Name: a cell culture model of resistance arteries
Uploaded: 2017-09-08T12:00:00.000+00:00
Duration: 10 min 54 s
Description: Watch this Scientific Journal Video about A Cell Culture Model of Resistance Arteries at JoVE.com

この作品は、各国用の中心によって支えられた、肺、血液研究所 R01088554、T32HL007284、アメリカの心臓協会の交わりを経て 14PRE20420024 を付与します。我々 は特に、聖ジュード細胞イメージング共有研究コア、ランドール ウェイク フィールドと電子顕微鏡画像のリンダ ・ ホーナー、アダム ストラウブについては代表的な蛍光画像彼女の地下鉄 Bagher などを感謝します。原稿プロトコルに関する貴重なフィードバック。

1.、VCCC のめっきセル <ol><li>文化大 225 cm 2 のフラスコ EC と SMC 70-90% 合流までの 37 ° C で滅菌条件の下で。SMC よりより多くの EC が使用されていることを確認します。プライマリ人間 EC と SMC、SMC の 1 大規模なフラスコに EC の通常 3 の大きなフラスコを使用して場合。
	<ol><li>下通路で一次電池を使用して、過去の 10 の通路を使用しないでください。共培養する細胞に基づく文化メディアを選択します。主なひと冠動脈 EC、EGM2 MV 成長因子と EBM 2 EC 基底メディアを使用します。プライマリのひと冠動脈 SMC SmGM 2 成長因子と SmBM SMC 基底メディアを使用します。培養細胞で使用する前に、メディアに成長因子を追加します</li>。
	</ol></li> <li>VCCC 日 1 の構築: 消毒剤 (例えば Cavicide) と大 (150 mm) ペトリ皿と蓋をスプレーし、ペーパー タオルや糸くずのおしりふきで拭いて。70% エタノール (エタノール) ペトリ皿をスプレーし、乾燥空気にフードにそれらを置く</li>。
	<li>は無菌条件下でフィルター挿入 (材料の表 を参照してください) のプレートを開き、フィブロネクチン溶液で挿入の SMC 側 (フィルターの底側) をコートします。提供 6 ウェル プレートの挿入を維持する、ピペット、フィブロネクチン溶液側フィルターの半分は下カバー (1 mL 以上) であることを確かめて、プレートの底にスリットします</li>。
	<li>細胞文化フードでは、室温でフィブロネクチン溶液の後 30 分は、過剰なソリューションや反転、きれいなペトリ皿の半分を下部に配置する真空プレートから挿入を取る。脇を設定します。余分な溶液を吸引するときにフィルター挿入膜を邪魔しないようにこれはプレートの傾斜および挿入の端からソリューションを掃除機をゆっくりによって回避できます</li>。
	SMC のセルが、プレートを持ち上げた後のあらかじめ加温の 2 X トリプシン-EDTA 溶液 3 ml Trypsinize 225 cm 2 フラスコを <li>は、トリプシン (3:1、メディア: トリプシン) を中和するために 9 mL SMC メディアを追加します。円錐管に転送、まあと携帯番号を確認する検定にピペット 10 μ L をミックスします。
	<ol><li>5 × 5 のセルの個数のカウント ボックス (すなわち 18)、掛けて 10,000 1 mL (180,000) 内のセルの数を取得します。12 mL (216 万 SMC 細胞懸濁液の容量) を乗算します。十分な SMC プレートに井戸の数があることを確認します 。
		注: たとえば、1 よくべきである 75,000 SMC を有し従って 18 井戸だろう 135 万合計 SMC。1 mL あたり 180,000 細胞によって必要な 135 万セルを分割してめっきに必要となる細胞の懸濁液の 7.5 mL でこの結果します。必要な最終巻を計算 (18 井戸 x 750 μ L = 13.5 mL)。したがって、細胞懸濁液の 7.5 mL を取る、ボリュームをもたらすに予め温めておいた SMC メディアとよく混ぜるまで 13.5 mL</li>。
	</ol></li> <li>セル ・ メディア ソリューションのいずれかをこぼさないように注意しながら各フィルターの下側に約 75,000 SMC を含む細胞懸濁液のプレート 750 μ L。上ふたを閉めて一晩 37 ° C の定温器に挿入でシャーレを慎重に転送します 。
	注: 他の細胞型の最適なセル密度が経験的に決定される必要があります</li>。
	<li>2 日目、2 ml の新鮮な予め温めておいた SMC メディアのきれいな 6 よく料理を埋めます。インキュベーターからプレートを削除します。1 つずつ挿入を取ると吸引、メディアを削除し、SMC メディア 6 よく皿に配置します</li>。
	<li>37 で少なくとも 30 分間 0.5% 牛ゼラチン溶液の 1 ml フィルター (EC 側) の反対側のコート ° C</li>
	<li>Trypsinize 3 ml に予め温めておいた 2 の EC の 225 cm 2 flask(s) X トリプシン-EDTA (10 倍希釈滅菌ダルベッコの &#39; s PBS) ソリューション、プレートから細胞を持ち上げるためにフラスコの穏やかなタップで 。
	注: 場合によっては、フラスコを 37 ° C で 1 〜 3 分のためのインキュベーターに配置する必要があります。
	トリプシン (3:1) を中和するために 9 mL EC メディアを追加 <ol><li>一度細胞が、プレートを持ち上げた。円錐管、プールに一緒に転送、まあと携帯番号を確認する検定にピペット 10 μ L をミックスします。
		<ol><li>5 × 5 のセルの個数のカウント ボックス (すなわち 60)、掛けて 10,000 1 mL (600,000) 内のセルの数を取得します。12 mL (720 万 EC 細胞懸濁液の容量) を乗算します。十分な EC プレートに井戸の希望の番号があることを確認します 。
			注: 約 360,000 EC は、ウェルあたり必要があります。たとえば、18 の井戸は 648 万理事会を必要と1 mL あたり 600,000 EC に必要な 648 万セルを分割し、10.8 mL めっきに必要となる細胞の懸濁液のこの結果します。必要な最終巻を計算 (18 の井戸 1 mL = 18 mL x)。したがって、細胞懸濁液の 10.8 mL を取る、最大ボリュームをもたらす 18 mL EC メディアで加温し、優しく再懸濁します</li>。
		</ol></li></ol></li> <li>1 mL 各挿入フィルターの上側に 360,000 EC のプレートします。4 日目に 24 時間交換媒体のための 37 ° C で邪魔されずインキュベート細胞ができます。5 日目で収穫</li>。
</ol> 2。収穫、VCCC から分数 <ol><li>4 ° C の部屋の分離を行い氷のすべての楽器を維持</li>
	<li>は隔離される個々 の挿入数を留意し、MEJ (分離フィルター) のラベル 50 mL チューブに換散バッファーを追加します。その後、ラベル 2 つの 10 mm の料理;EC と smc</li>。
	<li>調整方法集中ライセートする必要があります; 15 挿入によっては換散バッファーのボリューム、MEJ 管の換散バッファーを 500 μ l 添加し、シャーレあたり 250 μ L をお勧めします</li>。
	<li>作業時間での挿入 1 つ 6 ウェル プレートで。細胞文化フードとし、氷の冷たい部屋への輸送中に吸引 
	メモ: 次の手順は、1 挿入分離します</li>。
	<li>ピペット 10 μ L の挿入の SMC 側に 1 × PBS</li>。
	<li>こすりに挿入の一方の端のセルをセル リフターを使用し、換散バッファーの品詞がある SMC ペトリ皿にリフターからセルを転送</li>
	<li>セル リフターは、皿の中の換散バッファーに触れた後はそれまで完全に一掃し、ペーパー タオルで乾燥した挿入フィルターに触れることを許可しません。スクレイピング SMC フィルターの少なくとも 1 ~ 2 回残りの SMC 細胞の残骸を完全に削除するを繰り返します</li>。
	<li>は、挿入フィルターの上部/EC 側に上の挿入および 1x PBS のピペット 10 μ L を回します。2.6 および 2.7 細胞スクレーパーと EC のペトリ皿の手順を繰り返します</li>。
	<li>は、換散バッファーと EC ペトリ皿に、ピペットと転送フィルターの EC 側から残りのセルと PBS スラリーを収集します。MEJ 分数で EC/SMC 混入を最小限にしてフィルターの両側から細胞をこするの非常に徹底的な</li>。
	<li>は、メスを使って慎重にプラスチック製の挿入からフィルターをカットします。プラスチックからそれの切断 70-80% がこれを行うし、鉗子を使用して、プラスチック製の挿入構造完全にフィルターを引き出します。換散バッファー付け MEJ 50 mL の円錐管にフィルターをかけるし、それは換散バッファーに常駐し、ウェットのままを確認します</li>。
	<li>の挿入をすべてが処理されるまで、6 のグループで、分離手順を繰り返します</li>。
	<li>の個別のチューブや料理別の治療グループを維持します</li>。
	<li>渦 15 の完全な強さの 50 mL MEJ 円錐管 s またはよく混合されるまで。鉗子、管にタンパク質と液体の最大量を残す、チューブの側の壁に沿ってドラッグすることで円錐 MEJ からフィルターを削除します。このポイントの後、フィルターを破棄します</li>。
	<li>MEJ 円錐管からすべてのフィルターを取り外した後は、チューブの底にすべての液体とタンパク質をプルする遠心分離機のクイック スピン (最大速度 〜 16,000 x g で 10-15 秒)。独立した小さい遠心チューブに MEJ チューブと EC と SMC ペトリネットの料理の内容を転送します</li>。
	<li>オプション: MEJ/SMC/EC チューブの内容を超音波氷上 4 3 10 s のパルスを設定または手で優しくホモジナイズしてください</li>。
	<li>4時 15 分最大速度 (~ 16,000 x g) 遠心 ° C. ピペット上清しビシンコニン アッセイ法を用いたタンパク質含有のライセート アッセイします</li>。
</ol> 3。アン顔 蛍光の収穫をして、VCCC <ol><li>日 5 の VCCC の孵化はインキュベーターからドライブベイ カバーを取り外します。各 1 × PBS で 2 回すすぎ挿入フィルターの下の井戸から SMC メディア オフ吸引細胞文化フードで作業している。EC 側と繰り返し</li>。
	<li>穏やかなシェーカーでウォークイン 4 ° C の部屋で 10 ~ 20 分 4% パラホルムアルデヒド (PFA) を追加そのままに、プレートの挿入を維持します。5 分の 1 × PBS で挿入フィルターの両側を洗って、2 回繰り返します 。
	注意: PFA は毒性があり慎重に処理する必要があります</li>。
	<li>プレート、抗体でフィルターの両方の側面を保つ + ソリューションをブロックでブロック、第一次および二次抗体の孵化を実行 (9 mL PBS、25 μ L トリトン X100、50 mg ウシ血清アルブミン、血清 500 μ L)</li>
	<li>メスとプラスチック製の挿入からフィルターをカット、スライドにフィルターをマウントするマウント ハンドルとメディアをマウントを顕微鏡スライド上の場所</li>。
</ol> 4。横の蛍光の収穫をして、VCCC <ol><li>日 5 の VCCC の孵化はインキュベーターからドライブベイ カバーを取り外します。細胞文化フード挿入フィルターの両側からメディアを吸引し、1x PBS で 2 回フィルターの各側面をすすいでください</li>。
	<li>そのまま 6 ウェル プレートの挿入を維持追加 4 %pfa 穏やかなシェーカーのウォークイン 4 ° C の部屋で一晩インキュベートと 。
	注意: PFA は毒性があり慎重に処理する必要があります</li>。
</ol> 5。埋め込み、VCCC 横の蛍光 <ol><li>フィルター後の挿入は、およそ 4% で 24 h のため修正されている PFA、70% への転送 EtOH、少なくとも 24 時間は数週間まで</li>。
	<li>は、自動化されたティッシュ プロセッサ上で実行 (一晩) 長い上フィルターを処理します。プログラムを使用して、次のように: 70% EtOH 30 分、85% エタノール 30 分、95% エタノール 30 分、100% エタノール 30 分、100% エタノール 45 分、100% エタノール 45 分、キシレン 30 分、キシレン 30 分、キシレン 45 分、60 ° C のパラフィン 30 分、60 ° C パラフィン 30 分、60 ° C パラフィン 45 分</li>
	<li>処理後、収穫されたフィルターを埋め込み駅流動パラフィン、カセット フィルターを維持するのに転送します</li>。
	<li>は、慎重に鉗子を使用して端にちょうどそれを保持するために、カセットからフィルターを削除します。ハサミで半分にカット フィルター 2 つの半円形の半分の形を形成します。液体パラフィンが付いて金型埋め込みを埋めるのに十分な長いだけ埋め込み駅のコールド プレート部分に 2 の半分の各レイアウトします</li>。
	こうと、断面、中に 1 つ切断されるエッジに向かってフィルターの中心から <li>一度流動パラフィンで満ちている非常に簡単にコールド プレートに埋め込み型に触れる、フィルターの各半分埋め込む埋め込み型 (側をカット);.</li>
	<li>埋め込み、中にパラフィンが単独でする彼らのために十分に涼しいまでお互いに落ちるから半分を防ぐためにフィルターを保持するために鉗子を使用します。完全に固化するまでのコールド プレートに埋め込み型に移動します 。
	注: 製氷皿上のブロックを冷却しわセクションを防ぐことができます</li>。
</ol> 6。区分および横の蛍光染色、VCCC <ol><li>パラフィン ブロックの区分およびマウント スライド: 5 μ m のセクションをカット、42 ° C の水浴にセクションを配置および正荷電を使用してセクションを拾うスライド。乾燥する 42 ° C のオーブンで一晩のスライドを維持します</li>。
	<li>復元手順を開始する前にパラフィンを溶かすための 60 ° C でスライドを焼くし、スライドの VCCC セクションの付着を助けます。簡単に言えば、冷却し、キシレン、100% の 2 つの変更をスライドを水分補給 95% エタノール エタノール、70% の 1 つの変更 EtOH との 2 つの変更蒸留水します</li>。
	<li>マイクロ波またはスライドの外セクションが落ちることがあります暖房を含む抗原検索方法を避けてください</li>。
	<li>実行は 4 ° C で一次抗体と一晩インキュベートし、常温で 1 h の標準ブロック常温洗浄の手順と 1 h の二次抗体が続きます。メディアをマウントと coverslip マウントします</li>。
</ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+expression+of+an+Ins(1,4,5)P3+receptor+at+the+myoendothelial+junction+selectively+regulates+heterocellular+Ca2++communication.">Localized expression of an Ins(1,4,5)P3 receptor at the myoendothelial junction selectively regulates heterocellular Ca2+ communication.</a> J Cell Sci. 121 (Pt 21), 3664-3673 (2008).</li><li>Axel, D. I., 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=Induction+of+cell-rich+and+lipid-rich+plaques+in+a+transfilter+coculture+system+with+human+vascular+cells.">Induction of cell-rich and lipid-rich plaques in a transfilter coculture system with human vascular cells.</a> J Vasc Res. 33 (4), 327-339 (1996).</li><li>Hastings, N. E., Simmers, M. B., McDonald, O. G., Wamhoff, B. R., Blackman, B. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+injury+induces+vascular+smooth+muscle+cell+proliferation+in+highly+localized+regions+of+a+direct+contact+co-culture+system.">Endothelial injury induces vascular smooth muscle cell proliferation in highly localized regions of a direct contact co-culture system.</a> Cell Biochem Biophys. 52 (1), 37-46 (2008).</li><li>Scott-Drechsel, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+flow+co-culture+system+for+studying+mechanobiology+effects+of+pulse+flow+waves.">A new flow co-culture system for studying mechanobiology effects of pulse flow waves.</a> Cytotechnology. 64 (6), 649-666 (2012).</li><li>van Buul-Wortelboer, M. F., 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=Reconstitution+of+the+vascular+wall+in+vitro.+A+novel+model+to+study+interactions+between+endothelial+and+smooth+muscle+cells.">Reconstitution of the vascular wall in vitro. A novel model to study interactions between endothelial and smooth muscle cells.</a> Exp Cell Res. 162 (1), 151-158 (1986).</li><li>Wilhelm, I., Fazakas, C., Krizbai, I. A. <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+models+of+the+blood-brain+barrier.">In vitro models of the blood-brain barrier.</a> Acta Neurobiol Exp (Wars). 71 (1), 113-128 (2011).</li><li>Heberlein, K. R., 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+novel+mRNA+binding+protein+complex+promotes+localized+plasminogen+activator+inhibitor-1+accumulation+at+the+myoendothelial+junction.">A novel mRNA binding protein complex promotes localized plasminogen activator inhibitor-1 accumulation at the myoendothelial junction.</a> Arterioscler Thromb Vasc Biol. 32 (5), 1271-1279 (2012).</li><li>Straub, A. C., 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=Compartmentalized+connexin+43+s-nitrosylation/denitrosylation+regulates+heterocellular+communication+in+the+vessel+wall.">Compartmentalized connexin 43 s-nitrosylation/denitrosylation regulates heterocellular communication in the vessel wall.</a> Arterioscler Thromb Vasc Biol. 31 (2), 399-407 (2011).</li><li>Cucina, A., 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=Vascular+endothelial+growth+factor+increases+the+migration+and+proliferation+of+smooth+muscle+cells+through+the+mediation+of+growth+factors+released+by+endothelial+cells.">Vascular endothelial growth factor increases the migration and proliferation of smooth muscle cells through the mediation of growth factors released by endothelial cells.</a> J Surg Res. 109 (1), 16-23 (2003).</li><li>Heberlein, K. R., 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=Plasminogen+activator+inhibitor-1+regulates+myoendothelial+junction+formation.">Plasminogen activator inhibitor-1 regulates myoendothelial junction formation.</a> Circ Res. 106 (6), 1092-1102 (2010).</li><li>Isakson, B. E., Best, A. K., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+protein+on+actin+bridges+between+endothelium+and+smooth+muscle+in+arterioles+demonstrates+heterogeneous+connexin+expression+and+phosphorylation.">Incidence of protein on actin bridges between endothelium and smooth muscle in arterioles demonstrates heterogeneous connexin expression and phosphorylation.</a> Am J Physiol Heart Circ Physiol. 294 (6), H2898-H2904 (2008).</li><li>Biwer, L. A., Isakson, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum-mediated+signalling+in+cellular+microdomains.">Endoplasmic reticulum-mediated signalling in cellular microdomains.</a> Acta Physiol (Oxf). 219 (1), 162-175 (2017).</li></ol>

著者が明らかに何もありません。

VCCC は MEJsの in vitro、さたの面で利点の数が特定のアプリケーションのため、VCCC を利用できるかどうかを決定する際の議論のポイントがあります。たとえば、異なる種類の細胞をこのモデルで使用される場合は、最適化するそれ必要があります。ひと初代細胞を用いてウシ大動脈24、または野生型やノックアウト マウスから細胞を分離し、VCCC1,</...

小径抵抗血管の薄い内弾性板 (IEL) は、内皮細胞 (EC) と平滑筋細胞 (SMC) を分離します。セルはこの弾性マトリックスの穴を介して投影し、ギャップ結合チャネル1,2経由でダイレクトに細胞質接続できます。このユニークな構造は、myoendothelial のジャンクション (MEJ) と呼ばれます。MEJ です小 (血管床によって、0.5 μ m x 約 0.5 μ m)、細胞突起主に内皮細胞から成る3,4同様の平滑筋から発生させてよい。調査官の数はシグナル伝達 MEJ、内皮細胞と平滑筋5 間双方向のシグナル化を促進するため特に重要な場所、それはレンダリングに選択的に発生するネットワークの巨大な複雑さを示しています。 ,6,7,8,9,10。
ただし、そのまま動脈、MEJ のシグナル伝達経路の機構的郭清はむずかしい。MEJ から細胞の投影こそ体内MEJ 血管壁から分離することは現在はないです。このため、VCCC モデル1が開発されました。重要なは、VCCC 複製生理的血管内皮形態11 ・頂間の信号の偏波とセル12MEJ 部分。アルファのヘモグロビンは、血管内皮細胞、MEJ に偏光の発見を容易にするこのユニークなモデルだった。これはアルファのヘモグロビン13を表現しない従来培養内皮細胞とは対照的です。微小血管内皮細胞に興味がある研究者は、特に小径抵抗血管で発生するシグナル伝達経路を分析することが目的の場合、VCCC で培養した血管内皮細胞を使用するより適切な場合があります。
2 つ共培養細胞型に小径孔 (直径 0.4 μ m) を持つフィルターを含む頑丈なプラスチック製の挿入を使用すると、セルで層間の移行できなくなります。それはin vivoよりはるかに時間がまだ MEJs、蛋白質のローカリゼーション、シグナルの1二次メッセンジャーなどの生体内の特性の多くを複製セル間の 10 μ m 間隔の結果します。,14します。 さらに、、VCCC セル アゴニストまたは特定の細胞内コンパートメントに拮抗薬の追加を介して信号の種類に固有のターゲットにできます。たとえば、カルシウムをキレート化するためこれら AM で EC を読み込みとフェニレフリン14SMC を刺激します。これにより、共培養モデル15,16,17,18,19,20,21の他の記述と対照をなしてmRNA および蛋白質、分数を含む、異なる MEJ フィルター孔内の異なる画分を分離することについて説明します。この手法、VCCC を添加できます蛋白質活動12蛋白質のリン酸化の12,23, や転写22mRNA の局在の変化の具体的な調査。異なる立体構造11,18,242 つの細胞の種類の培養が可能だが、底部には、フィルターと SMC の上に EC のめっきが、この資料に記載されます。

<table><tbody><tr><td>24 mm diameter, 0.4 &amp;micro;m Transwell Polyester membrane filter insert</td><td>Corning</td><td>3450</td><td>This is one 6 well plate of inserts</td></tr><tr><td>225 cm flask</td><td>Corning</td><td>431082</td><td></td></tr><tr><td>6 well plates (without filter inserts)</td><td>Corning</td><td>3506</td><td></td></tr><tr><td>Primary human coronary artery endothelial cells</td><td>Lonza</td><td>CC-2585</td><td></td></tr><tr><td>Primary human coronary artery smooth muscle cells</td><td>Lonza</td><td>CC-2583</td><td></td></tr><tr><td>EBM-2 EC Basal media</td><td>Lonza</td><td>CC-3156</td><td></td></tr><tr><td>EC growth factors (EGM2 MV Single quots)</td><td>Lonza</td><td>CC-4147</td><td>Add to basal media before use</td></tr><tr><td>SmBM SMC basal media</td><td>Lonza</td><td>CC-3181</td><td></td></tr><tr><td>SMC growth factors (SmGM-2 SingleQuot)</td><td>Lonza</td><td>CC-4149</td><td>Add to basal media before use</td></tr><tr><td>0.5% Trypsin-EDTA 10X</td><td>Gibco</td><td>15400-054</td><td>Dilute to 2X with sterile dPBS</td></tr><tr><td>Hemocytometer</td><td>VWR</td><td>15170-208</td><td></td></tr><tr><td>large petri dishes for SMC Plating (150mm)</td><td>Corning</td><td>353025</td><td></td></tr><tr><td>Bovine gelatin</td><td>Sigma</td><td>G-9382</td><td></td></tr><tr><td>Human fibronectin</td><td>Corning</td><td>356008</td><td>5&amp;micro;g/ml of fibronectin and store at -20</td></tr><tr><td>10x Hanks&#039; Buffered salt solution (HBSS)</td><td>Gibco</td><td>14065-056</td><td>Dilute to 1x with sterile water</td></tr><tr><td>10x Dulbecco&#039;s Phosphate buffered saline (dPBS)</td><td>Gibco</td><td>14200-075</td><td>Dilute to 1x with sterile water</td></tr><tr><td>Disposable Curved Scalpel (30mm cutting edge) with handle</td><td>Amazon</td><td>MDS15210</td><td></td></tr><tr><td>Forceps</td><td>Fisher</td><td>17-467-201</td><td></td></tr><tr><td>Cell lifter</td><td>Corning</td><td>3008</td><td></td></tr><tr><td>Cell scraper</td><td>BD Falcon</td><td>353085</td><td></td></tr><tr><td>50 mL conical tube</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>10 mm petri dishes</td><td>Falcon</td><td>35-1008</td><td></td></tr><tr><td>Rneasy mini kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>RNA lysis reagent</td><td>Qiagen</td><td>79306</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>158127</td><td>Make 4% solution with dPBS</td></tr><tr><td>70% ethanol</td><td>Fisher</td><td>04-355-305</td><td></td></tr><tr><td>Cavicide disinfectant</td><td>Fisher</td><td>131000</td><td></td></tr><tr><td>RIPA protein lysis buffer</td><td>Sigma</td><td>R0278</td><td></td></tr><tr><td>Sonic dismembranator</td><td>Fisher</td><td>Model 50</td><td></td></tr><tr><td>1x PBS for harvesting EC/SMC and immunocytochemistry</td><td>Gibco</td><td>10010023</td><td>does not need to be sterile</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Embedding/Sectioning/Staining&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Paraffin</td><td>Fisher</td><td>P31-500</td><td></td></tr><tr><td>Automated tissue processer</td><td>Excelsior</td><td>ES</td><td></td></tr><tr><td>Embedding station + cold plate</td><td>Leica</td><td>HistoCore Arcadia</td><td></td></tr><tr><td>Tissue Tek Accu-Edge Microtome blade</td><td>VWR</td><td>25608-961 or 25608-964</td><td></td></tr><tr><td>Microscope slides</td><td>Thermo Fisher</td><td>22-230-900</td><td></td></tr><tr><td>Coverslips</td><td>Thermo Fisher</td><td>12-548-5E</td><td></td></tr><tr><td>Prolong Gold mounting medium</td><td>Thermo Fisher</td><td>P36931</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Other Solutions&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;**Perform all steps in sterile conditions if using the solution on live cells&lt;/strong&gt;</td></tr><tr><td>Sterile dPBS</td><td></td><td></td><td>Autoclave water to sterilize. Using the sterile water make DPBS (450mL sterile water: 50mL 10X DPBS), mix and filter sterilize (0.2&amp;micro;m filter).&lt;br/&gt; Final concentration: 1X</td></tr><tr><td>sterile HBSS</td><td></td><td></td><td>Autoclave water to sterilize. Using the sterile water make HBSS (450mL sterile water: 50mL 10X HBSS), mix and filter sterilize (0.2&amp;micro;m filter).&lt;br/&gt; Final concentration: 1X</td></tr><tr><td>Fibronectin stock solution</td><td></td><td></td><td>Use 50mL of sterile water to fibronectin to make stock of 1mL aliquots, then freeze until needed&lt;br/&gt; Final concentration: 100&amp;micro;g/mL</td></tr><tr><td>Fibronectin to coat SMC side of VCCC</td><td></td><td></td><td>thaw one aliquot and add 1mL of fibronectin (100&amp;micro;g/mL) to 19mLs of 1X HBSS to reach a final concentration of 5&amp;micro;g/mL.&lt;br/&gt; Final concentration: 5&amp;micro;g/mL</td></tr><tr><td>2x Trypsin EDTA solution</td><td></td><td></td><td>Dilute the 10X 0.5% Trypsin-EDTA to 2X with 1X dPBS (40mL DPBS: 10mL 10X Trypsin) .&lt;br/&gt; Final concentration: 2X</td></tr><tr><td>Blocking/antibody solution</td><td></td><td></td><td>(9mL 1X PBS, 25mL Triton X100, 50mg bovine serum albumin, 500mL normal serum)</td></tr><tr><td>Bovine gelatin to coat EC VCCC</td><td></td><td></td><td>Mix 0.5g bovine gelatin into 100mL distilled water. Autoclave prior to first use and store at room temperature. Keep in sterile conditions.&lt;br/&gt; Final concentration: 0.50%</td></tr><tr><td>1x PBS for harvesting cells</td><td></td><td></td><td>does not need to be sterile</td></tr></tbody></table>

a cell culture model of resistance arteries

Myoendothelial ジャンクション (MEJ) 小径抵抗血管でユニークなシグナリングの創製は、血管緊張や血圧を制御することができますシグナリング プロセス特定の蛋白質の局在を展示します。内皮細胞や平滑筋細胞からの投影は、その小型のためサイズ (〜 1 μ m2の面積平均)、上、MEJ と単独で勉強することは困難です。ただし、我々 は MEJ 形成体外、内皮細胞の極性形成とシグナル伝達タンパク質と抵抗の血管壁内のプロセスの郭清では、血管細胞共培養 (VCCC) と呼ばれる細胞培養モデルを開発しました。動脈。VCCC 多数のアプリケーションには、異なる種類の細胞に合わせて適応することができます。モデルは、0.4 μ m 孔体外でMEJs することができますフォームをフィルターの両側に成長して 2 種類の細胞で構成されています。ここでセルのめっきと内皮細胞の分離を介して VCCC を作成する方法について述べる MEJ、および蛋白質の隔離や活動のため使用することができます平滑筋分数の試金。埋め込み、および免疫蛍光分析用断面、そのまま細胞層でフィルターを固定できます。重要なは、このモデルからの発見の多くは生理学的な関連性を強調し、そのまま抵抗動脈を使用して確認されています。

VCCC 用フィルター挿入が小、0.4 μ m 穴。図 1 a、VCCC フィルター挿入の正面ビューは、毛穴、MEJ が生体外で形成できますを示しています。回路図は、一日前に、内皮細胞を反対側 (図 1 b) めっきフィルターの下側にメッキの平滑筋細胞を示しています。セルはメッキ後、EC、MEJ、および SMC の成分は分離の 3 日後?...

Watch this Scientific Journal Video about A Cell Culture Model of Resistance Arteries at JoVE.com

A Cell Culture Model of Resistance Arteries

抵抗血管の細胞培養モデルを説明することで、シグナル伝達経路の内皮細胞、平滑筋や内皮細胞と平滑筋 (myoendothelial ジャンクション) 間の解剖を可能にします。アゴニストまたは蛋白質の隔離、電子顕微鏡、蛍光抗体の選択的なアプリケーションは、この細胞培養モデルを使用して利用できます。

抵抗血管の細胞培養モデル

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...

a-cell-culture-model-of-resistance-arteries

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Medicine

7.8K ビュー.  University of Virginia School of Medicine. 抵抗血管の細胞培養モデルを説明することで、シグナル伝達経路の内皮細胞、平滑筋や内皮細胞と平滑筋 (myoendothelial ジャンクション) 間の解剖を可能にします。アゴニストまたは蛋白質の隔離、電子顕微鏡、蛍光抗体の選択的なアプリケーションは、この細胞培養モデルを使用して利用できます。

ビデオ: A Cell Culture Model of Resistance Arteries

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

血管細胞リモデリングの研究への応用生理学的および病理学的血流のインビトロモデル

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

生物工学

Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations and development of microstructurally motivated mathematical models for understanding the mechanical properties of human resistance arteries in health and disease have the potential to aid understanding how disease and medical treatments affect the human microcirculation. To develop these mathematical models, it is essential to decipher the relationship between the mechanical and microarchitectural properties of the microvascular wall. In this work, we describe an ex vivo method for passive mechanical testing and simultaneous label-free three-dimensional imaging of the microarchitecture of elastin and collagen in the arterial wall of isolated human resistance arteries. The imaging protocol can be applied to resistance arteries of any species of interest. Image analyses are described for quantifying i) pressure-induced changes in internal elastic lamina branching angles and adventitial collagen straightness using Fiji and ii) collagen and elastin volume densities determined using Ilastik software. Preferably all mechanical and imaging measurements are performed on live, perfused arteries, however, an alternative approach using standard video-microscopy pressure myography in combination with post-fixation imaging of re-pressurized vessels is discussed. This alternative method provides users with different options for analysis approaches. The inclusion of the mechanical and imaging data in mathematical models of the arterial wall mechanics is discussed, and future development and additions to the protocol are proposed.

We describe simultaneous mechanical testing and 3D-imaging of the arterial wall of isolated, live human resistance arteries, and Fiji and Ilastik image analyses for the quantification of elastin and collagen spatial organization and volume densities. We discuss the use of these data in mathematical models of arterial wall mechanics.

ラベル無料蛍光顕微鏡によるライブ、人間の抵抗血管のコラーゲンとエラスチンの圧力依存性アーキテクチャを評価します。

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations ...

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

血管平滑筋細胞と大動脈石灰化と炎症のイメージングの石灰化

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

医学

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of various important processes in the cardiovascular system. Recently, although several biological studies have been performed using such 3D models of human arteries, they have not been applied to study vascular targeting. This paper presents a new method to fabricate real-sized, reconstructed human arterial models using a 3D printing technique, line them with human endothelial cells (ECs), and study particle targeting under physiological flow. These models have the advantage of replicating the physiological size and conditions of blood vessels in the human body using low-cost components. This technique may serve as a new platform for studying and understanding drug targeting in the cardiovascular system and may improve the design of new injectable nanomedicines. Moreover, the presented approach may provide significant tools for the study of targeted delivery of different agents for cardiovascular diseases under patient-specific flow and physiological conditions.

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

Here, we present a new protocol to study and map the targeted deposition of drug carriers to endothelial cells in fabricated, real-sized, three-dimensional human artery models under physiological flow. The presented method may serve as a new platform for targeting drug carriers within the vascular system.

流れの下で標的とする血管薬を研究するためのin Vitro 3D細胞培養動脈モデル

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

分離抵抗性ラットの動脈のビデオ顕微鏡を用いた血管制御機構の評価

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection (TAAD). However, human TAAD sometimes cannot be characterized by animal models. There is an apparent species gap between clinical human studies and animal experiments that may hinder the discovery of therapeutic drugs. In contrast, the conventional cell culture model is unable to simulate in vivo biomechanical stimuli. To this end, microfabrication and microfluidic techniques have developed greatly in recent years, providing novel techniques for establishing organoids-on-a-chip models that replicate the biomechanical microenvironment. In this study, a human aorta smooth muscle cell organ-on-a-chip (HASMC-OOC) model was developed to simulate the pathophysiological parameters of aortic biomechanics, including the amplitude and frequency of cyclic strain experienced by human aortic smooth muscle cells (HASMCs) that play a vital role in TAAD. In this model, the morphology of HASMCs became elongated in shape, aligned perpendicularly to the strain direction, and presented a more contractile phenotype under strain conditions than under static conventional conditions. This was consistent with the cell orientation and phenotype in native human aortic walls. Additionally, using bicuspid aortic valve-related TAAD (BAV-TAAD) and tricuspid aortic valve-related TAAD (TAV-TAAD) patient-derived primary HASMCs, we established BAV-TAAD and TAV-TAAD disease models, which replicate HASMC characteristics in TAAD. The HASMC-OOC model provides a novel in vitro platform that is complementary to animal models for further exploring the pathogenesis of TAAD and discovering therapeutic targets.

Here, we developed a human aorta smooth muscle cell organ-on-a-chip model to replicate the in vivo biomechanical strain of smooth muscle cells in the human aortic wall.

大動脈壁における生体力学的ひずみを再現するためのヒト大動脈平滑筋細胞器官チップモデルの構築

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

マウス抵抗動脈から毛細血管内皮の管の隔離

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

生物学

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

冠動脈血管新生のインビトロモデル

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

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 ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

ネズミ大動脈挫傷：効率的<em&gt;インビボ</em&gt;平滑筋細胞の増殖と内皮機​​能のモデル

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...