ACD is supported by a grant from the National Institute of Health (R01-CA177875). LX is a fellow in the HHMI-funded translational medicine program at UNC Chapel Hill. JVM is supported by a T32 pre-doctoral fellowship from the Integrative Vascular Biology Program at UNC Chapel Hill. We thank Clayton Davis for assistance with confocal microscopy.

以下のプロトコルは、ノースカロライナ大学チャペルヒル校で実験動物医学科によって確立されたガイドラインに従って実施されます。 1.開始する前に、以下の材料および試薬を準備します<ol><li> 50ミリリットルの熱不活性化ウシ胎児血清、50ミリリットルのNu-血清IV、抗生物質 - 抗真菌5ミリリットルと400ミリリットルを低グルコースを補充することにより、ECのメディアを準備します（1グラム/ LのD-グルコースまたはLG）ダルベッコ改変イーグル培地（DMEM）そして、のhFGF、VEGF、のhEGF、R3-IGF-1、および市販のキットからのヘパリン成分は。 </li><li> 500 mLのFACS緩衝液（0.5％BSA、PBS中の2mM EDTA）を調製。滅菌0.22μmのフィルターカップを通して濾過します。 </li><li>滅菌またはボードを解剖消毒。 </li><li>解剖ピン、手術用はさみ、および解剖器具を滅菌します。 </li></ol> 2. EC単離（1日目〜5時間） <ol><li>二酸化炭素または他の方法に準拠し機知にマウスを安楽死させます時間制度動物実験委員会（IACUC）方針。 注：サイズが異なる複数の乳腺腫瘍（5 - 直径15mm）は、このようなC3-TAGのような単一の遺伝的に改変マウスで開発することがあり、TECの分離のためにそれらのすべてを収穫してください。単一のEC単離のために同所性マウスに移植している腫瘍、プール2 1 3センチ3の腫瘍を使用した場合。ここでは、デモンストレーションとしての乳腺腫瘍を使用したが、プロトコールは、腫瘍の他のタイプに変更することができます。 </li><li>噴霧または、75％（v / v）のエタノールの十分な量で、マウス腹側を拭いて、マウスを消毒します。 </li><li>無菌フード内やクリーンベンチで無菌技術を用いて、ハサミや解剖の1組で腫瘍を切除します。 <ol><li>伸ばし解剖ボード上のマウスの四肢を固定します。腹膜を開かずにハサミで正中切開腹を行います。皮膚腫瘍が配置されている乳腺に向かって腹膜の間に横方向に解剖。腹膜を開けないでください。 </li><li>消費税​​のみ腫瘍乳腺組織から、正常な乳房余白を残します。慎重に、皮膚や筋肉などの非腫瘍組織を切り落とすと、氷上でLG-DMEM 30 mlを含むコニカルチューブに解剖した腫瘍を配置します。 </li></ol></li><li>組織培養フードに腫瘍サンプルを持参。 2回 - 無菌LG-DMEM 1で組織を洗います。 </li><li>無菌の組織培養シャーレへのコニカルチューブから転送腫瘍は、皿にLG-2mlのDMEMを追加し、片に滅菌ハサミ&lt;5 mmのミンチ。 </li><li> PBS中、ディスパーゼの1ミリリットルを（ストック=ハンクス液中の2mg / mlの、HBSSを以後）コラゲナーゼの5ミリリットルを追加します（ストック= HBSS中2.5 U / ml）およびデオキシ75μlの（株= 1 mg / mlの）ペトリ皿に。トータルボリュームは今〜10ミリリットルです。 </li><li> （予め設定された解離PR組織解離管にペトリ皿からコラゲナーゼ/組織ミックスを移し、二回60秒のための組織解離で実行解離にogram：1回あたり1,270合計ラウンド）。光は37℃で75分間シェーカー上で振盪しながらインキュベートします。 </li><li>フィルターを50mlコニカルチューブの上に100μmのセルストレーナーを通して組織を消化しました。残りの細胞を洗浄するためにFACS緩衝液5mlでフィルターを洗浄します。 5分間280×gでスピンし、慎重に細胞ペレットを乱すことなく、上清を吸引除去します。 </li><li>滅菌水9mlの株式RBC溶解バッファー（10倍）の1ミリリットルを希釈します。溶解緩衝液（1×）の10 mlの溶解赤血球、すぐ280 X gで5分間回転さ。注：少し血が表示されている場合、この手順はスキップすることができます。 </li><li> FACS緩衝液10ml中に再懸濁。トリパンブルーの10μlの細胞懸濁液10μlを混合し、血球計数器を使用して生細胞を数えます。 </li><li> 〜10 7細胞 /100μlので細胞を再懸濁します。 100μlの細胞懸濁液あたりのFcRブロックの10μlを添加し、氷上で10分間インキュベートします。 </li><li>ラット抗マウスPE結合CD31抗体を追加。たまに15分とフリックチューブ- 表1に10氷上でインキュベートします。 </li><li> 5分間280×gでチューブとスピンにFACS緩衝液10mlのを追加します。注意深く上清を除去し、FACS緩衝液5mlで再び細胞ペレットを洗浄します。細胞をペレット化するために遠心分離。細胞ペレットを乱すことなく、上清を吸引。 </li><li> 表2に記載の FACS緩衝液および抗PEマイクロビーズを追加10氷上でインキュベートする- 。15分。時折フリックチューブ。 </li><li> 5分間280×gでFACS緩衝液とスピンのサンプルの10ミリリットルを追加します。再びFACS緩衝液および遠心5mlで1回洗浄。ペレットを乱すことなく上清を吸引。 </li><li> FACS緩衝液中で300μlにボリュームを持参してください。 280 X gで5分間、35ミクロンの細胞ストレーナーキャップ付きチューブを介してスピン。必要に応じて2チューブと、より大きなボリュームを使用してください。 </li><li> 、フード内で磁気multistandと磁気カラムを設定し、セパレータに列を添付し、FACS緩衝液2mlでカラムを平衡化。 </li><li> AspiratE上清と0.5で細胞ペレットを再懸濁 - FACS緩衝液1ml。 </li><li>平衡化した磁気カラムを通して細胞懸濁液を渡します。 </li><li> FACS緩衝液2mlでカラムを3回洗浄し、15mlチューブ（FT画分）にフロースルー（FT）を収集。 </li><li>セパレーターオフ列を取り、別の15mlチューブ（溶出画分）にFACS緩衝液2mlで溶出します。すべてのセルが列をオフであることを確認するために、プランジャーを使用してください。毎回バッファーFACS 2mlの溶出をさらに2回繰り返します。 </li><li> 5分間280×gで溶出液をスピン。 </li><li>上清を除去し、EC培地10mlにペレットを再懸濁。 </li><li>同様に10cmのゼラチンコートディッシュに溶出画分（〜6ml）に分割します。 3 1 cm 3の腫瘍については、プレートは、少なくとも4つのプレートで細胞を溶出しました。複数のプレートで、異なる濃度の代わりに、プレートを溶出した細胞（ 例えば 、0.5ミリリットル、1ミリリットル、1.5ミリリットル、及び4枚のプレートに溶出液の3ミリリットルをシード）ことを確実にするためにトン少なくとも一つのプレートがまばらに溶出細胞を播種されます。 （ - 2.0×10 5細胞が付着すなわち、約1.0）付着した細胞の密集度が〜1％で、次の日であることを確認してください。 注：細胞は、ECのコロニーは、他の細胞種によって汚染されることなく形成することができるように、スパースメッキされる必要があります。 </li><li> 1 10cmディッシュでプレートのFT画分は、細胞は、O / Nを回復させます。翌日、100％コンフルエント - プレートが80であることを確認してください。細胞（-80℃）250μlの細胞凍結培地中にクライオチューブ内を下に凍結し、翌日、液体窒素に保管してください。注：FTフラクションは、後の段階で腫瘍細胞の単離および/または単離されたECクローンのEC遺伝子発現解析のための陰性対照として使用することができます。 </li><li> 3日 - ごとに2メディアを変更します。 10日 - コロニーは7の後に形成し始めます。小型のECコロニーが皿の底に先の細いマーカーで早くも3日目としてマークするコロニーを同定することができます。 </li><li>非特異的なCを削り取ります無菌の200μlのpippetチップで識別されたコロニーを取り巻くells。 </li></ol>クローニングリングを使用3.コロニーセレクション<ol><li> 3日 - ごとに2メディアを変更します。 7日 - ECの純度は約5でLDL（DII-AC-LDL）を添加することによって確認することができます。 10ミリリットルのEC媒体ごとのLDLの50μLを加え、3インキュベート - 蛍光顕微鏡下で細胞をチェックする前に4時間。 注：複数のLDL + ECクローンは、〜7日目に観察することができました。 </li><li>大きさが5ミリメートル - 彼らは3の直径に達したときのECコロニーを収穫開始。 （最良の結果を得るための小型LDL +細胞が充てんされて大きなコロニーを選択してください。） </li><li>クローニングリング、0.5％ゼラチンでプレコート数6ウェルプレートでECを回収する前に。吸引しゼラチン、各ウェルにECメディアの2ミリリットルを追加し、必要になるまでインキュベーターにプレートを保ちます。 </li><li>他の細胞型はクローニングリング内に捕捉されませんを確認するために、コロニーの縁に非ECを削り取ります。 </li><li>使い方位相差顕微鏡（4Xまたは10X目的）は、培養皿の底に先の細いマーカーで概説ECコロニーを含む領域。 </li><li> 10mlのPBSでプレートを洗浄し、吸引したときに、プレートにPBSの非常に薄い層を残します。 （重要：少量のPBS（〜0.5ml）をクローニングリング手順中に生きている細胞を維持します。また、組織接着剤は、結合に水を必要とします。） </li><li>適切なサイズのクローニングリングを選択してください。リングを拾うために解剖ピンセットを使用し、10μlのピペットチップを均等にクローニングリングに組織接着剤の少量を適用します。 注：リングのクローニングに組織接着剤の最小限の量（小さなリングのための〜0.2μl）を使用し、組織接着剤は、良好なシールを確実にするために、底面の周りに均一に広がることを確認してください。過剰な組織接着剤は、熱を生成し、細胞を死滅させることができるフィルムを形成します。 </li><li> ECコロニーの上クローニングリングを置きます。優しく里を接着するためにクローニングリングを押し下げますプレート上にngの。コロニーがリングを接着前に乾燥されていないことを確認してください。 </li><li>すぐにクローニングリングに酵素的細胞剥離溶液25μlをピペットと〜1分間インキュベートするか、細胞が緩く付着されるまで。 </li><li>クローニングリング滴下予熱EC培地を含む6ウェルプレートの1ウェル中にピペットで細胞。 50でクローニングリングを洗う - できるだけ多くの細胞を収集するために、100μlのECメディア、および同じ6ウェルにすべての洗浄を転送;：（。重要ECはタイトなクラスターに成長することを好む細胞を分散させるためにプレートを振らないでください） 。 </li><li> 10cmディッシュ内のいくつかのコロニーが収穫するには小さすぎる場合、コロニーはさらに数日間増殖させ、10ミリリットル新鮮な培地を追加し、再びクローニングリングの手順を繰り返します。 </li><li> 10cmの組織培養皿でそれらを拡大する前に、6ウェルプレートの3ウェルに細胞を100％コンフルエントと転送 - 80まで6ウェルプレート中に採取したコロニーを成長させます。汚染物質の細胞を削り取ります。繰り返し必要に応じて - リングの手順（3.10ステップ3.5）をクローニングの別のラウンド。拡張時 - （70％〜60）は比較的コンフルエント細胞を保管してください。あまりにもまばらなメッキ場合ECが成長している停止することがあります。 </li><li>など、FACS、染色、PCR法により、ECを特徴づけるはディル-AC-LDLは蛍光性であるし、FACSのためにPEまたは他の蛍光抗体を妨げる可能性があることに注意してください。 </li></ol>

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The authors have nothing to disclose.

これにより、純粋な一次TEC培養、例えば、ヒト臍帯静脈EC（HUVEC）13として市販されているEC線や主要EC を用いたin vitro の研究で多くの代替TECを得ることが困難に。しかし、正常組織からこれらのEC集団は、それらの正常な対応物とは著しく異なるTECのためのプロキシとして機能することができます。例えば、TEC は 、生体内で表現型および機能的異常である?...

内皮細胞（EC）は、固形腫瘍の開発中に必要不可欠です。休眠腫瘍における血管新生スイッチの開始から離れた部位に転移の普及と播種に、ECは、血液を提供導管を形成する酸素および栄養素は、腫瘍増殖1を維持します 。最近示唆したように、ECはまた、灌流独立した機能を有しており、がん幹細胞および他の腫瘍間質細胞2-5の増殖を支持するニッチを形成します。このように、高度に精製された腫瘍特異的EC（TEC） のin vitro培養のための腫瘍血管新生および腫瘍細胞とのクロストークを仲介する新たな分子メカニズムの解明に役立つであろうルーチン機能研究を可能にします。 ECの起源6の組織に応じて高度に専門化しています。異なる腫瘍タイプの不均一な性質及び腫瘍微小環境に、TECはまた、腫瘍特異的分業Oを反映して独自の機能が表示されることがあります血管系F。例えば、異なる種類または腫瘍7,8のグレードから分離されたTECにおける遺伝子発現シグネチャの著しい変動があります。しかし、TECと、特に腫瘍関連線維芽細胞および腫瘍細胞、非ECの同時精製頻繁には、ゲノムワイドな発現解析を混乱することができます。これらの望ましくない細胞型は、TEC培養物のインビトロでの拡大を長期的に依存している研究では特に問題です。 ここで説明するには、一貫して腫瘍および他の組織からの純粋なEC培養を生成し、高忠実度の方法です。 EC画分と共精製された非ECの除去の免疫列濃縮した後、追加のクローニングリングステップは9を使用している純粋なECコロニーをキャプチャします。各コロニーは、非ECを汚染することなく、複数出現継代培養で増殖することができます。また、この方法は、遠藤の研究のための理想的である単一の単離手順、から複数のECクローンをもたらしthelial異質。 ZsGreen リットル/秒/リットルレポーターマウス文化10でZsGreen蛍光を維持する「運命マッピングされた」と消えないマークされたECを生成するための貴重なツールです。また、Cdh5 CREがいることを示しています。プロトコルへの微調整では、この方法は、異なる腫瘍タイプまたは正常組織に適応する必要があります。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s medium (1 g/L D-glucose) (LG-DMEM)</td>
			<td>Gibco</td>
			<td>11885-084</td>
			<td></td>
		</tr>
		<tr>
			<td>EGM-2 Bullet Kit&#160;</td>
			<td>Lonza</td>
			<td>CC4176</td>
			<td>Not all components used</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (Hyclone)</td>
			<td>Thermo Scientific</td>
			<td>SH30071.03</td>
			<td>Heat inactivated at 56&#176;C for 30 min</td>
		</tr>
		<tr>
			<td>Nu-Serum IV</td>
			<td>Corning</td>
			<td>CB-51004</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBBS)</td>
			<td>Gibco</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS buffer&#160;</td>
			<td>-</td>
			<td>-</td>
			<td>0.5 % BSA and 2 mM EDTA in PBS, filtered through a 0.22 &#956;m filter</td>
		</tr>
		<tr>
			<td>75% v/v ethanol for disinfection</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PE microbeads&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-048-801</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA) fraction V, 7.5%</td>
			<td>Gibco</td>
			<td>15260-37</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell freezing media (Bambanker)</td>
			<td>Wako Chemicals</td>
			<td>302-14681</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type II&#160;&#160;</td>
			<td>Worthington Biochemical</td>
			<td>LS004176</td>
			<td>Make stock concentration 2 mg/ml in HBSS</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I (DNase)</td>
			<td>Worthington Biochemical</td>
			<td>LS002004</td>
			<td>Make stock concentration 1 mg/mL in PBS</td>
		</tr>
		<tr>
			<td>Dil-Ac-LDL</td>
			<td>Biomedical Technologies</td>
			<td>BT-902</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, 0.5M, pH 8.0</td>
			<td>Cellgro</td>
			<td>46-034-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzymatic cell detachment solution (Accutase)</td>
			<td>Sigma-Aldrich</td>
			<td>A6964-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin, 2 % in water, tissue culture grade</td>
			<td>Sigma-Aldrich</td>
			<td>G1393-100ML</td>
			<td>Dilute in PBS to make 0.5 % gelatin solution</td>
		</tr>
		<tr>
			<td>Mouse FcR Blocking Reagent&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-092-575</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral protease (Dispase)</td>
			<td>Worthington Biochemical</td>
			<td>LS02104</td>
			<td>Make stock concentration 2.5 U/mL in HBSS</td>
		</tr>
		<tr>
			<td>PE-rat anti-mouse CD31 antibody</td>
			<td>BD Pharmingen</td>
			<td>553373</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC lysis buffer (BD Pharm Lyse)</td>
			<td>BD Pharmingen</td>
			<td>555899</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue, 0.4 %&#160;</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mm tissue culture dishes</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes (sterile)</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes (sterile)&#160;</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plates</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-dissociator tubes (gentleMACS) C tubes)&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Separator&#160; (MidiMACS)</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 100 &#956;m&#160;</td>
			<td>Corning</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Cloning rings (assorted sizes)</td>
			<td>Bel-Art Products</td>
			<td>378470000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryotubes</td>
			<td>Thermo Scientific</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting board</td>
			<td>-</td>
			<td>-</td>
			<td>Sterilize or disinfect with 75% v/v ethanol before use&#160;</td>
		</tr>
		<tr>
			<td>Dissecting forceps and scissors</td>
			<td>-</td>
			<td>-</td>
			<td>Sterilize before use&#160;</td>
		</tr>
		<tr>
			<td>Dissecting pins 2&#34;</td>
			<td>-</td>
			<td>-</td>
			<td>Sterilize before use&#160;</td>
		</tr>
		<tr>
			<td>FACS tubes with 35 &#956;m filter cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter cup (Stericup, 0.22 &#956;m)</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine-tip marker</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Multistand</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue adhesive (Vetbond)</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>Or a centrifuge with similar capacity for 15 mL and 50 mL conical tube centrifugation</td>
		</tr>
		<tr>
			<td>Tissue culture hood</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue dissociator (gentleMACS)</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-235</td>
			<td>Preset program &#34;m_impTumor_01&#34; used for tissue dissociation&#160;</td>
		</tr>
		<tr>
			<td>Liquid nitrogen freezer</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate or rotary shaker</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase contrast light microscope</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture expansion of tumor-specific endothelial cells

新しく単離した腫瘍特異的内皮細胞（TEC）は、腫瘍の血管新生の分子機構を探索し、癌の新規血管新生阻害剤を開発するためのインビトロモデルとしての役割を果たすために使用することができます。しかし、マウスの内皮細胞（EC） の in vitroでの展開での長期起因非ECに表現型の文化のドリフト（内皮-間葉移行）や汚染に困難です。これは容易に培養液中の共精製された線維芽細胞または腫瘍細胞によってoutcompetedされているTECに特に当てはまります。ここでは、コロニーの選択にインビトロで結合された拡張免疫磁気濃縮を利用する高忠実度の分離方法について説明します。このアプローチは、間質細胞または腫瘍細胞の汚染の全く自由である純粋なECの画分を生成します。また、その系譜トレースCdh5のCREを示している：本明細書で説明するプロトコルで使用ZsGreen リットル/秒/リットルレポーターマウスは、セルを検証する貴重なツールですこれらのマウスから単離したECコロニーとして純度は、文化の中で、耐久性と鮮やかなZsGreenの蛍光を示します。

ECは、ほとんどの成人組織11の総細胞集団のわずかな割合を表しています。これは、完全に細胞外マトリックス（ECM）および結合組織からECの最大放出を確実にする単一細胞懸濁液中に採取した組織を消化することが重要です。我々の経験では、CD31媒介免疫磁気選択は、濃縮されただけではなく、純粋なEC画分を提供します。従って、別の重要なステップは、クローニングリング（ <st...

Watch this Scientific Journal Video about Isolation and Culture Expansion of Tumor-specific Endothelial Cells at JoVE.com

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

腫瘍特異的内皮細胞の単離および培養拡大

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

isolation-and-culture-expansion-of-tumor-specific-endothelial-cells

Research

JoVE Journal

Medicine

11.1K ビュー.  University of North Carolina at Chapel Hill. We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models. 

ビデオ: Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

単離、特徴付けと二つの異なる方法を使用して永久歯に由来するヒト歯髄幹細胞の分化の比較

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

医学

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

CD133の患者由来の細胞培養および分離<sup&gt; +</supメラノーマから&gt;推定されるがん幹細胞

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

ヒト胎児膵臓細胞クラスターの単離、培養、およびイメージング

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

臨床応用のためのヒト羊膜上皮細胞の単離、凍結保存と文化

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

マルチパラメータ蛍光活性化細胞選別によるヒトリンパ内皮細胞の単離

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

犬の動脈と静脈からの初代内皮細胞の単離および培養

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

健康なボランティアからの内皮前駆細胞の単離および心臓手術後の血清試料の影響を受け彼らの渡り鳥の可能性

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

準備と<em&gt;インビトロ</em磁化のmiR修飾内皮細胞の&gt;キャラクタリゼーション

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the &#34;natural&#34; anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

We present an in vitro model which allows the study and analysis of coagulation in whole, non-anticoagulated blood. Anticoagulation in the system depends on the natural anticoagulation effect of healthy endothelial cells and endothelial cell activation will result in clotting.

3 D 細胞培養と非高血圧症全血を用いた血管内皮細胞の抗凝固および抗炎症作用の評価

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite of these eccentric hamstring actions are concentric quadriceps actions, where the larger and likely stronger quadriceps straighten the knee. Therefore, to stabilize the lower limbs during movement, the hamstrings must eccentrically combat against the strong knee-straightening torque of the quadriceps. As such, eccentric hamstring strength expressed relative to concentric quadricep strength is commonly referred to as the &#34;functional ratio&#34; as most movements in sports require simultaneous concentric knee extension and eccentric knee flexion. To increase the strength, resiliency, and functional performance of the hamstrings, it is necessary to test and train the hamstrings at different eccentric speeds. The main purpose of this work is to provide instructions for measuring and interpreting eccentric hamstring strength. Techniques for measuring the functional ratio using isokinetic dynamometry are provided and sample data will be compared. Additionally, we briefly describe how to address hamstring strength deficiencies or unilateral strength differences using exercises that specifically focus on increasing eccentric hamstring strength.

The hamstrings are a group of muscles that are sometimes problematic for athletes, resulting in soft tissue injury in the lower limbs. To prevent such injuries, functional training of the hamstrings requires intensive eccentric contractions. Additionally, hamstring function should be tested in relation to quadricep function at different contraction speeds.

筋肉の不均衡: テストと競技人口の機能的な偏心ハムスト リングの筋力トレーニング

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...