この研究は米国心臓協会の助成金0950118G.to MC - Wによって部分的にサポートされていました。

1。抗PECAM - 1抗体結合磁気ビーズ（Dynabeads）を準備<ol><li> BSAが溶解するまでボルテックスを使用して50 mlのPBSで50mgのBSAを混合することにより、PBS中0.1％のBSAを準備します。 0.22μmのシリンジフィルターを通して濾過することにより滅菌する。 4℃で保存します。 </li><li> TCのフードでは、1.5mlのエッペンドルフチュー​​ブに加え再懸濁し、ヒツジ抗ラットIgGのダイナビーズの200μlを転送し、ビーズを洗浄：マウントチューブをMPCにし、堆積物にビーズを可能にするために約1分間放置します。上清を吸引し、MPCからチューブを取り出して、無菌の0.1％BSA / PBSを1 mlでビーズを再懸濁します。ビーズを再懸濁するために上下にピペッティングし。 </li><li> four洗浄の合計のために、さらに3回洗浄繰り返します。 </li><li> MPCからチューブを外し、0.1％BSA / PBS500μlにビーズを懸濁します。 </li><li>チューブに10μlのラット抗マウスPECAM - 1（CD - 31）抗体を追加。 </li><li>室温で低温室または2時間で4℃で一晩タンブル。 </li><li>としてステップ1.2の4倍をで説明されている無菌の0.1％BSA / PBSでビーズを洗浄する。 </li><li> MPCからチューブを外し、200μlの0.1％BSA / PBS中でビーズを再懸濁します。ストア抗PECAM - 1抗体コンジュゲート4のダイナビーズ·最大1ヶ月のためのC。 </li></ol> 2。新生仔マウスから分離するマウス肺内皮細胞組織の精製プロトコールに進む前に、手順のIACUC委員の承認が必要です。 <ol><li>以下を準備します。 <ul><li> DMEMで1 mg / mlのC / D溶液15ml。 37にフィルタと暖かい0.22μmのシリンジとフィルター℃に</li><li> 15ミリリットルDMEM、氷の上に店舗を持つ50 mlコニカルチューブ</li><li> DMEM 20％FBSおよび1xペニシリン/ストレプトマイシンを含む分離メディア（IM） </li><li>ゼラチン2％。 4で100ミリリットルのdd水とゼラチンウシ皮膚2gの、オートクレーブで15分、ストアを混ぜる℃に37℃くらいまで温めコーティングTCフラスコの前に液化する。 </li></ul></li><li>ケタミン（100 mg / kg体重）及びキシラジン（10 mg / kg）のIP注射を使用して、3つの六から八日目の子犬を麻酔。一つずつを麻酔の有効性をチェックし、動物を解剖し、次のように：ピンの子犬をボードに、70％エタノールで皮膚を浸して滅菌解剖鋏で胸の部分から皮膚を取り除く。肺を公開する物品胸郭。 </li><li>無菌的に物品の個々の肺葉、気管支及び肺の周辺で目に見える結合組織を分析しないように注意してください。氷冷DMEMで全肺を組み合わせる。子犬を安楽死させる。 </li><li> TCのフードでの作業、DMEM、100ミリメートルTC皿の場所から肺の葉を取り除き、過剰なDMEMを吸引除去する。 </li><li>滅菌済みのハサミを使用して、〜100倍をカットして細かく組織をミンチ。酵素消化のために15ミリリットル暖かいC / Dソリューションに転送する。ローテーターで37℃で45分間インキュベートする。 </li><li> TCのフードでは、単一の細胞懸濁液中に14グラムカニューレ接続されていると粉薬塊を20 mlの注射器の中に少なくとも12回を消化した組織懸濁液を吸引除去する。 </li><li> 70μmのセルストレーナーを通して組織懸濁液を渡すと消化を停止するには、15ミリリットルのIMとストレーナーを洗浄。 </li><li> 5分間400 × gで遠心分離によってペレットの細胞懸濁液。 </li><li> 3ミリリットル0.1％BSA / PBSで上清を吸引し、ペレットを再懸濁します。 </li><li> 5ミリリットルの丸底ポリスチレンチューブに細胞懸濁液を移し、22.5μlの抗PECAM - 1抗体コンジュゲートのDynabeadsを加える。 12分間室温でタンブル。 </li><li>ゼラチン2 mLの2％ゼラチンおよび吸引過剰とT75フラスココート。プレーティング前の細胞に約30分間、TCのフードの内側に乾燥するためにゼラチンを許可。 </li><li>ビーズインキュベーションが完了したら（ステップ2.10）、三エッペンドルフチュー​​ブにも同様に細胞懸濁液を分割し、MPCをマウントします。 </li><li>ビーズは、MPCを（〜1分）を使用して沈殿したら、上清を収集、MPC上で再マウントして〜1 mlの0.1％BSA / PBSでビーズを洗浄、MPCからチューブを取り外し、と。 </li><li> 4回（合計5回洗浄を）洗う繰り返す</li><li>よく混合したチューブあたりEnGS - MV生活要因キットおよび1xペニシリン/ストレプトマイシン（VL）とVascuLife 1ml中に再懸濁しビーズ、。 </li><li>プレコートT75フラスコ上プレートとVLで10mlに各フラスコの総容積をもたらす。 </li><li>翌日のメディアを変更する、成長の1つの完全な一日を可能にし、一日おきに培地の半分を変更する。細胞が&gt; 70から80コンフルエント％以上（通常、文化の3〜4日後）である場合、それらは抗ICAM - 2ダイナビーズを並べ替えることができます。 </li></ol> 3。抗ICAM - 2抗体コンジュゲートのDynabeadsの準備<ol><li>抗ICAM - 2抗体結合ダイナビーズはさらにコンフルエントになるまで抗PECAM - 1のDynabeads抗体結合し、培養で選択されている細胞を精製するために使用されています。この手順は、これは抗マウスICAM - 2（CD - 102）抗体、抗PECAM - 1抗体の代わりに使用されている場合を除き、抗PECAM - 1抗体結合ダイナビーズ（1、上記）で説明したように実行されます。 </li><li>抗ICAM - 2抗体結合ダイナビーズは、最大で4℃で保存することができます1ヶ月まで。 </li></ol> 4。抗ICAM - 2ダイナビーズとマウス肺内皮細胞のソート<ol><li>ゼラチン2％のコートT75組織培養フラスコ。 </li><li>細胞をコンフルエントT75フラスコから培地を吸引除去し、8 mlのPBSで洗浄する。 </li><li>吸引PBSは、2 mlの0.05％トリプシン/ EDTAを加えると（約5分）細胞がデタッチできます。切り離しを支援するために軽くフラスコをタップします。 </li><li>細胞がはがれるしたら、残りの接着細胞を分離するためにトリプシンとこすり細胞を抑制するために2mlのIMを追加。 15 mlのチューブに細胞懸濁液を移し、400 × gで5分間スピンダウン</li><li> 2ミリリットル0.1％BSA / PBS中でメディアを再懸濁します細胞ペレットを吸引除去する。 </li><li> 5 mlのポリスチレン丸底チューブに移し、10μlの抗- ICAM - 2のDynabeadsを加える。室温で12分間タンブル。 </li><li> MPCの2つの1.5mlエッペンドルフチュー​​ブとマウントに細胞懸濁液を分割する。 </li><li>ビーズを1分間付着した後に上清を吸引除去する。 MPCからチューブを外し、1 mlの0.1％BSA / PBSで各チューブを再懸濁します。 MPCで再マウント。 </li><li> 4回（5回洗浄の合計）洗い繰り返します。 </li><li> 1 mlのVLで各チューブを再懸濁し、細胞数を実行する。 </li><li> T25フラスコおよび/またはT75フラスコあたり750K - 1万個の細胞あたり250 - 350Kの細胞で細胞を​​プレート。 </li><li> T25フラスコに5 mlのVL、およびT75フラスコで10mlに容積をもたらす。 </li><li>一日おきにメディアを変更します。文化は通常2〜3日で、約80％コンフルエントに達したときに細胞が実験に使用することができます。 </li></ol> 5。代表的な結果一般的に、細胞の初期準備から6-7日後に、我々は、約1.2取得 ​​することができます- 3 6〜8日齢の仔から1.5 × 10 6肺内皮細胞は。細胞は光学顕微鏡下で典型的な&quot;敷石&quot;形態を表示し、内皮細胞（図1）の特性であるVE -カドヘリン（CD144）細胞間の接合部において染色を、示す。 FACS分析（図2）により実証されるようにMLECsエクスプレスVE -カドヘリンおよびVEGFR2の大半。通常、我々は最初のMLEC分離後二週間以内に実験のためにそれらを使用してください。 <img src="/files/ftp_upload/2316/2316fig1.jpg" alt="図1" /> 図1。コンフルエントMLECの単層の顕微鏡分析。 （A）光学顕微鏡の画像は、内皮細胞の典型的な培養細胞の石畳の形態を示しています。多くの細胞の核周辺部に位置して均一な円形構造物は、MLEC分離のために使用される磁気ビーズです。 （B）血管内皮特異的VE -カドヘリンは、共焦点顕微鏡を用いて示される細胞間の接合部に局在している。バーは、20μmの代表です。 <img src="/files/ftp_upload/2316/2316fig2.jpg" alt="図2" /> 図2血管内皮に特異的なマーカーに特異的な抗体で標識された培養MLECsのFACS分析：。フィコエリスリン標識二次抗体が続くVE -カドヘリン（CD144）またはVEGFR2、として起訴、、孤立MLECsの内皮IDを確認する。特定の抗IgG - 赤のラインがアイソタイプの特定のコントロールを示し、緑の線は、抗VE -カドヘリンまたは抗VEGFR2を示している。

<ol>
	<li>Maruyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+endothelial+cell+in+tissue+culture.">The human endothelial cell in tissue culture.</a> Z Zellforsch Mikrosk. Anat. 60, 69-79 (1963).</li><li>Lim, Y. C., Luscinskas, F. 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+and+culture+of+murine+heart+and+lung+endothelial+cells+for+in+vitro+model+systems.">Isolation and culture of murine heart and lung endothelial cells for in vitro model systems.</a> Methods Mol Biol. 341, 141-154 (2006).</li><li>Aird, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+heterogeneity+of+the+endothelium:+I.+Structure,+function,+and+mechanisms.">Phenotypic heterogeneity of the endothelium: I. Structure, function, and mechanisms.</a> Circ Res. 100, 158-173 (2007).</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> Am J Physiol Lung Cell Mol Physiol. 296, L1096-L1103 (2009).</li><li>Miebach, S., Grau, S., Hummel, V., Rieckmann, P., Tonn, J. C., Goldbrunner, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+microvascular+endothelial+cells+from+gliomas+of+different+WHO+grades.">Isolation and culture of microvascular endothelial cells from gliomas of different WHO grades.</a> J. 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Commun. 281, 827-834 (2001).</li><li>Kajimoto, K., Hossen, N., Hida, K., Ohga, N., Akita, H., Hyodo, M., Hida, Y., Harashima, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+microvascular+endothelial+cells+from+murine+inguinal+and+epididymal+adipose+tissues.">Isolation and culture of microvascular endothelial cells from murine inguinal and epididymal adipose tissues.</a> J. Immunol. Methods. 357, 43-50 (2010).</li><li>Breviario, F., Caveda, L., Corada, M., Martin-Padura, I., Navarro, P., Golay, J., Introna, M., Gulino, D., Lampugnani, M. 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動物実験はプロトコルAUA＃00001206下ウィスコンシンIACUC委員会の医科大学が定めるガイドラインおよび規則に従って行った。

微小血管内皮は、血管生物学の多くの分野で有用なモデルであることが証明されていると、より生理的に広く研究されているHUVECを3以上（例えば、血管新生）勉強に関連すると考えられている。以前に、それは微小血管内皮は腎臓、心臓、皮膚、網膜、脳、神経膠腫、脂肪組織や、肺2、4-7、10、11から得られることが報告されている。しかし、微小血管内皮単離するための一貫性と信頼性の高い方法が依然として必要です。ここに示す手順は、以前に発行されたプロトコルバージョン2の変形例であり、それは子犬ではなく、成熟した動物を使用する点で最も公開された手順とは異なります。若い動物からの細胞は高い増殖能を持ち、彼らの組織由来の培養は、細胞数の増加をもたらす傾向があるので、これはアイソレーションを成功させるために重要です。一週間古い動物が最適であると思われるが、より若いマウス（4日齢）を使用することもできます。私達は1つの子犬からMLECsを単離することに成功しているとしても、仔の上位または下位の数値は、必要に応じて使用することができます。しかし、分離のプロセスをスケールアップまたはダウンしながら、細胞の播種密​​度は、これがまた、細胞増殖に重要であるとして、変更されないままにしておく必要があります。 2ステップ精製抗PECAM - 1に結合した第一磁気ビーズを使用してMLECsの過程と抗ICAM - 2抗体よりは、前述のシングルステップ精製法よりも純粋なECの文化を得ることをはるかに効率的です。このプロトコルは、非常に手間のかかる手動のテクニック、勾配遠心分離し、繊維芽細胞、血液細胞と平滑筋細胞などの廃棄汚染セルの並べ替えを少なく効率的なFACSを使用する必要がなくなります。結果MLEC人口は血管新生反応、血管透過性及び白血球遊出のin vitroでの解析で 、癒しだけでなく、シグナル伝達経路の生化学的解析を巻かに使用することができます。必要に応じて、MLECsはそのようなトランス内皮抵抗（TER）測定のための免疫蛍光または電極アレイ用のフィブロネクチンまたはゼラチンコートしたカバースリップのような別の表面にめっきすることができる。得られた結果を直接ノックアウトおよびトランスジェニックマウス系統の増加のコンテキスト内で特に重要な生体内で観察された表現型、比較することができます。 in vivoで観察された欠陥の基礎となる細胞メカニズムのin vitroでの解析では、このメソッドの実装を成功させるには、すでに12を提示されている。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>Spring scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15032-13</td>
<td>&nbsp;</td>
<tr>
<td>forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11223-20</td>
<td>&nbsp;</td>
<tr>
<td>14G cannula</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>1482516N</td>
<td>&nbsp;</td>
<tr>
<td>20 ml syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309661</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich </td>
<td>A7030-100G</td>
<td>&nbsp;</td>
<tr>
<td>anti-rat IgG Dynabeads</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>110.35</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic Particle Concentrator (MPC)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>123-21D</td>
<td>&nbsp;</td>
<tr>
<td>anti-mouse PECAM-1 antibody (CD-31)</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>553369</td>
<td>&nbsp;</td>
<tr>
<td>anti-mouse ICAM-2 antibody (CD-102) </td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>553326</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase/Dispase (C/D)</td>
<td>&nbsp;</td>
<td>Roche Applied Science</td>
<td>11097113001</td>
<td>100mg/ml stock solution can be prepared and stored at -20&deg;C</td>
</tr>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Cellgro Mediatech</td>
<td>10-013-CV</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Skin Gelatin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>#G9391-100G</td>
<td>&nbsp;</td>
<tr>
<td>Vasculife EnGS-Mv complete kit (VL)</td>
<td>&nbsp;</td>
<td>Lifeline Cell Technology</td>
<td>LL0004</td>
<td>&nbsp;</td>
<tr>
<td>0.05% Trypsin/EDTA </td>
<td>&nbsp;</td>
<td>Mediatech</td>
<td>25-052-CI</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer 70 &mu;m nylon</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>tissue culture flask 75 cm2</td>
<td>&nbsp;</td>
<td>TPP</td>
<td>90076</td>
<td>&nbsp;</td>		</tbody>
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isolation and culture of pulmonary endothelial cells from neonatal mice

内皮細胞は血管生物学の多くの分野で有用な研究モデルを提供しています。その最初の単離1日以来、ヒト臍帯静脈内皮細胞（HUVEC）を入手し、文化に容易、便利であることが示され、したがって、最も広く研究されている内皮細胞であるしている。しかし、血管新生、透過性や他の多くのようなプロセスに焦点を当てた研究のために、微小血管内皮細胞（ECS）は、2を勉強するより生理学的に妥当なモデルです。さらに、ノックアウトマウスから単離されたECは、タンパク質の機能のex vivoでの解析のための有用なツールを提供しています。微小血管は異なる起源のEC分離と文化にいくつかのアプローチが3-7をこれまでに報告したが、純粋なECの一貫性のある分離および培養は、まだ多くの研究室の主要な技術的な問題です。れましたここで、我々は、単離および培養マウス肺内皮細胞（MLECs）の信頼性と比較的簡単な方法をステップバイステップのプロトコルを提供しています。このアプローチでは、6から得られた肺組織 - 8日齢の仔には、最初のコラゲナーゼ/ディスパーゼ（C / D）ソリューションで消化し、単一細胞懸濁液に機械的に分散し、小片に切断される。 MLECSは、磁性粒子のコンセントレータ（MPC）を使用してダイナビーズに結合した抗PECAM - 1抗体を用いてポジティブセレクションを使用して細胞懸濁液から精製されています。彼らがコンフルエントになるまで、そのような精製された細胞は、ゼラチンコート組織培養（TC）皿上で培養されています。その時点で、細胞はさらに抗ICAM - 2抗体に結合ダイナビーズを用いて精製されています。このプロトコルで得られたMLECsは、石畳の表現型を示す位相差光学顕微鏡などによって可視化、およびそれらの内皮表現型は、抗VE -カドヘリン8および抗VEGFR2 9抗体およびVE -カドヘリンの免疫蛍光染色とFACS分析を使用して確認されている。私たちの手で、この二段階の分離手順は、一貫性と信頼性をさらに培養することができるMLECs、の純粋な集団が得られます。このメソッドは、研究者が直接in vivoで観察された血管の表現型の分子機構を明らかにするために役立つため、孤立したMLECs上で実行し、in vitroでの実験の結果をin vivo試験で相関するノックアウトおよびトランスジェニックマウスの成長数の利点を受けることができるようになる。

Watch this Scientific Journal Video about Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice at JoVE.com

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

ここで、我々は、マウス肺内皮細胞の単離と培養のためのプロトコルについて説明します。このメソッドは、メカニックと酵素肺組織の解離と同様に抗PECAM - 1とほとんどが微小血管起源の純粋な内皮細胞集団を生成する磁気ビーズへの結合抗ICAM - 2抗体を用いた2ステップ精製プロセスを含む。

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-culture-pulmonary-endothelial-cells-from-neonatal

Research

JoVE Journal

Biology

46.9K ビュー.  BloodCenter of Wisconsin. ここで、我々は、マウス肺内皮細胞の単離と培養のためのプロトコルについて説明します。このメソッドは、メカニックと酵素肺組織の解離と同様に抗PECAM - 1とほとんどが微小血管起源の純粋な内皮細胞集団を生成する磁気ビーズへの結合抗ICAM - 2抗体を用いた2ステップ精製プロセスを含む。

ビデオ: Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

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 ...

生物学

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

ヒト皮膚から大人上皮幹細胞の単離と培養

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

新生仔マウス心筋細胞の単離および培養

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

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 Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

新生児マウスから肺動脈平滑筋細胞の単離

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

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 ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

ヒト末梢血からの血液伸長内皮細胞の生成と文化

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

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 Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

新生児および成人マウス皮膚由来の初代マウスケラチノサイトの単離および培養

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...