胞やDNAの正確​​ローカライズ転送する方法

デュアル赤外線蛍光スキャンを用いてラットオリゴデンドロサイト前駆細胞培養およびOligodendrogenesisの定量化の準備

マウス胚性造血内皮細胞の単離

ラット胚神経細胞の分離と文化：クイックプロトコル

ニューロンとグリアの開発研究ラットの脳の集計文化の調製

元ウテロ マウス胚の採虫前から高度器官形成までの培養

腱板断型ボトルの培養システムを使用してげっ歯類全胚培養の方法

無血清全胚培養系で、マウス胚発生

早期着床後のマウス胚への細胞やDNAの正確ローカライズ転送する方法

元ウテロマウス胚の採虫前から高度器官形成までの培養