著者は、原稿に有益なコメント、ダニエラターレルとマルガレッタ神父hwirth技術支援のための、言語編集用のモニカファレル、ウーラング、マルギットホルツアップフェルと産科の部でサンドラEppichためpHPL、エヴァローデとニコールホフマンを提供するためのカタリーナSchallmoserに感謝コー​​ドを提供する。 この作品は、オーストリアの研究財団（FWF、DSへの助成金N211 - NAN）、オーストリアの研究推進機構（FFG、DSへの助成金N200）で、成体幹細胞研究財団（AR）によってサポートされています。 ARは、グラーツの医科大学の博士課程のプログラム分子医学の仲間です。

パート1：セットアップ 1。細胞培養培地の調製メディアの準備を開始する前に、必要なすべての材料や道具を集める。 雪解け2 ×プールしたヒト血小板溶解液の56 mlのアリコート（pHPL、自作：リファレンス1＆Joveの＃1523）、100倍ペニシリン/ストレプトマイシン液、L -グルタミンの2 × 5mLの（両方​​ともSigma社）を10ml。 EGM - 2培地（ロンザ）の1瓶（500 mL）を補足するために調整（、アムホテリシンは&quot;単一のquots&quot;として提供されているVEGF、bFGFは、EGF、IGF、ヒドロコルチゾン、アスコルビン酸、ヘパリン）サイトカインと成長因子のアリコートを解凍。 冷蔵庫からの各（i）1つ500mlボトルを取る最小必須培地（α- MEM）および（ii）内皮基礎培地（EBM）のα​​-改変。 次の手順のすべては、無菌条件下で層流の組織培養フードで実行されます。 滅菌水で1000 IU / mlの最終濃度に粉末を溶解することにより防腐剤フリーヘパリン（例えば、Biochrom）準備。 MSC -ミディアム： 、- MEMの500mlを使用してください（詳細は1を参照することも参照）融解pHPL 56 mLを加え、防腐剤フリーのヘパリンの2 IU / mLの（ストック溶液の= 224μL）が（の凝集によって培地の凝固を避けることができます10％のpHPLの最終濃度になるように血漿中のフィブリノゲン）。また、ペニシリン（100U/mL）/ストレプトマイシン（100μg/mL）ソリューションとL -グルタミンの2mMの（両方ともSigma社）を追加します。 20μmの孔径真空フィルター（ミリポア）を介してメディアをフィルタします。 （内容、日付）を適切にボトルにラベルを付けます。 ECFC -ミディアム： サイトカイン - アリコート、防腐剤フリーヘパリンのpHPL、10 IU / mlの（ストック溶液の=1120μl）の56 mLを、ペニシリン（100U/mL）/ストレプトマイシン（100を追加し、EBMの一瓶（500 mL）を使用してG / 20μlの細孔サイズの真空フィルター（ミリポア社）で基礎培地とフィルタへのL -グルタミンの添加）ソリューションおよび2mM。 （内容、日付）を適切にボトルにラベルを付けます。 2。手術器具の滅菌鉗子、外科用ハサミやメスホルダーの鋭いペアは、乾熱滅菌鉱石無菌使い捨てにする必要があります。 3。コー​​ドのコレクションのためのチューブの準備 50 mlのポリプロピレンチューブ（ファルコン）に無料のヘパリンとPBSで20mLの最終的な音量に調節する - 防腐剤500 IUを追加。分娩室にチューブを移す。 4。インフォームドコンセント（最寄りの倫理委員会またはIRBで確認）。 彼らはコードの一部を寄付し、十分にそれがサービスを提供する目的を通知したい場合は、配信前に親を求める。 5。コー​​ドの寄贈胎盤と臍帯の配信と検査後10 cmの部分を切断してすぐに脊髄血管内に残った血液の凝固を避けるために、ヘパリンであらかじめ入力してチューブに移す。できるだけ早く、細胞分離の処理を進める。 パート2：セルの分離<ol><li> Incidinまたは70％エタノールで表面を洗浄により、層流の組織培養フードを準備します。場所滅菌150センチメートル2細胞培養プレート、75センチの細胞培養フラスコ（コーニングの両方）、PBSは、手術器具、セルスクレーパー、チューブホルダー、25 mLのstripettesとクリーニングフードで適切に平滑末端の針を備えた5 mLシリンジを滅菌。 </li><li> 37〜プレ暖かいご準備α- MEMとEGM - 2細胞培養培地℃の水浴インチ</li><li>分娩室からの細胞培養のフードを装備してラボにコードをもたらす。層流中にコードを転送した後汚染血液細胞を取り除くために（新しいプレートに移すこと）PBSで2回洗ってください。コー​​ドのもう一方の端を出てくる液体が（赤は血液の汚染を示す）完全に透明になるまでPBSで平滑末端針を装備した滅菌5mLをシリンジで一方の側にそれをcanulating後に臍静脈をすすぎます。 </li></ol> ECFCs： <ol start="4"><li> 75センチメートル2フラスコ（コーニング）を準備し、それにラベルを付け、予め温めておいたEGM - 2培地の15〜20 mLで埋める。 </li><li>滅菌PBSで満たされた新しいプレートに電源コードを置きます。 外科はさみを取り、長さが約5cm（臍血管がその軸に沿ってねじれているので、短い作品は、処理しやすい）の2つの部分に臍帯を切る。 </li><li>臍静脈（大血管）を見つけることを試みる、あなたの外科はさみいずれかのブレードを挿入し、縦方向に静脈をカット。あなたがさらにカットしながらアシスタント二つ鉗子で既にカット静脈を保持できる場合は、この時点で、それが有用である可能性があります。 </li><li>コー​​ドのパイを配置PBSのない新たなプレートのカット静脈とCEは、端から端まで拓本によって臍静脈の内側（内皮）表面のセルスクレイパーとこすりしてください。培地中のスクレーパーの先端を清掃して、準備したフラスコに細胞を移す。この手順を少なくとも10回を繰り返します。 </li><li>あなたのフラスコを閉じ、インキュベーター（37℃、5％CO 2、湿度95％）に入れて。 </li></ol> MSCは： <ol start="4"><li> 150センチメートル2培養プレート（コーニング）を準備してラベルを付ける。 </li><li>コー​​ドの一部を（内皮層を掻き後）に乗り、滅菌済みのハサミと滅菌メスを使用すると、非常に小さな部分に全体のコードを切断。コー​​ド片の最大サイズは1〜2 mmでなければなりません。 </li><li>お使いの事前標識培養プレートに滅菌ピンセットを使用してコードの部分を転送する。プレート培地で一杯にする前に5分間開いておくことで、プラスチック表面へ片の付着が強化される。 </li><li>コー​​ド片をプラスチックに適切に接続したら、静かにMEMを修正予め温めておいたアルファの30〜35 mLを加える。すべてのピースが接続されたままであることを確認するために最低速度を使用するようにしてください。 </li><li>プレートを閉じて、インキュベーター（37℃、5％CO 2、湿度95％）に入れて。 </li></ol>パート3：セルの拡張とフローサイトメトリーによる免疫表現型の確認セルの拡張 EFCFs： <ol><li>すべての非接着細胞や粒子を取り除くために全体EGM - 2培地を倒立顕微鏡で、内皮細胞の付着翌日を確認し、交換。 </li><li>週二回合流し、交換するまで培地の3分の1をセルを展開します。コンフルエンスに達したときに、0.05％Trypsin/0.7 mMのEDTAで細胞を切り離し、新しい培養容器に移す。あなたのT75フラスコ内の細胞の数に応じて、さらなる拡大のための低播種密度を保証するために、新しいフラスコの大きさと数を選択してください。 </li><li>細胞の拡大時に培地の交換を3分の1週二回。 </li></ol> MSCは： <ol><li>コー​​ドからのMSCsの伸長は、多くの時間がかかりますので、添付された組織片の周囲に紡錘形細胞の出現のために倒立顕微鏡で確認するまでは、少なくとも3-4日を待つことができます。十分な細胞外増殖がある場合には、作品は、プラスチックとの接触を失うので、デタッチする傾向がある。 </li><li> 10-12日は、組織片を除去した後、0.05％Trypsin/0.7 mMのEDTAでプラスチックからMSCSを切り離し、新しい培養容器に移す。お皿の中で細胞の量に応じて、さらなる拡大のための低播種密度を保証するために、新しいフラスコの大きさと数を決定する必要があります。 </li><li>細胞の拡大時に培地の交換を3分の1週二回。 </li></ol>フローサイトメトリー必要な部品： フローサイトメーター（BD FACSキャリバー）、micronicチューブ（コーニング）、チューブホルダー、ヒツジ血清（ブロッキング試薬）、Eppifuge（エッペンドルフ）、モノクローナル抗体 MSCは：CD45、CD14、CD19、HLA - DR、CD31、CD73、CD90、CD105、アイソタイプコントロール ECFCs：CD45、CD14、CD19、HLA - DR、CD31、CD34、CD90、CD105、CD144、CD146、アイソタイプコントロール<ol><li>コンフルエントに達した後、4℃で7分間℃で300グラム0.05％Trypsin/0.7 mM EDTAおよび遠心機で細胞を切り離す</li><li>変更されたPBSと4で20分間、10％v / vのヒツジ血清℃のブロック非特異的抗体結合で1 × 10 6細胞/ mLの濃度に細胞を再懸濁し</li><li> micronicチューブにラベルを付け、蛍光標識抗体の適切な濃度を追加してください。 </li><li>各チューブに細胞懸濁液（ブロック）の50μlを加え、4℃で30分間インキュベート℃に</li><li>非結合抗体を除去するために修正されたPBSを添加し、細胞を洗浄する。 </li><li>フローサイトメーターにラベルされた細胞に移動して、分析を行います。 </li></ol>パート4：ECFCコロニーの成長、固定、染色し、階層分析<ol><li>単一のコロニーの成長を可能にする細胞培養プレートの平方センチメートル当たり3または10の細胞の非常に低い播種密度でシード実質的に純粋な収穫ECFCsを。 </li><li>交換培地の三分の一週二回。 </li><li> 14日は（文化の終わり）培地を除去した後、PBSで2回洗浄し、氷冷固定液（アセトン2：：メタノール= 3）でコロニーを固定し、冷蔵庫で15分間。 </li><li>固定液を捨て、プレートを10分間乾燥するために開いたままにしておきます。 </li><li> 10分間蒸留水で細胞を再水和。 </li><li>ハリスヘマトキシリン​​溶液を追加し、12分間固定コロニーを染色。 </li><li> H matoxylin溶液を除去し、残りの染色液を取り除くために水道水でプレートをすすぎます。 </li><li>して、各コロニーの写真を撮る実体顕微鏡とインポートを使用して*. jpgまたは*. tif形式のファイルをコンピュータに。 </li><li> ImageJのソフトウェアで写真を開いて、アニメーションで説明したように各コロニーの正確な細胞数を分析する。 </li><li> ImageJのソフトウェアを開き、プルダウンメニューで&quot;ファイル\開く&quot;機能を使用してコロニーのイメージをインポートします。 </li><li>機能&#39;\減算バックグラウンドプロセス&quot;を使用して進む</li><li>コロニーのマージンをマークするImageJのメニューで&#39;*楕円*またはブラシの選択&quot;機能を使用してください。 </li><li> &quot;編集​​は\クリア外&quot;の関数と作物&#39;\トリミング画像&quot;を選択することで画像を使用して周囲の画像を消去します。 </li><li>すべての細胞が完全に赤色に着色されるように、関数の&#39;\ \しきい値の調整イメージ&quot;を使用して手動で閾値を調整します。 </li><li> &quot;分析\分析粒子&quot;を選択することにより、コロニーの細胞数をカウントします。適切な設定を（各セルのタイプに最適化された）を選択し、&quot;OK&quot;を選択します。細胞数と計数した細胞の対応するアウトラインを含む新しいイメージを含む結果ボックスが、表示されます。 <img src="/files/ftp_upload/1525/1525op2.jpg" alt="図1" /></li></ol>

<ol>
	<li>Schallmoser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+platelet+lysate+can+replace+fetal+bovine+serum+for+clinical-scale+expansion+of+functional+mesenchymal+stromal+cells.">Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells.</a> Transfusion. 47, 1436-1446 (2007).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+system+to+propagate+cord+blood-derived+multipotent+mesenchymal+stromal+cells+for+clinical+application.">Humanized system to propagate cord blood-derived multipotent mesenchymal stromal cells for clinical application.</a> Regen Med. 2, 371-382 (2007).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+large-scale+expanded+endothelial+colony-forming+cells+function+in+vitro+and+in+vivo.">Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo.</a> Blood. 113, 6716-6725 (2009).</li><li>Strunk, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+characterization+and+preclinical+production+of+human+lineage-negative+cells+for+regenerative+stem+cell+therapy.">Phenotypic characterization and preclinical production of human lineage-negative cells for regenerative stem cell therapy.</a> Transfusion. 45, 315-326 (2005).</li></ol>

著者は宣言するために興味のある金銭的競合を持っていない。

骨髄、脂肪組織、臍帯血、等を含むMSCSまたはECFCsの取得元となる、さまざまなソースがありますそれは、直接組織と血管再生や腫瘍の進行への寄与のようなプロセスでの異なる細胞型の関与を比較するために、同じソースから前駆細胞の両方のタイプを取得する必要があります。 分離し、その後同じドナーからECFCsとMSCの自家ペアを研究する際の容?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>alpha modified MEM</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>L-GLutamin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>EBM</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Single cytokine quots</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>75 cm2 flasks</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>150 cm2 plates</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cell scraper</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin/EDTA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Monoclonal antibodies</td>
<td>&nbsp;</td>
<td>Becton Dickenson</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50 ml tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>500 mL filter 20 &mu;m pore size </td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Preservative free heparin</td>
<td>&nbsp;</td>
<td>Biochrom</td>
<td>&nbsp;</td>
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isolation and animal serum free expansion of human umbilical cord derived mesenchymal stromal cells (mscs) and endothelial colony forming progenitor cells (ecfcs)

へその緒は、間葉系幹細胞（とも呼ばれる間葉系幹細胞は、MSC）および内皮コロニー形成前駆細胞（ECFCs）を含む、高い増殖能を持つ前駆細胞の豊富な情報源となります。両方の細胞型は、組織の整合性を維持するためのキープレーヤーであり、おそらくまた、再生のプロセスと腫瘍形成に関与している。 比較的にそれらの生物学と機能を研究するためには、同じドナーから入手可能な両方の細胞型を持つことが重要です。また、同じ組織からのMSCsとECFCsを導出するために再生目的のために有益であるかもしれません。 細胞治療は、最終的にベンチからベッドサイドへの道を見つける必要があるので我々は分離する新しい方法を確立し、さらに動物性タンパク質を使用せずに前駆細胞を展開します。プールされたヒト血小板溶解液（pHPLは）完全に異種蛋白質への細胞の接触を避けるために、我々のプロトコルのすべての段階においてウシ胎児血清を取り替えた。 このビデオ一臍帯からの前駆細胞の分離と拡大のための方法論を示しています。 すべての材料および手順について説明する。

Watch this Scientific Journal Video about Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

このプロトコルは、間葉系幹細胞および実験的移植の目的のために自己のペアを生成するために動物血清を使用することなく、内皮コロニー形成細胞の分離とその後の展開について説明します。

ヒト臍帯由来間葉系幹細胞（MSCs）と血管内皮コロニー形成前駆細胞（ECFCs）の単離および動物血清自由膨張

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

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Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

15.3K ビュー.  Medical University of Graz, Austria. このプロトコルは、間葉系幹細胞および実験的移植の目的のために自己のペアを生成するために動物血清を使用することなく、内皮コロニー形成細胞の分離とその後の展開について説明します。

ビデオ: Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

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 Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.

ラット間葉系幹細胞（MSC）とMSCを派生シングルコロニーの分離の分離と濃縮

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

ヒト臍帯動脈平滑筋細胞の単離（HUASMC）

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

ヒト臍帯血由来血管内皮コロニー形成細胞の表現型および機能解析

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPAR&gamma; agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

ベージュ/ブライト細胞間質血管細胞の単離と分化

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

間葉転換を誘導し、上皮の分析

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

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 of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

CD146の単離<sup&gt; +</supラット肺から&gt;常駐肺間葉系幹細胞

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

オルガノイド培養のためのヒト肺上皮前駆細胞の単離と濃縮

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...