この研究は、NSFのキャリア賞、ハートウェル財団、米国心臓協会（＃0830384N）によってサポートされています。

1。準備<ol><li>対象となる機器のトレイに以下の項目をオートクレーブ： <ol><li>鋸歯状の組織鉗子 - リーフレットの組織の処理について</li><li>組織のはさみ（8センチ） - リーフレットの組織と尖をトリミングする</li><li>綿棒 - リーフレットまたはカスプから内皮層を分離するための</li></ol></li><li>無菌コラゲナーゼ溶液を作る<ol><li> 18MΩ250mlの水に粉末DMEMの4.0グラムを追加。 </li><li>重炭酸ナトリウムの1.11グラムを追加。 </li><li> （600 U / mL）をコラゲナーゼの18万台を追加。 </li><li> 1％（3ML）ペニシリン/ストレプトマイシンを追加。 </li><li> 7.2〜溶液のpHを調整します。 </li><li> 270 mLに解決策を持ち出す。 </li><li> 0.2μmのフィルターを通過することで解決策を滅菌する。 </li><li>滅菌ウシ胎児血清の10％（30ml）を追加。 </li></ol></li><li>滅菌内皮細胞の培地を作る<ol><li> 18MΩ水400mlに粉DMEMの6.7グラムを追加します。 </li><li>重炭酸ナトリウムの1.85グラムを追加。 </li><li> （50 U / mL）をヘパリンの25,000台を追加。 </li><li> 1％（5ml）をペニシリン/ストレプトマイシンを追加。 </li><li> 7.2〜溶液のpHを調整します。 </li><li> 450ml中に解決策を持ち出す。 </li><li> 0.2μmのフィルターを通過することで解決策を滅菌する。 </li><li>滅菌ウシ胎児血清10％（50mLの）を追加します。 </li></ol></li><li>無菌の間質細胞の培地を作る<ol><li> 18MΩ水800mlに粉DMEMの13.37グラムを追加。 </li><li>重炭酸ナトリウムの3.7グラムを追加。 </li><li> 1％（10ml）をペニシリン/ストレプトマイシンを追加。 </li><li> 7.2〜溶液のpHを調整します。 </li><li> 900mLのためのソリューションを立ち上げます。 </li><li> 0.2μmのフィルターを通過することで解決策を滅菌する。 </li><li>滅菌ウシ血清の10％（100mLの）を追加します。 </li></ol></li></ol> 2。心臓弁尖の分離<ol><li>消費税​​の犠牲の後に心臓から直ちに無菌的大動脈根。 </li><li>徹底的に冷滅菌DPBSで血液の大動脈根を洗う。それは、VECの死と細菌汚染を制限するために、できるだけ早くすべての血液成分を除去することが不可欠です。彼らはVECに潜在的に有害であるため、抗生物質と抗真菌薬は、この段階でお勧めされていません。 </li><li> 12 mLの冷DPBSで満たされた無菌の15 mLコニカルチューブのルートと場所から直接（3バルブあたり）弁尖を分離します。新鮮なDPBSで破片とリフィルを削除するには、数回振る。 </li><li>氷の上で研究室に戻って輸送する。 </li><li>実験室に到着すると、無菌フードの下で組織を持つコンテナを配置します。 </li></ol> 3。内皮層の分離<ol><li>バルブごとに寒さコラゲナーゼ溶液（3リーフレット）の3mlの滅菌35mmディッシュを埋める。 </li><li>コラゲナーゼ溶液で満たされた皿に15 mLの試験管からすべての3つのリーフレットを置きます。 </li><li> 37℃で5〜10分間組織をインキュベート℃に</li><li>ゆっくりとリーフレットの表面に乾いた滅菌綿棒を回転させることにより内皮層を取り除く。せん断応用の回転と量の方向は、試料の純度が非常に重要です。綿棒の回転が制御されたせん断を作成するあなたの手の直線運動に反対方向にする必要があります。このせん断は、組織から内皮細胞を持ち上げるものです。力の適用量は、組織の抵抗を感じるのですが、基底膜を貫通しないように十分なはずです。 </li><li>時折、軽く先端繊維から細胞を取り除くために、コラゲナーゼ溶液内に綿棒。スワブが完了すると、内皮層の質感は以前より少しスムーズに感じるはず。 </li><li>細胞懸濁液/コラゲナーゼを収集し、新しい滅菌15mlのチューブに移す。 </li><li>ペレットへ5分の任意の単離された細胞を1000 rpmでチューブを遠心し、上清を吸引除去する。同様に間質細胞を単離する場合、これらの細胞を遠心分離されている間に、そのプロトコルを実行します。 </li><li> 、15 mLの試験管に内皮ブタの培地3 mlを添加し、もう一度遠心し、吸引メディア。この第二の遠心分離は、そのような先端繊維などの不要な物質のいくつかをフィルタするのに役立ちます。 </li><li>コラーゲンをプレコートT - 25フラスコ（遠心分離管あたり1フラスコを使用）で内皮ブタの培地とプレートの細胞を5mLで遠心分離した内皮細胞を再懸濁する。 </li><li>細胞は内皮培地を変更する前に、少なくとも2〜3日に成長しましょう​​。これは、細胞が回復し、分離プロセスはかなり厳しいですし、細胞の収率が低くなる可能性があるので分割するのに役立ちます。接触阻害が細胞形質転換につながる可能性があるので、それは合流の近くに継代細胞にとって非常に重要です。 </li></ol> 4。サイド特定のアイソレーション用の60 mmの培養皿の準備<ol><li>アルミ箔（leafleあたり2ガラスの皿とライン60 mmのガラスシャーレをt）を。アルミ箔は、ガラスシャーレのが、他のアイソレーションのために再利用できるように、パラフィンを除去するのに役立ちます。 </li><li>半分程度、料理中にパラフィンのビーズを配置し、オートクレーブ用カバー。 </li><li>オートクレーブが完了するとパラフィンは溶融になると、クールな平らな面に皿のアンサンブルを移動する。パラフィンは冷えると、それは硬くなりますし、針穿刺をサポートするレイヤーを作成します。 </li><li> 30分後、滅菌シャーレは、リーフレットの組織を固定するために隔離室として使用することができます。 </li></ol> 5。サイド特定の内皮層の分離<ol><li>側の特定の分離のための準備60ミリメートル培養皿上で15mlのチューブと場所からチラシを取り外します。 </li><li> ventricularis側が露出線維の側を離れるパラフィンの表面上に下向きになるようにリーフレットを操作する。内皮層を露出するリーフレットの端を固定。 </li><li>各（上向きに面した）内皮表面に冷たいコラゲナーゼの数滴を置き、37℃で5〜10分のための組織をインキュベート℃を</li><li>前と同じように、静かにリーフレットの表面に乾いた滅菌綿棒を回転させることで内皮層を取り除く。せん断応用の回転と量の方向は、試料の純度が非常に重要です。綿棒の回転が制御されたせん断を作成するあなたの手の直線運動に反対方向にする必要があります。このせん断は、組織と他には何から内皮細胞を持ち上げるものです。力の適用量は、組織の抵抗を感じるのではなく、組織内に浸透していない十分なはずです。 </li><li>時折、軽く先端繊維から細胞を取り除くために、コラゲナーゼ溶液内に綿棒。スワブが完了すると、内皮層の質感は以前より少しスムーズに感じるはず。 </li><li>細胞懸濁液/コラゲナーゼを収集し、新しい滅菌15mlのチューブに移す。ラベルのサイド特異性を示している（同じバルブからチラシを一緒にプールすることができます）。 </li><li> ventricularis側は現在公開され、繰り返し行ってください（5.3から5.6まで）されるように、新しい培養皿にリーフレットを転送する。 </li><li>一度にすべての細胞懸濁液/コラゲナーゼが収集され、任意の単離された細胞ペレットに5分間、1000 rpmでチューブを遠心し、上清を吸引除去する。同様に間質細胞を単離する場合、これらの細胞を遠心分離されている間に、そのプロトコルを実行します。 </li><li> 、チューブに内皮ブタの培地3 mlを添加し、もう一度遠心し、培地を吸引除去する。この第二の遠心分離は、そのような先端繊維などの不要な物質のいくつかをフィルタするのに役立ちます。 </li><li>コラージュをプレコートT - 25フラスコ（遠心分離管あたり1フラスコを使用）で内皮ブタの培地とプレートの細胞を5mLで遠心分離した内皮細胞を再懸濁する。 </li><li>細胞は内皮培地を変更する前に、少なくとも2〜3日に成長しましょう​​。これは、細胞が回復し、分離プロセスはかなり厳しいですので、分割するのに役立ちます。あなたが収率が極めて低いことに気付いた場合、細胞間接着は、細胞増殖を促進するので、小さなフラスコやプレートに移行することを検討。接触阻害が細胞形質転換につながる可能性があるため合流点近くに通過することを忘れないでください。 </li></ol> 6。間質細胞の単離<ol><li>バルブあたりのコラゲナーゼ溶液（3リーフレット）10mLで滅菌15 ml遠心チューブを埋める。 </li><li>内皮細胞のリーフレットを拭き取り後、すぐにコラゲナーゼ溶液を用いて適切な15mlのチューブに配置します。 </li><li> （必要に応じて軽く攪拌）約12〜18時間インキュベートする。 </li><li>細胞懸濁液/コラゲナーゼが均質化になるまで血清ピペットで軽く劣化した組織を混在させる。この均質化は、組織を分割し、間質細​​胞を放出するのに役立ちます。 </li><li> 1000 rpmで5分間消化組織を遠心し、上清を吸引除去する。 </li><li> 15mlの試験管に5 mLの間ブタの培地を追加し、もう一度遠心し、上清を吸引除去する。 </li><li>間質ブタ媒体と板T - 75フラスコ中の細胞（遠心分離管あたり1フラスコを使用する）5mLに遠心分離した内皮細胞を再懸濁する。 </li><li>細胞が間質細胞の培地を変更する前に少なくとも1〜2日に成長しましょう​​。期待されている内皮細胞が、よりもはるかに組織破片が存在します。細胞はまた、より高速なので、細胞収量とその性質の内皮細胞よりも合流して成長してください。 </li></ol> 7。代表的な画像<img src="/files/ftp_upload/2158/2158fig1.jpg" alt="図1" /> 図1 2〜3日の後の分離で単離された細胞の形態。 （A）VECは、典型的な内皮細胞形態とフォームの成長を促進するためのクラスタを示す。 （B）VICの形態は、一般に紡錘形、フラスコ全体に均等に分散している筋線維芽細胞に似ています。 <pクラス=&quot;jove_content&quot;&gt; <img src="/files/ftp_upload/2158/2158fig2.jpg" alt="図2" /> 図2。コンフルエントで単離された細胞の形態。 （A）VECは、一般的に石畳と成長の接触禁止されている典型的な内皮細胞形態を示す。 （B）VICの形態は、一般に紡錘形としない成長の接触禁止されている筋線維芽細胞に似ています。 <img src="/files/ftp_upload/2158/2158fig3.jpg" alt="図3" /> 図3。単離された細胞の機能特性6。 （）VECは、アセチル化LDLの取り込みの高いレベル（赤）に関連付けられています。 （B）VICは、アセチル化LDLの取り込みの低レベルに関連付けられています。 <img src="/files/ftp_upload/2158/2158fig4.jpg" alt="図4" /> 図4。単離された細胞の細胞マーカー6。 （A）VECの表現型は、von Willebrand因子（緑）、青（核）のための陽性染色に貢献しています。 （B）VICの表現型は、von Willebrand因子の負の染色に貢献しています。 <img src="/files/ftp_upload/2158/2158fig5.jpg" alt="図5" /> 図5。単離された細胞の細胞マーカー。 （A）VECの表現型は、αSMA（緑）、青（核）のためのネガティブ染色に貢献しています。 （B）VICの表現型は、アルファSMA陽性の染色に貢献しています。 <table border="1"><tbody><tr><td> 解離エージェント </td><td> 解離テクニック </td><td> 細胞コレクション </td><td> セルの数量 </td><td> セル 純度 </td><td> 汚染 </td></tr><tr><td>のCaCl 2のないEDTA（3MM） </td><td> 5、20、60分。 CaCl 2の添加前に</td><td> 20、60、120分。コレクションの前に</td><td> - </td><td> + / - </td><td> + + + </td></tr><tr><td>のCaCl 2のないEDTA（6MM） </td><td> 5、20、60分。 CaCl 2の添加前に</td><td> 20、60、120分。コレクションの前に</td><td> + / - </td><td> + </td><td> + + + </td></tr><tr><td>トリプシン- EDTA（0.5グラム/ L） </td><td> 5、10、15分。 非活性化の前に</td><td>培地は、直ちに収集</td><td> + </td><td> + </td><td> + + </td></tr><tr><td>コラゲナーゼII（300 U / mL）を</td><td> 5、10、15分。 非活性化の前に</td><td>培地は、直ちに収集</td><td> - 、+、+ + </td><td> - 、+ +、+ </td><td> + </td></tr><tr><td>コラゲナーゼII（600 U / mL）を</td><td> 5、10、15分。 非活性化の前に</td><td>培地は、直ちに収集</td><td> +、+ +、+ + + </td><td> + +、+ +、 - </td><td> - </td></tr></tbody></table> 表1。予備弁膜内皮細胞の分離プロトコルの結果。

<ol>
	<li>Thompson, R. P., Fitzharris, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+of+the+truncus+arteriosus+of+the+chick+embryo+heart:+the+formation+and+migration+of+mesenchymal+tissue.">Morphogenesis of the truncus arteriosus of the chick embryo heart: the formation and migration of mesenchymal tissue.</a> Am J Anat. 154, 545-556 (1979).</li><li>Johnson, C. M., Fass, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porcine+cardiac+valvular+endothelial+cells+in+culture:+A+relative+deficiency+of+fibronectin+synthesis+in+vitro.">Porcine cardiac valvular endothelial cells in culture: A relative deficiency of fibronectin synthesis in vitro.</a> Lab Invest. 49 (5), 589-598 (1983).</li><li>Manduteanu, I., Popov, D., Radu, A., Simionescu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calf+cardiac+valvular+endothelial+cells+in+culture:+production+of+glycosaminoglycans,+prostacyclin+and+fibronectin.">Calf cardiac valvular endothelial cells in culture: production of glycosaminoglycans, prostacyclin and fibronectin.</a> J Mol Cell Cardiol. 20 (2), 103-118 (1988).</li><li>Cheunyg, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+isolating+and+purifying+porcine+aortic+valve+endothelial+cells.">Techniques for isolating and purifying porcine aortic valve endothelial cells.</a> JHVD. 17 (6), 674-681 (2008).</li><li>Paranya, G., Vineberg, S., Dvorin, E., Kaushal, S., Roth, S. J., Rabkin, E., Schoen, F. J., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortic+valve+endothelial+cells+undergo+transforming+growth+factor-beta-mediated+and+non-transforming+growth+factor-beta-mediated+transdifferentiation+in+vitro.">Aortic valve endothelial cells undergo transforming growth factor-beta-mediated and non-transforming growth factor-beta-mediated transdifferentiation in vitro.</a> Am J Pathol. 159 (4), 1335-1343 (2001).</li><li>Butcher, J. T., Penrod, A., Garcia, A. J. M., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+morphology+and+focal+adhesion+development+of+valvular+endothelial+cells+in+static+and+fluid+flow+environments.">Unique morphology and focal adhesion development of valvular endothelial cells in static and fluid flow environments.</a> Arterioscler Thromb Vasc Bio. 24 (1), 1429-1434 (2004).</li><li>Simmons, C. A., Grant, G. R., Manduchi, E., Davies, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+heterogeneity+of+endothelial+phenotypes+correlates+with+side-specific+vulnerability+to+calcification+in+normal+porcine+aortic+valves.">Spatial heterogeneity of endothelial phenotypes correlates with side-specific vulnerability to calcification in normal porcine aortic valves.</a> Circ Res. 96, 792-799 (2005).</li><li>Butcher, J. T., Tressel, S., Johnson, T., Turner, D., Sorescu, G., Jo, H., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+Profiles+of+Valvular+and+Vascular+Endothelial+Cells+Reveal+Phenotypic+Differences:+Influence+of+Shear+Stress.">Transcriptional Profiles of Valvular and Vascular Endothelial Cells Reveal Phenotypic Differences: Influence of Shear Stress.</a> Arterioscler Thromb Vasc Biol. 26, 69-69 (2006).</li><li>Parachuri, S., Yang, J. H., Aikawa, E., Melero-Martin, J. M., Khan, Z. A., Loukogeorgakis, S., Schoen, F. J., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Pulmonary+Valve+Progenitor+Cells+Exhibit+Endothelial/Mesenchymal+Plasticity+in+Response+to+Vascular+Endothelial+Growth+Factor-A+and+Transforming+Growth+Factor-&#946;2.">Human Pulmonary Valve Progenitor Cells Exhibit Endothelial/Mesenchymal Plasticity in Response to Vascular Endothelial Growth Factor-A and Transforming Growth Factor-&#946;2.</a> Circ Res. 99 (8), 861-869 (2006).</li><li>Shi, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+circulating+bone+marrow-derived+endothelial+cells.">Evidence for circulating bone marrow-derived endothelial cells.</a> Blood. 92, 362-367 (1998).</li><li>Rehman, J., Li, J., Orschell, C. M., March, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+blood+endothelial+progenitor+cells+are+derived+from+monocyte/macrophages+and+secrete+angiogenic+growth+factors.">Peripheral blood endothelial progenitor cells are derived from monocyte/macrophages and secrete angiogenic growth factors.</a> Circulation. 107, 1164-1169 (2003).</li></ol>

利害の衝突は宣言されません。

弁膜生物学の理解は、弁内皮細胞の純粋な集団を分離して培養する技術的な問題によって損なわれている。典型的な分離方法は、基礎となる基礎行列または内皮接着剤の結合2,3の化学的解離の酵素消化を伴う。予備分離実験を定性的に解離剤と潜伏期間を変化させることによって評価した。これらの実験の結果は、最大60分の場合とEDTA（またはトリプシン- EDTA）インキュベーション?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dulbecco&#x2019;s Modified Eagle Medium</td>
<td>&nbsp;</td>
<td>Mediatech</td>
<td>50-103-PB</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>26140</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin Streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>0.25% Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25200</td>
<td>&nbsp;</td>
<tr>
<td>Heparin Sodium Salt</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>H4784-1G</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase Type 2</td>
<td>&nbsp;</td>
<td>Worthington Biochemical</td>
<td>LS004176</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>21300-058</td>
<td>&nbsp;</td>
<tr>
<td>Rat Tail Collagen</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354236</td>
<td>&nbsp;</td>
<tr>
<td>Critical Swabs</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89031-270</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>55761</td>
<td>&nbsp;</td>
<tr>
<td>T25 Flasks</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>353018</td>
<td>&nbsp;</td>
<tr>
<td>T75 Flasks</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>353136</td>
<td>&nbsp;</td>
<tr>
<td>24 Well Plate</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>353047</td>
<td>&nbsp;</td>
<tr>
<td>60x15 mm Dishes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>25384-092</td>
<td>&nbsp;</td>
<tr>
<td>60x15 Glass Dishes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89000-310</td>
<td>&nbsp;</td>
<tr>
<td>Paraffin Embedding Wax</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>19304-01</td>
<td>&nbsp;</td>
<tr>
<td>Precision Glide Needles</td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>305165</td>
<td>&nbsp;</td>
<tr>
<td>500 mL Nalgene Filters</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>73520-985</td>
<td>&nbsp;</td>
<tr>
<td>1L Nalgene Filters</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>73520-986</td>
<td>&nbsp;</td>
<tr>
<td>Tissue Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11023-15</td>
<td>&nbsp;</td>
<tr>
<td>FSC Tweezers #5</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11295-00</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation of valvular endothelial cells

心臓弁は、心臓血管系を介して単方向の血流を維持する責任を持つことになります。彼らは寿命にわたって数十億回も開いたり閉じたりするように、これらの薄い、繊維状の組織が重要な機械的ストレスにさらされる。これらの組織の驚くべき耐久性は、内皮常駐弁膜（VEC）と間質細胞によるものである（VIC）は、常に地元の機械的および生物学的信号に応答して、修理および改造。ごく最近我々 は in vitro の実験において重要な役割を果たしているため、これらの細胞のユニークな行動を、理解し始めている。特に困難は、VECの分離と文化です。特別な注意を払ってその組織が、最終的なメッキを介してホストから削除された瞬間から使用する必要があります。ここでは直接分離、側面の特定のアイソレーション、文化、およびVECの純粋な集団の検証のためのプロトコルを提示する。我々は表面の細胞を取り除くために穏やかな綿棒スクレイピング技術に続いて酵素消化を使用してください。これらの細胞をチューブに回収し、ペレットに遠心分離されています。ペレットを再懸濁し、コラーゲンI、マトリックスをプレコート培養フラスコに播種しています。 VECの表現型は、接触によって確認され成長し、血管内皮特異的などPECAM1（CD31）のようなマーカー、フォンヴィレブランド因子（vWF）、及びα-平滑筋アクチン（α- SMA）の負の発現の発現を阻害した。 VECの機能的特性は、アセチル化LDLの高レベルに関連付けられています。血管内皮細胞とは異なり、VECは通常、胚弁形成1の間に発生する間充織、に変換するユニークな能力を持っている。これはまた、in vitro培養における有意に延長後の合流時に発生する可能性がありますので、注意が合流点付近で通過するためになされるべきである。 VECの単離後、VICの純粋な集団は、簡単に取得することができます。

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

Isolation of Valvular Endothelial Cells

我々は、心臓弁内皮細胞（VEC）の純粋な集団を分離して培養する方法を提供する。 VECがカスプやリーフレットのいずれかの側から分離し、直ちに以下のことができる、基本的な間質細胞（VIC）の分離は簡単です。

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 ...

Research

JoVE Journal

Biology

20.2K ビュー.  Cornell University. 我々は、心臓弁内皮細胞（VEC）の純粋な集団を分離して培養する方法を提供する。 VECがカスプやリーフレットのいずれかの側から分離し、直ちに以下のことができる、基本的な間質細胞（VIC）の分離は簡単です。

ビデオ: Isolation of Valvular 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

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

分離と成人ヒト内皮コロニー形成前駆細胞の大規模拡張

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

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 Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

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 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 and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

ミニチュアブタからの原発性大動脈内皮細胞の単離と培養

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

マウス初代肝臓正弦波内皮細胞の単離と特性評価

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...