1 EX -オボ文化プロトコル。： <ol><li>受精鶏卵をインキュベート<ol class="lroman"><li>受精鶏卵は13℃で保存できますCを最大5日間インキュベーション前に開発を開始せずに。赤のワインクーラーは、この温度を維持することができます。 </li><li> 72時間の37.5での連続的なロッキングの間に60％恒湿インキュベーター° Cでインキュベーション卵鈍い面を上に。 </li></ol></li><li>ハンモックを準備する（図1a） <ol class="lroman"><li>塗りつぶし¾ 9オンスのプラスチックのコップの暖かい滅菌水で。定期的な9オンスカップは、8cmの上部直径を有している。我々は、このサイズがここで紹介する長い時間培養アプリケーションに最適であることがわかった。 </li><li>約20cmのラップをしがみつくカット。円周のまわりのラバーバンドを配置することによって、カップの上に貼付。プラスチックは、水の上に分散させる必要があります。バンド周りの余分なプラスチックをカット。我々は、輸送中に卵黄の分割の結果、それは卵黄の付着を防止するため、このアプリケーションのための最も適切なものにラップをしがみつく発見。 </li><li> 70％エタノールで湿らせたキムワイプ（キンバリークラーク社）とプラスチックを拭きます。 </li></ol></li><li> 72時間のインキュベーション後にインキュベーターから卵を取り除く。 </li><li> 70％エタノール接液キムワイプで表面を拭いて卵を滅菌する。 </li><li>胚の適切な配置（図1b）のための1-2分のために水平に卵を産む。卵のカートンを水平に卵を産むために使用することができます。胚は、1〜2分後に上に回転します。 </li><li>層流フード内で無菌的にハンモックの上に胚を移す。 <ol class="lroman"><li>卵（図1c）の下側に小さなへこみがあるまでシャープなエッジ（金属製のバケツやガラスのビーカーの端など）を使用して、静かに卵をタップします。覚えておいて、胚は、上側に配置されている。 </li><li>くぼみの反対側に親指を置くと（図1d）離れて水平にシェルを引き出します。 </li><li>胚と同様にアルブミンハンモック（図1E）上に卵黄を置きます。アルブミンは、それが長い文化の実践のための胚に栄養を提供するだけでなく、システムを移動することによって誘発される衝撃を吸収するために卵黄の周りに湿し水環境を提供します。ハンモックの下の水は、水の循環のヒーターが使用されている場合、胚は、撮像時の輸送中に地面に平行に保ち、熱の伝導体として機能することを確実にします。 </li><li>転送が正しく実行されている場合、胚はすでに一番上に配置する必要があります。されていない場合は、アンシャープ、滅菌オブジェクト（閉じた曲線ハサミのすなわち鈍先端）と方向的に愛撫胚が上に回転するように卵黄を使用してください。 </li><li>胚を密封するためにカップの上に直径10cmのシャーレを置きます。 </li></ol></li><li> 37.5でのインキュベーターセットに配置カップの文化℃の携帯用インキュベーターを使用する場合は、蒸留水で満たされた1月2日9オンスのカップを置くと、〜で湿度を保つ60％（図1F）。 </li><li>胎生7日目を超えて培養胚の場合、粉砕卵殻片を骨の発達と成熟のためのカルシウム源として胚の周縁部に点在してください。 </li></ol>外インキュベーターイメージング/操作のためのアクセサリー機器： 循環水浴をインキュベーション温度を維持するために、家の中に建てられましたながらイメージングまたは操作（図3）。カップに合うことができる水浴は水の貯水池への適切なサイズのチューブを介して接続されている。抵抗ヒータは、水の温度に従って、手動で調節されている貯水槽の水を加熱する。ウォーターポンプは、水を循環させる。 Pictoral表現はultrasongraphy / florescent顕微鏡（図3）を介して画像処理しやすさを表しています。 <img src="/files/ftp_upload/2154/2125fig1.jpg" alt="図1" /> 図1。ハンモックとハンモックにニワトリ胚の転送の準備。 <img src="/files/ftp_upload/2154/2125fig2.jpg" alt="図2" /> 図2。代表的な結果- 。EX OVO培養技術を介して成長し、異なる段階のニワトリ胚胚HH 17以降からは、この手法を使って成長させることができる。 <img src="/files/ftp_upload/2154/2125fig3.jpg" alt="図3" /> 図3。理想的な開発の温度で胚を保つために元卵培養系と組み合わせて使用する水循環システムの概略図と代表的な写真。 2。選択したアプリケーション： I.マイクロインジェクションこの胚培養では、ソリューションがコントラストに基づくイメージング（すなわち、蛍光顕微鏡、マイクロ計算地形など）（図4）のために血管系に注入する必要があるマイクロインジェクションのアプリケーションに適しています。以下のマイクロインジェクションのプロトコルは、次のとおりです。 <ol><li>注射apparaを準備TUS <ol class="lroman"><li>ファッションは、マイクロフォージを使用して、斜め先端のマイクロニードルに0.75 IDのガラスキャピラリーチューブを引っ張った。必要なチップのサイズは、所望の標的静脈径/流量に依存します。 </li><li> 0.03インチIDのシリコンチューブにシリンジを接続してください。 </li><li>注入する溶液でチューブをロードし、チューブにマイクロニードルを接続してください。 </li><li>シリンジホルダーまたはシリンジポンプにマイクロマニピュレータと注射器の上にマイクロニードルを置きます。 </li></ol></li><li>カップに滅菌水を追加し、それによって胚を上げる。我々は、血管（すなわち、卵黄血管）に針の適切な浸透のために、水平方向の浸透が必要であることを経験した。これは、胚はカップの縁と同じ平面上にある必要があります。 <ol class="lroman"><li>胚の下にプラスチックを保持しているゴムバンドを取り出してください。 </li><li>優しくプラスチックの片側を持ち上げて、カップにいくつか滅菌水を加える。 </li><li>再びプラスチックの上にゴムバンドを固定します。 </li></ol></li><li>胚の血管への解決策を注入。我々の経験では、ソリューションが卵黄の容器に注入されると、マイクロニードルは出血を引き起こすことなく効率的な血流を維持するために、血管の直径の約1 / 10サイズにする必要がありました。 <ol class="lroman"><li>気泡のない形成を確実にしないマイクロニードルへの解決策をポンプ。 </li><li>選択された容器の上にマイクロニードルの先端を合わせます。卵黄血管の場合は、フォークのエリアを選択すると、容器を貫通する最大面積を与える。 </li><li>船に向かって針を押してください。船は最初にリトラクトします。針が貫通したら、灌流を開始します。流れる血の色は血流に変更する必要があります。色の変化が見られない場合、針が血管外に浸透している場合があります。容器の色の変化が見られるまで同時にソリューションをポンピングしながらその場合には、徐々にニードルを引っ込める。灌流が完了すると、容器の外マイクロニードルを引っ込める。 </li></ol></li></ol><img src="/files/ftp_upload/2154/2125fig4.jpg" alt="図4" /> 図4：EX - OVO培養ニワトリ胚の血管系への蛍光色素のマイクロインジェクション。 II。顕微手順 - 左心房ライゲーションこのEX - OVO文化のセットアップでは、胚への完全なアクセシビリティを提供するため、ニワトリ胚への外科的なアプリケーションの実行に最適です。以下の例のテクニック、左心房ライゲーション（LAL）（図5）ためのプロトコルです。手順は、約24時間培養期間（HH24）に行われます。 <ol><li>ノットをオーバーハンドの準備〜10から0ナイロン縫合糸を使用して、0.5 mmの直径を。 </li><li>細かい鉗子で、局所的に胚を介して絨毛膜と尿膜膜を除去し、胚の左側に表示されていることそれが垂直方向にそのような回転に戻ってから胚を持ち上げます。 </li><li>細かい鉗子を使用して、左心房を介して心膜を取り除く。 </li><li>左心房を介して結び目の位置を合わせ、それを締めます。左側に元の血流の約75％を禁止するように結び目は、その接続する必要があります。 microscissorsと結び目の端をカットし、慎重に余分な縫合糸を除去し捨てる。 </li><li>元の位置（上右側）に戻す胚を回転させる。 </li><li>偽のコントロールは、同じ手順が含まれますが、縫合糸は、ただ通過し、関連付けられていないされます。効果的なLAL治療（75％収縮）が24時間後に手術にてさせていただきます、と失格胚は除外されます。 </li></ol><img src="/files/ftp_upload/2154/2154fig5.jpg" alt="図5" /> 図5：コントロールと左心房の連結されたニワトリ胚矢印は、連結された胚の約75％小型である左心房を示しています。

<ol>
	<li>Zamir, E. A., Czirok, A., Cui, C., Little, C. D., Rongish, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesodermal+cell+displacements+during+avian+gastrulation+are+due+to+both+individual+cell-autonomous+and+convective+tissue+movements.">Mesodermal cell displacements during avian gastrulation are due to both individual cell-autonomous and convective tissue movements.</a> Proc Natl Acad Sci U S A. 103, 19806-19811 (1980).</li><li>Ehrman, L. A., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+regulation+in+the+heart+forming+region+of+avian+embryos.">Lack of regulation in the heart forming region of avian embryos.</a> Dev Biol. 207, 163-175 (1999).</li><li>Clark, E. B., Hu, N., Rosenquist, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+conotruncal+constriction+on+aortic-mitral+valve+continuity+in+the+stage+18,+21+and+24+chick+embryo.">Effect of conotruncal constriction on aortic-mitral valve continuity in the stage 18, 21 and 24 chick embryo.</a> Am J Cardiol. 53, 324-327 (1984).</li><li>deAlmeida, A., McQuinn, T., Sedmera, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+ventricular+preload+is+compensated+by+myocyte+proliferation+in+normal+and+hypoplastic+fetal+chick+left+ventricle.">Increased ventricular preload is compensated by myocyte proliferation in normal and hypoplastic fetal chick left ventricle.</a> Circ Res. 100, 1363-1370 (2007).</li><li>Nakamura, H., Funahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+DNA+into+chick+embryos+by+in+ovo+electroporation.">Introduction of DNA into chick embryos by in ovo electroporation.</a> Methods. 24, 43-48 (2001).</li><li>Butcher, J. T., McQuinn, T. C., Sedmera, D., Turner, D., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transitions+in+early+embryonic+atrioventricular+valvular+function+correspond+with+changes+in+cushion+biomechanics+that+are+predictable+by+tissue+composition.">Transitions in early embryonic atrioventricular valvular function correspond with changes in cushion biomechanics that are predictable by tissue composition.</a> Circ Res. 100, 1503-1511 (2007).</li><li>McQuinn, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+ultrasonographic+imaging+of+avian+cardiovascular+development.">High-frequency ultrasonographic imaging of avian cardiovascular development.</a> Dev. Dyn. 236, 3503-3513 (2007).</li><li>Korn, M. J., Cramer, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Windowing+chicken+eggs+for+developmental+studies.">Windowing chicken eggs for developmental studies.</a> J Vis Exp. , (2007).</li><li>Auerbach, R., Kubai, L., Knighton, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+procedure+for+the+long-term+cultivation+of+chicken+embryos.">A simple procedure for the long-term cultivation of chicken embryos.</a> Dev Biol. 41, 391-394 (1974).</li></ol>

利害の衝突は宣言されません。

光アクセスと鳥類の胚の実験は、卵の殻の制約のため困難である。のウィンドウが大幅に注入し、顕微鏡を使った手術のアプローチ8のアクセス可能な微小血管の数を制限します。結果としてのみ初期胚を操作して連続観測が不可能であることができる。長期的な文化9を防止胚での表面張力の不十分な制御のためのペトリ皿を使用して初期のEX - OVOの文化は、使用が制?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Fertile white Leghorn chicken eggs</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Model GB1, Avery Incubators, Hugo CO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Saran Wrap</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Kimwipes</td>
<td>&nbsp;</td>
<td>Kimberly-Clark, Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Rubber bands</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Warm sterile water</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>9 oz plastic cup</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>100 mm diameter Petri dish</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1602N thermal air</td>
<td>&nbsp;</td>
<td>GQF Manufacturing CO, Savannah GA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescein-conjugated dextran (2 MDa, 1% w/v in phosphate buffered saline)</td>
<td>&nbsp;</td>
<td>Sigma Aldrich Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microforge</td>
<td>&nbsp;</td>
<td>Glassworx, Inc, St Louis MO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glass capillary tubes (0.75 mm ID)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulator</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Sarasota FL</td>
<td>Model M3301L</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent microscopy</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>Z20</td>
<td>&nbsp;</td>
<tr>
<td>Fine 55-forceps</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Sarasota FL</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10-0 nylon surgical suture</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tubing</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>1mm OD</td>
</tr>
<tr>
<td>3 mL syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>200 &mu;L pipetter and pipette tips</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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an ex-ovo chicken embryo culture system suitable for imaging and microsurgery applications

正常と不正な形式の胚発生を促進遺伝子と微環境要因間の関係を理解することは新たな治療戦略を発見するための基本です。イメージング技術の進歩は、身体の計画の組織と成熟の定量的な調査を有効にしているが、後の段階の胚の形態形成にはあまり明確ではない。ニワトリ胚のための文化と手術操作の容易さのこのアプリケーションのための魅力的な脊椎動物の動物モデル系です。初期胚は、細胞のパターニングと運命学の1,2のための完全な光アクセスを可能にするろ紙のリング、上に短い時間のために培養することができる。このような心臓の形態形成などの勉強を高度な発達過程は伝統的に卵殻3-5の窓を通して行われますが、この手法は、ウィンドウのサイズに起因する光アクセスを制限している。我々は以前までの超音波検査6,7を経由して高分解能のイメージングを有効にして10日間、にのために六角形の重船の文化胚EX - OVO全体を簡単に計測する手法を開発した。これらの文化は、ライブの実験で使用可能なイメージングツールの種類を制限する、輸送することが困難でした。ここでは、費用対効果の高い、ポータブル環境室との改善されたシェルレス培養系を提示する。卵は、部分的に滅菌水で満たされたプラスチック製のコップに円周方向に貼付ポリウレタン膜（ラップしがみつく）によって作成されたハンモックに割れていた。ハンモックと水の下の力学が輸送によって誘発される振動を減衰させる助けながら、ハンモックの円周と深さの寸法は、両方の表面張力を維持するために重要でした。水浴を循環させ、小さなフットプリントは、実験中に連続的な温度制御を可能にするために開発されました。我々は、形態形成の欠陥または遅滞なく、少なくとも14日間この方法における培養胚に能力を発揮し、いくつかの顕微およびイメージングアプリケーションでこのシステムを採用しています。

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

この記事では、我々は長期的な有効にするシンプルな方法論を提示<em&gt; EX - OVO</em&gt;鳥類の胚培養。この手法は、鳥類胚における完全な光のアクセシビリティおよび/または無菌輸送を必要とする縦の実験に最適です。

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 ...

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

22.9K ビュー.  Cornell University. この記事では、我々は長期的な有効にするシンプルな方法論を提示 EX - OVO鳥類の胚培養。この手法は、鳥類胚における完全な光のアクセシビリティおよび/または無菌輸送を必要とする縦の実験に最適です。

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

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform routine activities, but prosthetic suspensions using traditional socket technology can prove to be cumbersome and uncomfortable for a person with limb loss.  Moreover, for those with high proximal amputations, limited residual limb length may prevent exoprosthesis attachment all together. Osseointegrated implant technology is a novel operative procedure which allows firm skeletal attachment between the host bone and an implant. Preliminary results in European amputees with osseointegrated implants have shown improved clinical outcomes by allowing direct transfer of loads to the bone-implant interface.  Despite the apparent advantages of osseointegration over socket technology, the current rehabilitation procedures require long periods of restrictive load bearing prior which may be reduced with expedited skeletal attachment via electrical stimulation.  The goal of the osseointegrated intelligent implant design (OIID) system is to make the implant part of an electrical system to accelerate skeletal attachment and help prevent periprosthetic infection. To determine optimal electrode size and placement, we initiated proof of concept with computational modeling of the electric fields and current densities that arise during electrical stimulation of amputee residual limbs.  In order to provide insure patient safety, subjects with retrospective computed tomography scans were selected and three dimensional reconstructions were created using customized software programs to ensure anatomical accuracy (Seg3D and SCIRun) in an IRB and HIPAA approved study.  These software packages supported the development of patient specific models and allowed for interactive manipulation of electrode position and size. Preliminary results indicate that electric fields and current densities can be generated at the implant interface to achieve the homogenous electric field distributions   required to induce osteoblast migration, enhance skeletal fixation and may help prevent periprosthetic infections.  Based on the electrode configurations experimented with in the model, an external two band configuration will be advocated in the future.

There is a need to develop alternative prosthesis attachment due to limb loss attributed to vascular occlusive diseases and trauma. The goal of the work is to introduce an osseointegrated intelligent implant design system to increase skeletal fixation and reduce periprosthetic infection rates for patients needing osseointegrated technology.

切断者のためのOsseointegratedインテリジェントインプラント設計システムの生体電気分析

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform ...

生物学

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

チック EX卵文化と EX卵 CAMアッセイ：それが実際にどのように動く

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

ニワトリ胚へのヒト幹細胞の異種移植

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

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 ...

Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice. HYPERStack vessels allow closed system scale up from the current stacked plate products and delivers >2.5X more cells in the same volumetric footprint. The HYPERStack vessels function via gas permeable material which allows gas exchange to occur, therefore eliminating the need for internal headspace within a vessel. The elimination of headspace allows the compartment where cell growth occurs to be minimized to reduce space, allowing more layers of cell growth surface area within the same volumetric footprint.
For many applications such as cell therapy or vaccine production, a closed system is required for cell growth and harvesting. The HYPERStack vessel allows cell and reagent addition and removal via tubing from media bags or other methods.
This protocol will explain the technology behind the gas permeable material used in the HYPERStack vessels, gas diffusion results to meet the metabolic needs of cells, closed system cell growth protocols, and various harvesting methods.

An Introduction into the technology, protocol and handling of the Corning HYPERStack Vessels and accessories used for high yield adherent cell culture. The protocol will show how to use the closed system vessels for increasing cell harvesting over current stacked plate products.

ガス透過性材料技術とHYPERStackの船舶を使用するクローズドシステムの細胞培養のプロトコル

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice.   ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

して、古いニワトリ胚への遺伝子導入<em&gt; EX OVO</em&gt;エレクトロポレーション

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

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 ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

アン<em&gt;生体外</em甲状腺の開発を研究する&gt;文化システム

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

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 ...

アン EX - OVOニワトリ胚培養システム