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

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

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No conflicts of interest are declared.

WECの実験における再現性のある結果は、高品質のICラット血清、正確胚解剖技法3を使用することに依存している。空腹時には、血液中のグルコース濃度を標準化する必要があるため、採血前に絶食期間を短くしないでください。ホルモンレベルが発情周期に係る雌の動物に変化するため、雌ラット血清の調製のために使用すべきではない、およびエストラジオールホルモンのこのような変化は、胚の培養には適していない。また、非常に脂肪雄ラットは、血液中の脂質汚染大量のを回避するために、血清調製のために使用すべきではない。 血液を収集する際、最初の動脈血の色は麻酔下で正常な呼吸を反映して、明るい赤色であることを確認してください。呼吸低下は、採取した血液の量の減少をもたらす。このような場合に復旧するには、一時的に注射器がPUする速度を下げるlled。血清中の準備の中で最も重大な問題は、溶血である。採取した血液サンプルは重度の溶血を示す場合、血液を遠心分離により二層に分離されない。軽度溶血血清は次善の開発をサポートする可能性があるため、このような血清は破棄されるべきである。溶血が強制圧力によって誘発される傾向にあるように、血液を収集する際、注射器をゆっくりとプルアップする必要があります。なお、採取した血液サンプルを溶血を増加させる、試験管に注ぎ入れているときに血液をバブリング避けることが重要である。 培養マウス胚に、雄のマウスから採取したIC血清が理想的であり;我々は、マウスから血液の十分に大きな容量を得ることができないためしかし、この要件は実用的ではない。我々はラット血清を準備するとき、私たちは、少なくとも10の雄ラットを繁殖引退準備。私たちの研究室では、採血を定期的に少なくとも2の担当者が行っている：個々の血液と個々の麻酔を収集し、実行する最初の遠心分離。 今日まで、化学的に明確な培地を組み合わせることによってWECの商業ラット血清を用いたいくつかの研究は、23〜25が報告されている。例えば、50％の市販のラット血清および50％のDMEMの混合物は、36時間23と40〜24時間のためのE9.75マウス胚の正常な成長のためのE8.5マウス胚の正常な成長をサポートしています。別のプロトコルでは、別の会社から入手できる50％のラット血清とN-2を補充した50％のF12培地からなる混合物は、24時間25 E7.5とE8.5マウス胚の適切な成長をサポートしています。しかしながら、これらのコンビナトリアル媒体は、マウスにおけるE11.5-E12.5およびラットにおけるE13.5-E14.5のように、後の段階でのラットおよびマウス胚の培養に適しているかどうかは不明のままである。代わりに、ラット血清の様々な代替メディアを使用してWECの研究はまた、26-28に報告されている。例えば、DMEM/F12、20％ウシ胎児血清（FBS）の組み合わせは、開発をサポート28時間26 E11.5からのラット胚のopment。しかし、この媒体は、28時間26 E10.5からのラットの胚培養には適していません。したがって、我々はFBSを含む培地中の胚の正常な成長が胚の胚の段階に依存していると信じています。 唯一市販​​されている胚性幹細胞培地、ウシ血清アルブミン、およびN-2サプリメントを含む定義された試薬からなる無血清培養培地を使用してWECが報告されている。この媒体は16〜40時間27、28のため、E10.5マウス胚の文化をサポートしています。Morreスコットら 24時間培養マウス胚のクラウン·ツー·臀部サイズはE11のそれよりもかなり小さいことを示した子宮内で成長した胚0.5。しかし、このような体節および後根神経節などの主要な構造は、これらの培養された胚では正常に表示されます。一方、Kalaskar及びローダーは、この培地中で試験した胚の60％がノルムを示したことを報告ら18時間後の開発が、追加の20〜22時間培養した胚では良い開発率は30から40パーセントに低下した。これらの研究とは対照的に、我々は、正常ラット6又はE10.5でE12.5からICラット血清2日間我々のプロトコルを用いて製造さ（ すなわち 、約48時間）は100％で培養したマウスおよびラット胚の良好な現像を再現することができ24時間後に一度培地を交換することにより、マウス8。これらの結果は、ICラット血清を調製するための私たちの方法は、哺乳動物のWECat異なる胚の段階を適用する種々の実験のために有用であることを示唆している。したがって、我々は我々のビデオプロトコルは、新しい研究者が自分の研究室にWECを用いた実験を紹介するのに役立ちますことを願っています。

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

<table border="0" cellpadding="0" cellspacing="0" width="892">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:156px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:401px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Isoflurane</td>
			<td>Abbott</td>
			<td>B506</td>
			<td>For anesthesia of rats.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Large scissors</td>
			<td>Napox</td>
			<td>B-7H</td>
			<td>Stelized scissors for cutting the skin and muscle of rats.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Curved forceps</td>
			<td>Napox</td>
			<td>A-3-2</td>
			<td>Stelized forceps for picking up the skin of rats and squeezing the fibrin clot.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sprague-Dawley (SD) rat</td>
			<td>Charles Rivers Laboratories</td>
			<td></td>
			<td>Retired male rats from colony in the lab or purchased retired male rats.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe (20 ml)</td>
			<td>TERUMO</td>
			<td>SS-29ESZ</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Needle (21G x 5/8&#34;)</td>
			<td>TERUMO</td>
			<td>NN-2116R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile test (spitz) tube (10 ml)</td>
			<td>ASIAKIZAI</td>
			<td style="width:119px;">1101C000B-10</td>
			<td>For collection of boold</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile disposable pipette</td>
			<td>Eiken Chemical</td>
			<td>CD2000 No.4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterilie disposable tube (15 ml)&#160;</td>
			<td>Falcon</td>
			<td>2196</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of rat serum suitable for mammalian whole embryo culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

図1は、血液細胞、血清を分離するための記載された手順の代表的な結果を示している。我々は、一般的に引退した雄性ラット（ 図1A）からの血液の15ミリリットルを得る。遠心分離によって、採取した血液、血清、矢印で示されているフィブリン塊、および血液細胞（ 図1C）を含む下層を含有する上層とに分離することができる。重要なことには、溶血血液試料は、血清、血液の層（ 図1B）とに分離することができない。 2時間4℃でチューブを出た後、フィブリン塊が固体になる。この固化は、湾曲鉗子（図1D）を使用したフィブリン塊から血清を分離するのに便利です。試験管は、その後、遠心分離した。フィブリン塊は、血清および血液細胞層（ 図1E）の間の界面に見える。 2本の試験管からの血清に集めた使い捨てピペットを用いて新たなチューブ。全体の手順が成功した場合、我々は、オスのラットからの血清の約6ミリリットルを入手することができます。さらに、赤血球を除去するために、採取した血清を再び遠心分離した。残りの赤血球は、チューブ（ 図1F）の底に沈殿した。私たちは、新しいチューブに上清を移して、我々は、遠心分離後、上層から血清を収集することができていても、溶血の程度を判断するために、精製された血清の色をチェックした。軽度の溶血とラット血清はピンクがかった色（ 図1Gに右管）示すが、WECのための理想的なラット血清は、一般的に、（ 図1（g）にチューブを残した）淡黄色を呈する。 ICラット血清（ 図1Hの左側）に-20℃で少なくとも6〜12ヶ月間保存することができる。凍結時に軽度の溶血との血清（ 図1Hに残って）理想的な血清より深い色を示した。 図1。血液を遠心分離し、雄ラットの血清を調製するための手順。 A）雄ラットから採取した血液サンプルを遠心分離するための2つの試験管に等分される。B、C）非分離された試料（B）および理想的な分離血清およびフィブリン塊（矢印C）を含有する上層に第一の遠心分離後の血液細胞を含有する下層は、D）フィブリン塊をピンセットを用いて圧搾される。E）は 、血清および血液細胞層を二回目の遠心分離後。血液細胞のフィブリン塊がインターフェイス（矢印）で観察される。F）第三の沈殿を遠心分離（矢印）。G）の後に、左チューブを淡黄色の色で理想的なラット血清を示す。右の管は軽度の溶血とラット血清を示しています。 <strong&gt; H）の最適な（左）と軽度の溶血（右）ラット血清凍結した。

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

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

哺乳類の全胚培養に適したラット血清の調製

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

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

Nanyang Technological University

Research

JoVE Journal

Biology

13.8K ビュー.  Jichi Medical University. Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

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

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

E18ラット脳の器官型スライス培養

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

生物学

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.  

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

遺伝子銃の箇条書きとスライス培養における神経細胞の微粒子銃トランスフェクションの準備

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens.  Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2.  Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers.  The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied.  Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques.  Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.

Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

最終分化を研究するための準備とラットの水晶体上皮外植片の培養

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  ...

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 ...

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 ...

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

生体力学とMechanobiological研究ラット尾腱の準備

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

培養ヒト胚および誘導多能性幹細胞に適したマウス胚性線維芽細胞の調製

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

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 ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

ホールマウントによるゼブラフィッシュ胚標本ステンドの観察および分析のためのフラットマウントの準備<em&gt;その場で</em&gt;ハイブリダイゼーション

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

マンモスフィアの自然発生の事前侵襲乳房病変の非酵素的、無血清組織培養

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...