我々は、ビデオを編集するためのビデオ録画および有用な助言のために氏肇輝に感謝。我々はまた、ビデオ録画のための親切なアシスタントのためDrs.Yuji恒川とカイチ吉崎に感謝。この作業は若手Bで、日本の文部科学省から重点領域 - 分子脳科学に関する科研費でサポートされています。我々は日本の文部科学省と日本の科学技術振興機構（JST）から日本の科学とテクノロジー社から戦略的創造科学技術（CREST）のためのコアの研究から、グローバルCOEプログラム&quot;グローバル脳科学のための基本とトランスレーショナルリサーチセンター&quot;のサポートを認める。

1。 WECシステムのセットアップ<ol><li> ）WECシステム内の入口シリコンチューブ〜0.22μmのメンブランフィルタージョイント。 </li><li> O 2（95％）とCO 2（5％）を含むガスのボンベの ​​バルブを開きます。オートクレーブ水を含む、バブルボトルを経由してドラム内に混合ガスを流し込む。 </li><li> 50ccのために混合ガスの流量を調整します。 </li><li>外は水のボトルにシリコンチューブを聞かせて混合ガスの流れをチェックアウト挿入。 </li><li> 20回転/分の速度でドラムを回転させる。 </li></ol> 2。培地の調製<ol><li>融解ラット直ちに遠心分離（IC）血清37.0℃においてB）。オートクレーブした100mlのビーカーに解凍した血清中にD -グルコース（2 mg / mL）を追加します。培地は初代培養のレベル（例えば、フードを使用して）で準備する必要があります。 </li><li> 400希釈：1の血清に抗生物質 - 抗真菌剤（100倍）液体を追加。 </li><li>室温で20分間血清（RT）c）から（採血時のラットの麻酔に起因する）、残りのイソフルランを気化。 </li><li> 0.45μmのメンブランフィルターを用いて培養液を滅菌する。 </li><li>各培養瓶の培地3.0 mlを注ぐとシリコンプラグでそれぞれのボトルの上部をシール、アルミホイルで覆われ（文献14参照）D）。 </li><li> ）タイロードの生理食塩水の電子を含む3つまたは4つの処分ペトリ皿を準備します。 </li></ol> 3。子宮のラットと分離の麻酔<ol><li>吸入または経口麻酔薬で深く時限妊娠ラットを麻酔。 </li><li> 70％エタノールで麻酔妊娠したラットの腹部領域をクリーニングします。 </li><li>ピンセットで皮膚を拾うと大きなハサミf）を使用して皮膚をカットして腹壁を露出させる。 </li><li>ピンセットで腹壁をピックアップして）胸椎レベルのgに大きなハサミで腹壁上にU字型の大規模な縦断的切開を占めている。 </li><li>ピンセットで左側に腸を持って行ってしまうと卵巣に接続されている子宮の一端を公開。 </li><li>ピンセットで子宮の端を拾うと大きなはさみで子宮と卵巣の間に位置をカット。 </li><li>子宮のもう一方の端まで、子宮の周りの脂肪をカットすることで妊娠ラットから子宮を取り出してください。 </li><li>タイロードの生理食塩水を含むペトリ皿に子宮を転送する。 </li><li>子宮を除去した後、麻酔薬の過剰投与や頸椎脱臼によりラットを安楽死させる。 </li></ol> 4。子宮から胚の除去<ol><li>簡単に言うとタイロードの生理食塩水で子宮をすすぎ、細かい鉗子H）のペアで2ペトリ皿に移す。 </li><li>双眼顕微鏡下で、微細なピ​​ンセットで子宮壁をピックアップし、眼科まっすぐはさみを使用して血管を使用して接続し子宮間膜の反対側で子宮壁をカット。 </li><li>子宮壁と脱落膜との間の空間に眼科ストレートはさみの先端を挿入します。 antimesometrial側に沿って長手方向に子宮壁をカット。 </li><li> mesometrial側の脱落膜および微細な鉗子i）の 2組を使用して子宮とは別のconceptuses塊。 </li><li>適切な直径を持つ滅菌ガラスピペット3番目のシャーレにconceptusesを転送する。 </li></ol> 5。ラットの胚の解剖<ol><li>脱落膜にmicroforcepsいずれかの先端を挿入し、鉗子の二組の受胎産物の周りに脱落膜上に横切開を行います。 </li><li>胎盤側に脱落膜に鉗子の先端を挿入し、上部に脱落膜上の2つの縦切開を行います。 </li><li>鉗子の二組のことをリッピングにより、胎盤側の脱落膜を取り外します。鉗子の二組の残りの脱落を削除します。 </li><li> 、ライヘルトの膜を拾うことで水平方向の切開を行うこと、および抗胎盤側で受胎産物から膜を分離する。 </li><li>鉗子の二組の胚の頭部近くに卵黄嚢に小さな穴を確認し、眼科曲線ハサミで卵黄嚢をカット。主要な血管を損傷しないことが重要です。 </li><li>微細な鉗子の二組の胚の頭の周りのサイトで羊膜をピックアップ、そして静かに）羊膜hを引き裂くことによって卵黄嚢から胚を引き出します。胚の体は妊娠中期の段階で胚のための酸素の供給を増やすために卵黄嚢から撤退すべきである。 </li></ol> 6。全胚培養<ol><li>胎盤、卵黄嚢、そして心臓の拍動と血液循環の状態で損害賠償を確認してください。 </li><li>へその緒をストレッチではないの自覚し、転送滅菌ガラスピペットで培養ボトルへの胚。培養ボトルにタイロードの生理食塩水のキャリーオーバーは可能な限り少ないはずです。 </li><li>回転体ドラムから非穴シリコンプラグを取り外します。垂直にWECシステムの回転子ドラムに穴をプラグで培養ボトルを接続してください。回転子のドラムが非常に微細な機器であるため、不適当な方向にドラムに力を入れるように注意してください。解剖胚を含むボトルは1つで文化いずれかに転送され、インキュベーターのフロントドアの開口部は、インキュベーターの温度を避けるために最小限に抑える必要がありますjを減少させる）。 </li><li>文化の10時間後、最大75 ccと24時間（表1）後に最大100 ccの混合ガスの流量を増加させる。 </li><li>培地は、調製したばかりの培地を使用して新しい培養瓶に培養胚を転送することにより、24時間のまわりに変更する必要があります。 34時間後に最大125 ccの流量を増加し、48時間後に最大150のcc（表1） </li><li>時折ハートビートレートを（すなわち、120-150/minが最適である）カウントすることにより、血液の循環を観測することによって培養胚の状態を確認してください。必要に応じてタイロードの生理食塩水で胚の成長を判断する体節の数を数えます。通常の開発では、一方の体節が2時間に追加されます。 </li><li> WECが終了すると、混合ガスの供給を停止し、入口管およびバブリング瓶からインレットチューブに逆流を防ぐために、フィルタ膜を切断。 </li></ol> 7。ノート<ol class="lalpha"><li> WECシステム内部の温度は常に37.0から37.5℃に保持され</li><li> 100パーセントラットIC血清は、日常的にラットおよびマウス胚のための私達のWECで使用され、使用前に-20℃に保たれている。 </li><li>我々は、イソフルランで麻酔したラットから収集されたラットIC血清を、購入。 </li><li>使い捨ての瓶は、池本理化からも利用できます。ボトルとシリコンのプラグはオートクレーブする必要があります。 </li><li>解剖中に低温または高温での胚の維持がWECで、その後の発展に影響を与えるので、胚の解剖は、室温で行ってください。 </li><li>解剖のためのツールは、乾熱で滅菌されています。 </li><li>皮膚と腹壁はできるだけ皿に毛の汚染を避けるために別々にカットする必要があります。我々は皮膚をカットし、子宮を分離するときに我々はそれぞれ、それらの新しいペアで大きなハサミとピンセットのペアを変更してください。 </li><li>そのヒントうまくある鉗子を使用すると、確かに組織を分析する非常に重要です。我々は、手動で石と機械油と鉗子の先端をシャープに。 </li><li>我々は、胚へ過剰な圧力を避けるために、ナイフのような鉗子の片側を使用してください。 </li><li> WECシステム内部の温度が何度も扉を開くことによって減少したとき、我々は、システム内の温度を保つためにライトをオンにし続ける。 </li></ol> 8。代表的な結果図1は、ラット胚と培養ラット胚の解剖の手順を示しています。 <img src="/files/ftp_upload/2170/2170fig1.jpg" alt="図1" /> 図1。E12.5ラット胚の全胚培養。 （A）胎児は、妊娠ラットの子宮から解剖。胎盤側の脱落膜の（B）の除去。ライヘルトの膜の（C）除去。 （D）卵黄嚢を開く。 WECの開始後6時間ボトルで（E）ラット胚の培養。 （F）ラットの胚は、42時間培養。 D、脱落膜、R、ライヒェルトの膜、P、胎盤、YS、卵黄嚢、電子、胚。 <table border="1"><tbody><tr><td></td><td> 0時間</td><td> 12時間</td><td> 24時間</td><td> 36時間</td><td> 48時間</td></tr><tr><td> E12.5ラット胚</td><td> 95パーセント50ccの</td><td> 95パーセント75のcc （10時間） </td><td> 95パーセントを100cc </td><td> 95パーセント125のcc（34時間） </td><td> 95パーセント150ccの</td></tr></tbody></table> 表1。最適な酸素状態。

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利害の衝突は宣言されません。

成功のための齧歯類のWECの2つの重要なステップがあります。最初に、解剖の手順は、しないように、特に被害の胚、血管に正確でなければなりません。酸素と栄養がもはや子宮からの分離後の胎盤を介して供給されていないので、第二、手続きはできるだけ早くする必要があります。これは古い胚にとって非常に重要です。 E12.5後のラットのWECのケースでは、我々は30分以内に培養ボトルに?...

<table border="1"><tbody><tr><td> の名前 </td><td> タイプ </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td colspan="5"> WECの設定 </td></tr><tr><td> WECシステム（10〜0310） </td><td>ツール</td><td>池本リカ</td><td> 010-0310 </td><td>別のスモールモデルも用意されています。 </td></tr><tr><td>穴のないシリコンプラグ</td><td>ツール</td><td>池本リカ</td><td> 010-032-08 </td><td></td></tr><tr><td>混合ガスのシリンダー</td><td>ツール</td><td>日光山荘</td><td> - </td><td>酸素95％と5％の二酸化炭素を含む。カスタムオーダー。 </td></tr><tr><td>ガスレギュレータ</td><td>ツール</td><td>小野製作所</td><td> WR - 11 </td><td></td></tr><tr><td> 0.22μmのフィルターマイレクスGS </td><td>ツール</td><td>ミリポア</td><td> SLGS033SS </td><td></td></tr><tr><td colspan="5"> 麻酔と子宮の分離 </td></tr><tr><td>大きなはさみ</td><td>ツール</td><td> Napox </td><td> B - 7H </td><td></td></tr><tr><td>鉗子</td><td>ツール</td><td> Napox </td><td> - 3 - 2 </td><td></td></tr><tr><td>廃棄ペトリ皿 （90ミリメートル× 15ミリメートル） </td><td>ツール</td><td>いわき市</td><td> SH90 - 15 </td><td>深型シャーレは、解剖に最適です。 </td></tr><tr><td>イソフルレン</td><td>試薬</td><td>アボット</td><td> B506 </td><td>麻酔のための</td></tr><tr><td>ペントバルビタールナトリウム</td><td>試薬</td><td>シェリングプラウアニマルヘルス</td><td> - </td><td>麻酔のための</td></tr><tr><td>タイロードの生理食塩水</td><td>試薬</td><td> - </td><td> - </td><td>文献[プロトコルとしてよると。 2。 4℃で保存℃、 </td></tr><tr><td>時限妊娠Sprague - Dawleyラット</td><td>動物</td><td>チャールズ川ラボラトリーズジャパン</td><td> - </td><td></td></tr><tr><td colspan="5"> 培地の調製 </td></tr><tr><td>文化ガラス瓶</td><td>ツール</td><td>池本リカ</td><td> 010-032-05 </td><td></td></tr><tr><td>処分の培養瓶</td><td>ツール</td><td>池本リカ</td><td> 010-032-06 </td><td></td></tr><tr><td>穴付きシリコン栓</td><td>ツール</td><td>池本リカ</td><td> 010-032-07 </td><td></td></tr><tr><td>アルミ箔</td><td>ツール</td><td>どのような企業</td><td> - </td><td></td></tr><tr><td>オートクレーブ袋</td><td>ツール</td><td>カル</td><td> HM - 26 </td><td>培養ボトル用</td></tr><tr><td>オートクレーブ袋</td><td>ツール</td><td>カル</td><td> HM - 14A </td><td>シリコンプラグ用</td></tr><tr><td> 0.45μmのフィルターマイレクスHA </td><td>ツール</td><td>ミリポア</td><td> SLHA033SS </td><td></td></tr><tr><td> 50 mlコニカルチューブ</td><td>ツール</td><td>ベクトンディッキンソン</td><td> 352070 </td><td></td></tr><tr><td> 100mlのビーカー</td><td>ツール</td><td>いわき市</td><td> TE - 32 </td><td></td></tr><tr><td>ラットIC血清</td><td>試薬</td><td>チャールズ川ラボラトリーズジャパン</td><td> - </td><td> 、氏邦彦森崎：を参照してください。 TEL：+81-（0）45-474-9336、FAX：+81-（0）45-474-9340 </td></tr><tr><td> D（+） - グルコース</td><td>試薬</td><td>和光</td><td> 041から00595 </td><td></td></tr><tr><td>抗生物質antimyotic液体</td><td>試薬</td><td>ギブコ</td><td> 15240 </td><td></td></tr><tr><td colspan="5"> 胚の解剖 </td></tr><tr><td>眼科ストレートはさみ</td><td>ツール</td><td> Napox </td><td> MB50 - 7 </td><td>子宮壁を切断するための</td></tr><tr><td>眼科曲がったはさみ</td><td>ツール</td><td> Napox </td><td> MB54 - 2 </td><td>卵黄嚢を切断するための</td></tr><tr><td>鉗子＃5 </td&gt; <td>ツール</td><td>活力</td><td> T6715 </td><td></td></tr><tr><td>鉗子＃5F </td><td>ツール</td><td>レギ</td><td> T6819 </td><td></td></tr><tr><td>ガラスピペット</td><td>ツール</td><td> - </td><td> - </td><td>パスツールピペットを用いて手で作ら。 </td></tr><tr><td>オートクレーブ袋</td><td>ツール</td><td>カル</td><td> HM - 4 </td><td>ガラスピペットを用</td></tr><tr><td>双眼顕微鏡</td><td>ツール</td><td>ライカ</td><td> Mz7s </td><td></td></tr><tr><td>軽</td><td>ツール</td><td>ライカ</td><td> CLS 150XD </td><td></td></tr><tr><td>石油石</td><td>ツール</td><td>内田洋行</td><td> 833-2000 </td><td>鉗子のシャープニング用</td></tr><tr><td>マシン油</td><td>ツール</td><td>内田洋行</td><td> 835-0000 </td><td>鉗子のシャープニング用</td></tr></tbody></table>

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

全胚培養（WEC）技術は、新しいと彼の同僚によって1950年代に開発され、発生生物学1に適用されている。開発と哺乳類の胚の成長は、胎盤の機能に深く依存しているが、WECの技術は、胚の日中の妊娠中期の段階に対応する限られた期間の培養マウスやラットの胚ex vivoでの条件（E）が6.5 - E12.5に連絡することができますマウスまたはラット2、3、4のE8.5 - E14.5。胚を顕微鏡下で操作できるので、WECでは、我々が直接微細なガラスキャピラリーを用いた胚の所望の領域をターゲットにすることができます。我々はpostimplanted哺乳類胚のダイナミックな発生過程を研究する場合したがって、齧歯類のWECは、非常に有用な技術である。最新の、WECシステムのいくつかのタイプは、1が開発されている。これらのうち、ローテーター型ボトル培養系は、それぞれ1マウス及びラットにおけるE9.5およびE11.5、後に、最も人気があり、妊娠中期、すなわちにおける胚の長期培養に適しています。このビデオプロトコルでは、我々は、新規およびコックロフト5で設計されたオリジナルのローテータのシステム、洗練されたモデルを使用して12.5日後に6をラットのWECの私達の標準的な手順を示し、そして哺乳類の発達の研究にWECの技術の様々なアプリケーションを紹介生物学。

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

全胚培養法は、培養マウスやラットの胚に連絡することができます<em&gt;生体外で</em妊娠中期の段階に対応する限られた期間&gt;条件。このビデオプロトコルでは、ローテータ型ボトルの培養系を用いて12.5日後にラットの全胚培養の私達の標準的な手順を示しています。

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 ...

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

Research

JoVE Journal

Biology

23.6K ビュー.  Tohoku University Graduate School of Medicine. 全胚培養法は、培養マウスやラットの胚に連絡することができます生体外で条件。このビデオプロトコルでは、ローテータ型ボトルの培養系を用いて12.5日後にラットの全胚培養の私達の標準的な手順を示しています。

ビデオ: 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.

初期のニワトリ胚をex vivo（新文化）の文化のための方法

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

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.

マウス胎児肺の文化、分岐の分子機構を評価するシステム

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.

アン EX - OVO</emイメージングとマイクロサーアプリケーションに適して>ニワトリ胚培養システム

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.

小説<em&gt;エクスビボ</emマウス胎児ハート&gt;文化法

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.

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 ...

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.

外植片培養での歯胚の開発に続いてのためのスライス培養法

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.

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 ...

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.

酸性細胞外 pH 培養系の確立

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.

3次元共培養システムNICO-1を用いた卵巣癌幹細胞様細胞の血管遺伝学的特性の評価

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.