Name: organotypic culture method to study the development of embryonic chicken tissues
Uploaded: 2018-08-25T12:30:00.000+00:00
Duration: 5 min 12 s
Description: Watch this Scientific Journal Video about Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues at JoVE.com

著者感謝したいグレゴリー ・ ハラー (サスカチュワン大学) プロトコルの開発に彼の準備作業のため。著者も感謝したいニコラス ・ ジョーンズ (マウント ・ サン ・ ヴァンサン ・大学) 彼の技術的専門知識と撮影と原稿のオーディオ/ビジュアル部分の生産に関するご案内のため。ダニエル ・ アンドリュースは、MSVU と自然科学工学研究会議のカナダ (レベル)経由から学部学生研究奨励賞の資金によって支えられました。タマラ A. フランツ スティーブンオデンダールスーターは、レベル検出補助金によってサポートされます。

注: 胚の段階、ハンバーガーとハミルトン12 (HH) ステージング テーブルを使用します。
1. 胚培養
<ol><li>受精鶏卵で 37 ° C ± 1 ° C ~ 40% 湿度、滅菌温度制御のインキュベーターで孵化させなさい。</li>
	<li>卵 1 を有効に 1 日あたり x HH34 に孵化するようにし、(8 日後受精)。</li>
</ol>2. 準備中と材料の殺菌
<ol><li>12 胚、蒸留水、0.85% ひよこ生理食塩水、12 ガラス ピペット、紙ペーパー、半多孔質フィルター紙の 24 2.5 cm × 2.5 cm の正方形および鋼線の 24 2.5 cm × 2.5 cm 正方形の 1 ボックスの 1 L の 2 L メッシュ下方に湾曲したエッジのオートクレーブ。材料を滅菌するオートクレーブで 121 ° C、15 分以上。 注: マテリアルのオートクレーブは事前に行うことができます。</li>
	<li>側面、棚、ドアを拭くことによって 70% エタノールでインキュベーターを滅菌します。オートクレーブを含む場所 2 プラスチック容器に蒸留水湿度を維持するために孵化器の中。37 ° C ± 1 ° C ~ 40% 湿度に文化のインキュベーターを prewarm します。インキュベーターが完全に暗い;ドアが透明の場合は、アルミ箔でカバーします。</li>
	<li>100 mL の培養液とペニシリンの 1 mL を prewarm 文化のインキュベーターでそれらを配置することによって。</li>
	<li>ペーパー タオルでワークスペースをカバーし、ベンチを 70% エタノールを噴霧して消毒します。同様の方法で 2 組の鉗子、解剖はさみ、かみそりの刃、70% エタノールとプラスチック スプーンを消毒します。</li>
	<li>場所 12 滅菌 100 mm ペトリ皿と 24 滅菌 35 mm シャーレ用ベンチ。滅菌バッグでの使用まで、料理を確認します。</li>
</ol>3. 胚の準備
注: この時点から文化の汚染を避けるために防塵マスクを着用します。細菌や真菌感染を台無しに文化とインキュベーターのすべての文化にすぐに広がっていることの危険性があります。
<ol><li>亀裂は、卵を開き、0.85% ひよこ生理食塩水を含む 100 mm のペトリ皿に胚を転送します。解剖学的特徴によると胚のハンバーガーとステージングのガイドライン12ハミルトンと胚を確認の段階は、HH34 にあります。</li>
	<li>首を切り、二等分する殺菌かみそりの刃を使用して正中線で胚の頭。滅菌鉗子を使用して、脳を削除します。くちばしをそのまま。</li>
</ol>4. 文化セットアップ
<ol><li>1 つの 100 mm ペトリ皿内 35 mm のペトリ皿の場所 2。</li>
	<li>各 35 mm のペトリ皿の中のスチール ワイヤ メッシュを配置します。</li>
	<li>鉗子を使用すると、上を向いて目を 2.5 cm × 2.5 cm 半多孔質フィルター紙に切断 2 分離の頭の 1 を転送します。組織が安全であり、それを少し傾けることによってオフにスリップしていませんを確認します。</li>
	<li>眼組織とろ紙を 1 上眼組織の発生段階を作成する 35 mm ペトリ皿のスチール ワイヤ メッシュの上に置きます鉗子を使用して、慎重に。</li>
	<li>4.3 や 4.4 他の半分の頭で手順を繰り返します。</li>
	<li>滅菌ガラス ピペットを使用して、フィルター ペーパーのレベルに到達するまでそれぞれ 35 mm のペトリ皿に直接培養液を追加することは慎重に。栄養培地で半分の頭を漬けないでください。</li>
	<li>10,000 U/mL ペニシリン-ストレプトマイシンの 50 μ L を各シャーレで培養液に追加します。</li>
	<li>小さな広場にティッシュ ペーパーの部分を折るし、100 mm ペトリ皿の中に置きます。オートクレーブの蒸留水で湿ら します。</li>
	<li>4 日間で 37 ° C ± 1 ° C ~ 40% 湿度滅菌、暗いインキュベーターに培養皿を配置します。</li>
	<li>それぞれの胚の手順 3 と 4 を繰り返します。 注: 胚の数必要によって異なります特定の実験;ここでは、12 の胚のプロトコルについて述べる。</li>
</ol>5 文化の維持
<ol><li>一日に一回、新鮮なオートクレーブ蒸留水でインキュベーターでプラスチック容器をトップします。</li>
	<li>細菌や真菌感染症のすべての文化を毎日確認します。文化の崩壊を持っているまたは媒体が色を変更された感染しています。感染しているすべての文化を破棄します。</li>
</ol>6. 固定
<ol><li>インキュベーターからすべての料理を削除次の養殖。</li>
	<li>ピンセットのペアを使用して、慎重組織を引き裂くように注意して、ろ紙から目を取り外します。</li>
	<li>4% のパラホルムアルデヒドの 4 ° C で一晩リン酸緩衝生理食塩水 x 1 または室温で一晩 10% 中性緩衝ホルマリンの眼組織を修正します。</li>
	<li>リン酸緩衝生理食塩水 x 1 に 4 ° C で組織を保存します。</li>
</ol>

<ol>
	<li>Franz-Odendaal, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+understanding+the+development+of+scleral+ossicles+in+the+chicken,+Gallus+gallus.">Towards understanding the development of scleral ossicles in the chicken, Gallus gallus.</a> Developmental Dynamics. 237 (11), 3240-3251 (2008).</li><li>Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+motor+proteins+in+echinoderm+embryos:+an+argument+in+support+of+antibody+inhibition+experiments.">Functions of motor proteins in echinoderm embryos: an argument in support of antibody inhibition experiments.</a> Cell Motility and the Cytoskeleton. 39 (4), 257-260 (1998).</li><li>Horakova, D., 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=Effect+of+FGFR+inhibitors+on+chicken+limb+development.">Effect of FGFR inhibitors on chicken limb development.</a> Development, Growth &amp; Differentiation. 56 (8), 555-572 (2014).</li><li>Gerlach, G. F., Morales, E. E., Wingert, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbead+Implantation+in+the+Zebrafish+Embryo.">Microbead Implantation in the Zebrafish Embryo.</a> Journal of Visualized Experiments. (101), e52943 (2015).</li><li>Luz-Madrigal, A., Grajales-Esquivel, E., Del Rio-Tsonis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+Embryonic+Chick+Eyes.">Electroporation of Embryonic Chick Eyes.</a> Bio-protocol. 5 (12), e1498 (2015).</li><li>Roach, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+organ+culture+of+embryonic+chick+femora:+a+system+for+investigating+bone+and+cartilage+formation+at+an+intermediate+level+of+organization.">Long-term organ culture of embryonic chick femora: a system for investigating bone and cartilage formation at an intermediate level of organization.</a> Journal of Bone and Mineral Research. 5 (1), 85-100 (1990).</li><li>Jung, H. S., 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=Local+inhibitory+action+of+BMPs+and+their+relationships+with+activators+in+feather+formation:+implications+for+periodic+patterning.">Local inhibitory action of BMPs and their relationships with activators in feather formation: implications for periodic patterning.</a> Developmental Biology. 196 (1), 11-23 (1998).</li><li>McKinnell, I. W., Turmaine, M., Patel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+Hedgehog+functions+by+localizing+the+region+of+proliferation+in+early+developing+feather+buds.">Sonic Hedgehog functions by localizing the region of proliferation in early developing feather buds.</a> Developmental Biology. 272 (1), 76-88 (2004).</li><li>Smith, E. L., Kanczler, J. M., Oreffo, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+take+on+an+old+story:+chick+limb+organ+culture+for+skeletal+niche+development+and+regenerative+medicine+evaluation.">A new take on an old story: chick limb organ culture for skeletal niche development and regenerative medicine evaluation.</a> European Cells &amp; Materials. 26, 91-106 (2013).</li><li>Sato, T., 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=In+Vitro+Spermatogenesis+in+Explanted+Adult+Mouse+Testis+Tissues.">In Vitro Spermatogenesis in Explanted Adult Mouse Testis Tissues.</a> PLoS One. 10 (6), e0130171 (2015).</li><li>Ochoa-Villarreal, M., 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=Plant+cell+culture+strategies+for+the+production+of+natural+products.">Plant cell culture strategies for the production of natural products.</a> BMB Reports. 49 (3), 149-158 (2016).</li><li>Hamburger, V., Hamilton, H. 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+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Journal of Morphology. 88 (1), 49-92 (1951).</li><li>Yu, M., 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=The+developmental+biology+of+feather+follicles.">The developmental biology of feather follicles.</a> The International Journal of Developmental Biology. 48, 181-191 (2004).</li><li>Jabalee, J., Hillier, S., Franz-Odendaal, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+investigation+of+cellular+dynamics+during+the+development+of+intramembranous+bones:+the+scleral+ossicles.">An investigation of cellular dynamics during the development of intramembranous bones: the scleral ossicles.</a> Journal of Anatomy. 223 (4), 311-320 (2013).</li><li>Trowell, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+culture+of+mature+organs+in+a+synthetic+medium.">The culture of mature organs in a synthetic medium.</a> Experimental Cell Research. 16 (1), 118-147 (1959).</li><li>Pinto, C. B., Hall, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+an+understanding+of+the+epithelial+requirement+for+osteogenesis+in+scleral+mesenchyme+of+the+embryonic+chick.">Toward an understanding of the epithelial requirement for osteogenesis in scleral mesenchyme of the embryonic chick.</a> Journal of Experimental Zoology. 259 (1), 92-108 (1991).</li><li>Mbiene, J. P., MacCallum, D. K., Mistretta, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+cultures+of+embryonic+rat+tongue+support+tongue+and+gustatory+papilla+morphogenesis+in+vitro+without+intact+sensory+ganglia.">Organ cultures of embryonic rat tongue support tongue and gustatory papilla morphogenesis in vitro without intact sensory ganglia.</a> Journal of Comparative Neurology. 377 (3), 324-340 (1997).</li><li>Cloney, K., Franz-Odendaal, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+ex-ovo+culturing+of+chick+embryos+to+advanced+stages+of+development.">Optimized ex-ovo culturing of chick embryos to advanced stages of development.</a> Journal of Visualized Experiments. (95), e52129 (2015).</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> Journal of Visualized Experiments. (8), e306 (2007).</li></ol>

著者が明らかに何もありません。

このプロトコル (HH34) は 8 日目胚から鶏の目の成長を達成するために確立された組織培養技術を活用の in vitroの 4 日間。このグリッド培養法は、もともと Trowell15によって記述されていた。ニワトリ胚目15の分離組織層の間の誘導信号を研究するためにグリッドを持つ半多孔性膜を利用したピントとホールの 1991年研究16からプロトコ?...

ニワトリ (ギャラス) 生物学の分野でよく使用される優秀なモデル生物であります。その潜伏期間は約 21 日と迅速かつ効率的な実験を行う同時に多くの卵を孵化することができます。おそらく最も重要なは、胚はまた容易に操作、キーの発達過程とこれらのプロセスを駆動する蛋白質および遺伝子の広範な研究を有効にします。
ニワトリ胚目を介して数異なったティッシュのような他の多くのボディ システムの相互作用を開発する複雑な器官であります。このメソッドにより、研究開発の高度な段階で特にこれらの組織の開発のです。たとえば、多層の網膜は神経系の発達を学ぶものに特に関心のあります。眼瞼、強膜、レンズ vitreal 体、角膜などの他の眼組織の研究を有効にする別の方法は研究者に利点が。鶏胚の目には、平らな骨、膜内骨誘導および脊椎動物1の骨化の研究のためのモデルとして使用できる強耳小骨のシリーズも含まれています。
現在、胚を研究するために使用するメソッド数があります。誘発抑制抗体または他阻害分子の2,3, 外科的移植4阻害剤で浸したマイクロ ビーズとエレクトロポレーション5レセプター遺伝子を使用することができますすべてのメソッドまたは胚の興味の蛋白質。同様のメソッドは、リポタンパクリパーゼ蛋白質に使用されます。これらのメソッドは、制限ではないです。たとえば、萌芽期の開発を変更する化学物質を使用している場合は、胎児に対する致死効果を評価しなければならないとこれの生存を確保するため十分な低用量でのアプリケーションのローカライズされたサイトに前述のメソッドの使用を制限、胚。
体外の組織培養研究に有機体の広い範囲で使用されています、上記の制限の一部をバイパスに使用することができます。たとえば、大腿骨6羽芽7、8、および手足9鶏のすべてを調べたマウス10および根の精巣と11植物の茎を持っている組織培養の方法を使用しています。これらのメソッドは温度を変動し、栄養素の可用性を変更する機能など、組織開発の制御の高度科学者を付与します。全胚から組織の分離では、はるかに少ないより高い濃度でグローバル規模で操作研究ができ、化学物質の致死的な影響を受け。体外培養のもう一つの顕著な利点は、組織の細胞環境の保全組織の配置は比較的変わらない、異なったティッシュのタイプ9間の相互作用を研究することが可能となっています。したがって、体外養殖開きますドア追加実験アプローチ体内またはovo のモデルでは利用できません。
現在、化学物質を使用して萌芽期鶏眼研究開発は特に困難です。初期胚胚体外膜数カバー胚、ミクロ粒子のニキビトリートメントまたは化学薬品の適用が困難それにつれて、さらに複雑なメソッドがすでに困難胚にも卵の内で非常にアクティブです。このプロトコルは、目およびその周囲の組織、また眼内の発達過程を検討する新たな機会を提供しながら、これらの障壁を排除することに簡単にアクセスできます。このプロトコルは、萌芽期の目の強膜の小骨の誘導の研究に設立されました。

<table><tbody><tr><td>35 mm cell culture petri dishes</td><td>Corning</td><td>353001</td><td>easy grip tissue culture dish, polystyrene, non-pyrogenic</td></tr><tr><td>100 mm cell culture petri dishes</td><td>Corning</td><td>353003</td><td>tissue culture dish, polystyrene, non-pyrogenic</td></tr><tr><td>paper tissue</td><td>Kimtech</td><td>34155</td><td>Kimtech Science Brand Task Wipers, 280 per carton</td></tr><tr><td>wire mesh</td><td>n/a</td><td>n/a</td><td>stainless steel wire mesh (grid size 0.7 mm)</td></tr><tr><td>disposable glass pipettes</td><td>VWR</td><td>14673-010</td><td>Borosilicate glass disposable 5 3/4&quot;</td></tr><tr><td>nutrient medium</td><td>Gibco</td><td>12591-038</td><td>Fitton-Jackson Modification, [+] L-glutamine with phenol red (BGJB)</td></tr><tr><td>penicillin-streptomycin</td><td>Sigma-Aldrich</td><td>P4458</td><td>10000 units/mL penicillin streptomycin solution stabilised</td></tr><tr><td>filter paper</td><td>Whatman</td><td>1454 090</td><td>semi-porous filter paper 90mm</td></tr><tr><td>fertilized chicken eggs</td><td>Dalhousie University Agricultural College</td><td>n/a</td><td>can be obtained from local farms</td></tr><tr><td>sodium chloride (NaCl)</td><td>EMD</td><td>SX0420-3</td><td>sodium chloride crystals, reagent grade</td></tr><tr><td>1 L glass bottle</td><td>VWR</td><td>89000-240</td><td>1 L pyrex autoclavable glass bottle</td></tr><tr><td>ethanol</td><td>Fisher Scientific</td><td>BP82011</td><td>70% molecular biology grade</td></tr><tr><td>tupperware containers</td><td>n/a</td><td>n/a</td><td>store-bought and sterilized with EtOH</td></tr><tr><td>disposable razor blades</td><td>VWR</td><td>55411-050</td><td>single edge industrial razor blades (surgical carbon steel)</td></tr><tr><td>plastic spoons</td><td>n/a</td><td>n/a</td><td>store-bought and sterilized with EtOH</td></tr><tr><td>dust mask</td><td>3M</td><td>n/a</td><td>3M 8500 Comfort Mask</td></tr><tr><td>paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>paraformaldehyde, reagent grade, crystalline</td></tr><tr><td>neutral-buffered formalin</td><td>Fisher Scientific</td><td>72210</td><td>10% neutral buffered formalin</td></tr><tr><td>phosphate buffered saline (PBS)</td><td>n/a</td><td>n/a</td><td>10X phosphate buffered saline pH 7.4 (137mM NaCl, 2.5mM KCl, 4.3mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 1.4mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td></tr><tr><td>15 ml falcon tubes</td><td>VWR</td><td>21008-216</td><td>presterilized centrifuge tubes</td></tr><tr><td>forceps</td><td>FST</td><td>n/a</td><td>fine forceps</td></tr><tr><td>chick saline&amp;nbsp;</td><td>n/a</td><td>n/a&amp;nbsp;</td><td>0.85% NaCl</td></tr><tr><td>tinfoil</td><td>n/a</td><td>n/a</td><td>store-bought</td></tr><tr><td>paper towel</td><td>n/a</td><td>n/a</td><td>store-bought</td></tr></tbody></table>

organotypic culture method to study the development of embryonic chicken tissues

ニワトリ胚は、脊椎動物の発展のため信頼性の高いモデル有機体として使用されます。そのアクセシビリティと短い潜伏期間最適実験のためです。現在、鶏胚におけるこれらの発達の経路の研究は、阻害剤とローカライズされたサイトで、さまざまな方法を使用して低濃度で薬を適用することによって行われています。同時に全体の胚は、胚の感受性などを扱う際に問題の物理的な制限の多くを回避しながらホストの有機体から分離組織の研究を可能にする手法は、体外で細胞の培養潜在的に致命的な化学物質の高用量。培養ニワトリ胚半分頭、生体外で、現在確立された方法を超えて発達過程の検討のための新しい機会を提示するためのプロトコルを培養、切片を紹介します。

このメソッドを使用すると、ニワトリ胚目でく体外の開発 (HH34) の 8 日目から 4 日間培養します。Ovo の開発の 4 日間は、HH38 に対応します。
この培養方法は、まぶた (図 1 b) に目を囲む羽芽の開発をサポートします。これらの羽芽はありません現在ovo でHH34 で前培養 (<strong class="xf...

Watch this Scientific Journal Video about Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues at JoVE.com

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

ニワトリ胚組織の開発培養法

今回、切片培養ニワトリ胚の器官を成長するプロトコル体外。このメソッドを使用すると、ニワトリ胚のティッシュの開発を学べる、文化環境の制御の高度を維持しながら。

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

organotypic-culture-method-to-study-development-embryonic-chicken

Research

JoVE Journal

Developmental Biology

5.9K ビュー.  Mount Saint Vincent University. 今回、切片培養ニワトリ胚の器官を成長するプロトコル体外。このメソッドを使用すると、ニワトリ胚のティッシュの開発を学べる、文化環境の制御の高度を維持しながら。

ビデオ: Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

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

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

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

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

最適化された<em&gt;元OVO</em&gt;開発の進んだ段階にニワトリ胚の培養

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

発生生物学

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

長期用水中フィルターペーパーサンドイッチ<em&gt;例OVO</emニワトリ初期胚の&gt;タイムラプスイメージング

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

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

Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the formation of quail-chicken chimeras and the use of the chorioallantoic membrane (CAM) to sustain the development of ectopic tissues date back to the last century. Nowadays, the combination of these classical techniques with recent in vitro methodologies offers novel prospects to further explore organ formation.
Here we describe a two-step approach to study early- and late-stages of organogenesis. Briefly, the embryonic region containing the presumptive territory of the organ is isolated from quail embryos and grown in vitro in an organotypic system (up to 48 h). Cultured tissues are subsequently grafted onto the CAM of a chicken embryo. After 10 days of in ovo development, fully formed organs are obtained from grafted tissues. This method also allows the modulation of signaling pathways by the regular administration of pharmacological agents and tissue genetic manipulation throughout in vitro and in ovo developmental steps. Additionally, developing tissues can be collected at any time-window to analyze their gene-expression profile (using quantitative PCR (qPCR), microarrays, etc.) and morphology (assessed with conventional histology and immunochemistry).
The described experimental procedure can be used as a tool to follow organ formation outside the avian embryo, from the early stages of organogenesis to fully formed and functional organs.

This article provides an updated approach to the classical quail-chicken chimera system to study organ formation, by combining novel in vitro and in ovo experimental procedures.

初期と後期-段鳥モデルにおける器官形成を探索する 2 段階のアプローチ: 胸腺、副甲状腺腺器官パラダイム

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

ニワトリ胚脊髄スライス培養プロトコル

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

神経科学

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed contributions to unveiling key processes in developmental biology.
Herein is described an optimized method to isolate embryonic tissues from quail and chickens by microsurgery and enzymatic digestion while preserving its biological properties. After isolation, tissues from both species are associated in an in vitro organotypic assay for 48 h. Quail and chicken tissues can be discriminated by distinct nuclear features and molecular markers allowing the study of the cellular cross-talk between heterospecific association of tissues. This approach is, therefore, a useful tool for studying complex tissue interactions in developmental processes with highly dynamic spatial modifications, such as those occurring during pharyngeal morphogenesis and the formation of the foregut endoderm-derived organs. This experimental approach was first developed to study the epithelial-mesenchymal interactions during early-stages of thymus formation. In this, the endoderm-derived prospective thymic rudiment and mesoderm-derived mesenchyme, were isolated from quail and chicken embryos, respectively.
The capacity of the associated tissues to generate organs can be further tested by grafting them onto the chorioallantoic membrane (CAM) of a chicken embryo. The CAM provides nutrients and allows gas exchanges to the explanted tissues. After 10 days of in ovo development, the chimeric organs can be analyzed in the harvested explants by conventional morphological methods. This procedure also allows studying tissue-specific contributions during organ formation, from its initial development (in vitro development) to the final stages of organogenesis (in ovo development).
Finally, the improved isolation method also provides three-dimensionally (3D) preserved embryonic tissues, that can also be used for high-resolution topographical analysis of tissue-specific gene-expression patterns.

This article provides a method to isolate pure embryonic tissues from quail and chicken embryos that can be combined to form ex vivo chimeric organs.

萌芽期ティッシュと胸腺の例を使用してウズラ鶏キメラ器官の形成の分離

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed ...

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 ...

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

The soaked bead assay involves targeted delivery of test reagent at any developmental time point to study the regulation of cell differentiation and morphogenesis. A detailed protocol, applicable to any experimental animal model, for preparing three different types of soaked beads and implanting these in the interdigit of a chicken embryo is presented.

浸漬ビーズアッセイを用いたニワトリ胚発生時の細胞分化、形態形成、パターニングの解析

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of ...

鳥類胚性皮膚外植片培養プロトコルの包括的なレビュー

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin explant cultures to examine different aspects of skin development are described. First, ex vivo organ cultures and manipulations offer researchers opportunities to observe and study the development of feather buds directly. Skin explant culture can grow for 7 days enabling direct analysis of cellular behavior and 4D imaging at intervals during this growth period. This also allows for physical and molecular manipulations of culture conditions to visualize tissue response. For example, growth factor-coated beads can be applied locally to induce changes in feather patterning in a limited area. Alternatively, viral transduction can be delivered globally in the culture media to up or downregulate gene expression. Second, the skin recombination protocol allows researchers to investigate tissue interactions between the epidermis and mesenchyme that are derived from different skin regions, different life stages, or different species. This affords an opportunity to test the time window in which the epithelium is competent to respond to signals and its ability to form different skin appendages in response to signals from different mesenchymal sources. Third, skin reconstitution using dissociated dermal cells overlaid with intact epithelium resets skin development and enables the study of the initial processes of periodic patterning. This approach also enhances our ability to manipulate gene expression among the dissociated cells before creating the reconstituted skin explant. This paper provides the three culture protocols and exemplary experiments to demonstrate their utility.

著者スポットライト:現在の実験的鳥類皮膚外植片方法論への洞察

Here we describe protocols for three types of avian embryonic skin explant cultures that can be used to examine tissue interactions, 4D imaging timelapse movie (3D plus time), global or local perturbation of molecular function, and systems biology characterization.

鳥類の皮膚外植片を用いた組織パターン形成と器官形成の研究

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin ...

ニワトリ胚組織の開発培養法

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最適化された

長期用水中フィルターペーパーサンドイッチ

初期のニワトリ胚をex vivo（新文化）の文化のための方法

初期と後期-段鳥モデルにおける器官形成を探索する 2 段階のアプローチ: 胸腺、副甲状腺腺器官パラダイム

ニワトリ胚脊髄スライス培養プロトコル

萌芽期ティッシュと胸腺の例を使用してウズラ鶏キメラ器官の形成の分離

アン EX - OVOニワトリ胚培養システム

浸漬ビーズアッセイを用いたニワトリ胚発生時の細胞分化、形態形成、パターニングの解析

鳥類の皮膚外植片を用いた組織パターン形成と器官形成の研究

鳥類の皮膚外植片を用いた組織パターン形成と器官形成の研究