我々は、重要な原稿の読み取り、タイムラプスイメージングの支援のためのUNCの顕微鏡サービス研究所、および資金調達をサポートするためのNIH（助成金＃R01HL061656）がアンドレアPortburyのに感謝したいと思います。

1。抗体結合ビーズを準備<ol><li>前解剖へある日、製造元の指示に従って適切なダイナビーズ（ラットで育った抗体について例えばラットIgGダイナ、4×10 8ビーズ/ ml）で選択した抗体を兼ね備えています。 BDのラット抗CD31抗体（0.5μgの/μL、内皮細胞を分離するために使用される）の場合は、3μlの抗体は、最終1.5μgの抗CD31抗体の濃度及び/ 1×10 7ビーズのため、すべての25μlのビーズのために追加されます25μlのバッファー。 </li><li> 4℃でビーズと一晩抗体をインキュベート</li></ol> 2。コラーゲンプレートの準備<ol><li>単にコラーゲンコートプレートとは対照的に、コラーゲンゲル上で増殖させたときに単離された細胞は、より迅速に細管を形成する。 6で説明したようにそのため、コラーゲンゲルは、解剖前の晩に行われる。組織培養フード内で氷の上で、以下の試薬（十分に19の井戸のために）結合：206.4μlの滅菌Hを&lt;サブ&gt; 2 O、140.4μlのコラーゲンタイプI（4.08 mg / ml）で、38.4μlの10倍M199、及び1.15μlの5 MのNaOH。 </li><li> 384ウェル組織培養プレートのウェル減らすこのコラーゲンゲルのピペットを20μl。 30分間37℃の組織培養インキュベーターでプレートを置きます。 </li><li>各ウェルに80μlのDMEMを加え、37℃で15分間インキュベート℃、培地を削除し、さらに2回繰り返す。 </li><li>最終的には、すすぎ各ウェルに80μlの10％FBS / DMEMを加え、37℃で一晩インキュベートした後、℃、単離した細胞を追加する前に培地を取り除きます。 </li></ol> 3。物品税とハートを消化<ol><li>承認された安楽死法を用いて、所望の胎生で時限妊娠マウスを安楽死させる。全ての実験は、ノースカロライナ大学チャペルヒル校で制度動物実験委員会によって承認された。 </li><li>仰臥位でマウスを使って、自由に前に70％エタノールで雌の腹側をスプレー解剖。内臓から下腹部の皮膚や筋肉を持ち上げるために鉗子を使用して、内臓を可視化するために女性の下腹部空洞を切り開く。 </li><li>鉗子で子宮頸部を持って、慎重に鉗子に尾カットし、腹腔からの子宮角を持ち上げ始める。子宮角を持ち上げながら、所定の位置にホーンを保持している任意の結合組織を切り取る。子宮角を切除終了するには、子宮角を解放するために卵管と子宮角の接合部をカット。 </li><li>コー​​ルド1Xリン酸緩衝生理食塩水（PBS）表示し、必要に応じてすすぎを含むペトリ皿に子宮角に置きます。添付された胚嚢を露出する正中線を経由して子宮角を切り開く。 </li><li>胚を切断誤っ避け、胚を取得するために胎盤の接合部で胚嚢と胚を開いてカットします。冷PBSで二ペトリ皿に胚を置きます。氷に子宮角とペトリ皿を返します。 </li><li> D胸壁へのアクセスを改善するために胚をecapitate。ジェノタイピングが必要な場合、それを削除し、氷上に置いたエッペンドルフチュー​​ブに保存する尾を切った。 </li><li>その背中に胚では、心臓や肺を可視化するために胸壁を開いてカット。心臓を切断回避するために、肋骨の下部に水平カットに続いて前肢近く胸郭の側面に沿って垂直方向のカットを、確認してから、静かに道を持ち上げて胸壁を引っ張る。最大約胎生13.5を通じて、胸壁は、視覚化を助ける、透明になります。 </li><li>慎重にピンセットを用いて心臓を持ち上げ、下に血管を切った。その後、心を解放するために大血管の上にカット。肺血管が切断されていない場合は、肺、心臓を除去することができる。必要であればこのように、肺のように、余分な組織を除去する。 </li><li>心室から心房と大血管を分離して捨てる。はさみを使用して、小片（約1mm 3 &lt;に心室をミンチ/&gt;をSUP）、約500μlのコラゲナーゼ（滅菌PBSで1 mg / mlの）で開催。すべての心室がミンチとコラゲナーゼに配置されるまでサンプルを氷上で保管してください。 </li><li>心室が別々のエッペンドルフチュー​​ブにそれぞれの心を集め、すべての胚から摘出されるまで、上記の手順を繰り返します。すべての胚は同じ遺伝子型を持っている場合、心は単一のエッペンドルフチュー​​ブにプールすることもできる。単一一度に処理サンプルの数は、分離に使用される磁石に基づいて制限することができ、ここで使用DynaMag（123-21D）は、16エッペンドルフチュー​​ブの最大値を保持する。 </li><li> 45分間ロッキングと37℃でみじん切り心室を置きます。 15分毎に、そっとサンプルを削除して、機械的に細胞を解離するために上下にピペッティングし。 </li></ol> 4。内皮細胞を単離<ol><li>細胞の残りの塊を除去するためにフィルターが70μmを通して心室-コラゲナーゼ溶液を渡します。 </li><li> 10分間400 xgで細胞を遠心分離。捨てる上清は、DMEMで約200μlの10％FBSと交換し、細胞を解離する上下ピペット。 </li><li>一度前のステップを繰り返します。第二10％FBS / DMEM洗浄後10分間400 xgで細胞を遠心分離。 50μlの10％FBS / DMEMで細胞を再懸濁します。 </li><li>最後の遠心分離工程の間に、ビーズを準備します。 <ol><li> 1分間磁石上に抗体やビーズを含むエッペンドルフチュー​​ブを置きます。 </li><li>磁石の代わりにチューブで、上清を除去します。磁石からチューブを取り外し、約100μlの10％FBS / DMEMを追加します。 </li><li> 1分間磁石上にチューブを置きます。上清を除去し、10％FBS / DMEMでもう一度洗う。 3回洗浄の合計に対して、この手順を繰り返します。 </li><li>最終洗浄後、10％FBS / DMEM（サンプルあたり5μL）で抗体結合ビーズを再懸濁します。 </li></ol></li><li>各細胞試料に、抗体結合ビーズを5μlを追加します。ロッキングFOで4℃でビーズ細胞懸濁液を置きrは30分。 </li><li>層流フード中で、1分間マグネットに細胞および抗体結合ビーズを含むエッペンドルフチュー​​ブを置く。上清を取り除き、磁石からチューブを取り外し、約100μlの10％FBS / DMEMで洗浄する。 5回の洗浄のために繰り返します。 </li><li>最後の洗浄の後、1分間磁石上にチューブを置き、上清を除去し、磁石からチューブを取り外します。約100μlのDMEMを追加。 </li><li> 1分間磁石上にチューブを置き、上清を除去し、磁石からチューブを取り外します。 0.25％トリプシン-EDTAを温め100-200μLを加える。 37℃でサンプルをインキュベート℃、5分間、5％CO 2。 </li><li> 2分間磁石上にチューブを置きます。新鮮なエッペンドルフチュー​​ブに細胞を含む上清を慎重に移す。 400×gで5分間遠心分離します。 </li><li>上清を取り除きます。予想される収量に応じて、または384ウェル扱わ培養皿 - 80-100μlの成長培地とプレート96で細胞を再懸濁し（E14.5-E16.5胚の心室から約450〜600細胞）。内皮細胞は、内皮細胞増殖培地（以下に定義）を使用します。 </li></ol>

<ol>
	<li>Landecker, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Life: How Cells Became Technologies. , (2007).</li><li>Kan, C. Y., Wen, V. W., 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=Endothelial+cell+dysfunction+and+cytoskeletal+changes+associated+with+repression+of+p16(INK4a)+during+immortalization.">Endothelial cell dysfunction and cytoskeletal changes associated with repression of p16(INK4a) during immortalization.</a> Oncogene. , (2012).</li><li>Dyer, L. A., Patterson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+endothelium:+an+emphasis+on+heterogeneity.">Development of the endothelium: an emphasis on heterogeneity.</a> Semin. Thromb. Hemost. 36, 227-235 (2010).</li><li>Dong, Q. G., Bernasconi, 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=A+general+strategy+for+isolation+of+endothelial+cells+from+murine+tissues.+Characterization+of+two+endothelial+cell+lines+from+the+murine+lung+and+subcutaneous+sponge+implants.">A general strategy for isolation of endothelial cells from murine tissues. Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 17, 1599-1604 (1997).</li><li>van Beijnum, J. R., Rousch, M., Castermans, K., vander Linden, E., Griffioen, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+endothelial+cells+from+fresh+tissues.">Isolation of endothelial cells from fresh tissues.</a> Nat. Protoc. 3, 1085-1091 (2008).</li><li>Runyan, R. B., 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=Invasion+of+mesenchyme+into+three-dimensional+collagen+gels:+a+regional+and+temporal+analysis+of+interaction+in+embryonic+heart+tissue.">Invasion of mesenchyme into three-dimensional collagen gels: a regional and temporal analysis of interaction in embryonic heart tissue.</a> Dev. Biol. 95, 108-114 (1983).</li><li>Arnaoutova, I., Kleinman, H. K. <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+angiogenesis:+endothelial+cell+tube+formation+on+gelled+basement+membrane+extract.">In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract.</a> Nat. Protoc. 5, 628-635 (2010).</li><li>Zeisberg, E. M., Potenta, S. E., Sugimoto, H., Zeisberg, M., Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+in+kidney+fibrosis+emerge+via+endothelial-to-mesenchymal+transition.">Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition.</a> Journal of the American Society of Nephrology : JASN. 19, 2282-2287 (2008).</li><li>Chang, L., Noseda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+vascular+smooth+muscle+cells+from+local+precursors+during+embryonic+and+adult+arteriogenesis+requires+Notch+signaling.">Differentiation of vascular smooth muscle cells from local precursors during embryonic and adult arteriogenesis requires Notch signaling.</a> PNAS. 109, 5993-6998 (2012).</li><li>Fehrenbach, M. L., Cao, G., Williams, J. T., Finklestein, J. M., DeLisser, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+murine+lung+endothelial+cells.">Isolation of murine lung endothelial cells.</a> American Journal of Physiology: Lung Cellular and Molecular Physiology. 296, L1095-L1103 (2009).</li><li>Frauli, M., Ludwig, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+fibroblast+proliferation+in+a+culture+of+human+endometrial+stromal+cells+using+a+medium+containing+D-valine.">Inhibition of fibroblast proliferation in a culture of human endometrial stromal cells using a medium containing D-valine.</a> Archives of Gynecology and Obstetrics. , 241-96 (1987).</li><li>Lazzaro, V. A., Walker, R. J., 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=Inhibition+of+fibroblast+proliferation+in+L-valine+reduced+selective+media.">Inhibition of fibroblast proliferation in L-valine reduced selective media.</a> Research communications in chemical pathology and pharmacology. 75, 39-48 (1992).</li><li>Arima, S., Nishiyama, K., 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=Angiogenic+morphogenesis+driven+by+dynamic+and+heterogeneous+collective+endothelial+cell+movement.">Angiogenic morphogenesis driven by dynamic and heterogeneous collective endothelial cell movement.</a> Development. 138, 4763-4776 (2011).</li></ol>

著者は、彼らが競合する経済的利益を持っていないことを宣言します。

プライマリー胚細胞での作業小説実験は、開発のin vitro重要なステップに対処することができます。しかし、単離手順は簡単ではありません。細胞はどのような種類の単離するための重要なステップは、細胞が十分にコラゲナーゼ消化により分離されていること、およびそれらがよく洗浄工程中に懸濁されることを確実に挙げられる。ピペッティングによる機械的切開が大幅コ?...

不死化細胞株の出現は、基本的な細胞生物学1に革命をもたらした。現在入手可能な細胞株は広範囲の器官に由来し、すべての主要な細胞型を包含している。しかし、確立された細胞株はいくつかの制限があります。不死化の方法は、具体的に明らかに寿命および増殖に関して、細胞の挙動を変化させるだけでなく、例えば2細胞骨格タンパク質などの予期しないタンパク質の発現に影響を与えることができる。多くの異なる細胞株が利用可能であるが、さらに、全体生物内であっても単一の細胞型間に有意な多様性がある。特定の番組多様な行動の内皮細胞は、動脈や静脈と流れの種類、容器3に存在している、彼らはラインかどうかに基づいて。発達の観点から、さらに押圧しかしながら、（参照、 例えば、t利用できる細胞株の大部分は、成体組織から誘導されるということであるATCCから入手彼がコレクション）。これらの成人の細胞株は、おそらく彼らの胚性前駆体の動的な性質を要約するしないでください。開発中に観測された、急速に変化する時空間的遺伝子発現パターンはまた、与えられた発達段階で1つの器官からの内皮細胞は、異なる発達段階で、同じ臓器の内皮細胞と同じように動作しない可能性を示唆している。 市販の不死化細胞株を使用する代わりに、一次細胞は、関心のある特定の組織から単離することができる。この手法の利点の中に、これらの一次電池であっても、特定の器官または任意の特定の発生段階における臓器の一部から分離することができる。さらに、これらの一次細胞は、遺伝子ノックアウトのインビトロ試験で 、そのようなトランスフェクション効率のような他の問題を回避しながら、ノックインできるよう、利用可能なトランスジェニック動物の様々から単離することができる。驚くことではないが、多くの技術は、特定の細胞型は4,5単離する方法を詳細に公開されている。一般に、これらの技術は、タグ付け、細胞を解離、関心領域を収集する関心のある特定の細胞型を含む、そして更なる分析のために、これらの細胞を単離する。 冠状動脈の内皮細胞の初期胚人口を研究するために、我々は小さい臓器で使用するために以前に発行された技法4を縮小。このスケールダウン手順により、我々は特定胚の段階で胚心臓から冠動脈内皮細胞を分離することができます。これらの細胞は、その後、マイグレーション分析などの伝統的な内皮細胞アッセイで使用することができる。初期胚細胞株は、より一般的になるまで、一次細胞での作業は非常に重要な技術である。

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>Timed-pregnant mice</td>
			<td></td>
			<td></td>
			<td>To be dissected at the embryonic stage of interest</td>
		 </tr>
		 <tr>
			<td>Anti-mouse CD31</td>
			<td>BD Bioscience</td>
			<td>553370</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Rat IgG Dynabeads</td>
			<td>Invitrogen</td>
			<td>110-35</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Collagen</td>
			<td>BD Bioscience</td>
			<td>354236</td>
			<td></td>
		 </tr>
		 <tr>
			<td>PBS (1x)</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Collagenase, type I</td>
			<td>Worthington Biochemical</td>
			<td>LS004196</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Cellgro </td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>FBS</td>
			<td>Sigma-Aldrich </td>
			<td>F2442 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Endothelial cell growth medium</td>
			<td>(made in lab as described in notes)</td>
			<td></td>
			<td>DMEM containing 20% FBS, 5 &mu;M &beta;-mercaptoethanol, 50 &mu;g/ml ECG, and 1x penicillin/streptomycin</td>
		 </tr>
		 <tr>
			<td>&beta;-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td></td>
		 </tr>
		 <tr>
			<td>ECGS</td>
			<td>Biomedical Technologies, Inc.</td>
			<td>BT-203</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300</td>
			<td></td>
		 </tr>
		 <tr>
			<td>384-well culture plate</td>
			<td>Greiner</td>
			<td>T-3037-6</td>
			<td>Plate was prepared with a thin collagen gel, as described in 6; working surface area 10 mm2</td>
		 </tr>
		 <tr>
			<td>Collagen type I</td>
			<td>BD</td>
			<td>354236</td>
			<td>Use at a final concentration of 1.5 mg/ml</td>
		 </tr>
		 <tr>
			<td>M199, 10x</td>
			<td>Invitrogen</td>
			<td>11825-015</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Syto-16</td>
			<td>Invitrogen </td>
			<td>S7578 </td>
			<td>Used as directed in 13 </td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		 </tr>
		 <tr>
			<td>Stereoscopic microscope</td>
			<td>Nikon </td>
			<td>SMZ645</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Cell culture incubator</td>
			<td>Thermo </td>
			<td>3110 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>DynaMag</td>
			<td>Invitrogen</td>
			<td>123-21D</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Table-top centrifuge</td>
			<td>Thermo</td>
			<td>75002430</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

isolation of embryonic ventricular endothelial cells

細胞培養が大幅無数培養条件下で細胞の個々の集団を評価するために我々の能力を強化しています。不死化細胞株の使用の容易さのために重要な利点を提供するが、これらの細胞株は、すべての潜在的な細胞タイプでは使用できない。特にトランスジェニックマウスからの関心の特定の領域から一次細胞を単離することにより、より微妙な研究を行うことができる。基本的なテクニックは、臓器や、興味のある部分的器官（心臓や心臓の特定の領域など ）を解剖し、単一細胞に臓器を解離が含まれます。これらの細胞は、その後、その他の細胞型を認識する抗体に結合した磁気ビーズと共にインキュベートする。関心のある細胞を、次いで、ビーズから細胞をトリプシン解離短いインキュベーションでは、磁石を用いて単離することができる。これらの単離された細胞は、次いで培養し、必要に応じて分析することができる。この手法はもともと成体マウスオルガンのために設計されましたsがなく、簡単にここに実証されるように、胚の器官で使用するために縮小することができます。我々の関心は、途上冠状血管系であるため、我々は特定胚の段階で細胞のこの集団を勉強したかった。従って、オリジナルのプロトコルは、胚心室のサイズが小さく、これらの発達段階における内皮細胞の低電位収率と互換性を持つように変更しなければならなかった。このスケールダウンのアプローチを利用して、我々は、トランスジェニック胚心室内皮細胞における冠状動脈神経叢リモデリングを評価しました。

CD31を認識する内皮細胞特異的抗体を使用して、内皮細胞が胚（E）13.5（ 図1A）および18.5（ 図1B-1D）の心室胚から単離した。未処理の培養皿で増殖させた場合、これらの細胞は丸められたままと近コンフルエント（ 図1A）場合、石畳のパターンを形成するであろう。希薄コラーゲンはまた、ウェルコーティングするために使用されており、内皮細胞が（ <...

Watch this Scientific Journal Video about Isolation of Embryonic Ventricular Endothelial Cells at JoVE.com

Isolation of Embryonic Ventricular Endothelial Cells

初代細胞培養物は、特に、特定の発達段階におけるトランスジェニックマウス胚からの細胞の特定の集団を分析するのに有用な技術である。ここでは、胚の心室を切開し、解離、および抗体結合ビーズを認識し、さらなる分析のために内皮細胞を分離しています。

胚心室内皮細胞の単離

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

isolation-of-embryonic-ventricular-endothelial-cells

Research

JoVE Journal

Biology

9.3K ビュー.  University of North Carolina at Chapel Hill. 初代細胞培養物は、特に、特定の発達段階におけるトランスジェニックマウス胚からの細胞の特定の集団を分析するのに有用な技術である。ここでは、胚の心室を切開し、解離、および抗体結合ビーズを認識し、さらなる分析のために内皮細胞を分離しています。

ビデオ: Isolation of Embryonic Ventricular Endothelial Cells

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

ヒト臍帯静脈内皮細胞の単離（HUVEC）

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

生物学

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

後肢虚血の治療のための胚性幹細胞由来血管内皮細胞

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

マウスES胚細胞の単離と導出

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

弁内皮細胞の単離

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

鳥類胚弁膜前駆細胞の単離と培養

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

新生児マウスからの肺血管内皮細胞の単離と培養

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

ネズミ弁内皮細胞の単離

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

マウス大動脈内皮細胞の単離および初代培養

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

マウスの冠動脈内皮細胞の単離

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Ca2+過渡現象とL型カルシウム電流の同時測定のための高品質マウス心房および心室筋細胞の分離

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...