The authors would like to acknowledge Hans de Graaf and Tomas Veenendaal for their technical assistance in culturing the ECs.

倫理の声明：本研究で用いた血管は、新鮮な犬の死体から得た余剰材料として回収した（n = 4）は他の無関係の研究（大学3R政策）のために安楽死させ、健康な犬から。異常な血管（内および肝外門脈体循環シャント、n = 1のそれぞれ）は、ユトレヒト大学のコンパニオンアニマルのための大学クリニックに提示犬から所有者のインフォームドコンセント後の死後に採取しました。 1.単離および培養初代イヌ内皮細胞の<ol><li> 2ミリリットル/ウェルの0.5％重量/容量ゼラチンとでプレコート6ウェルプレートを37℃で5％CO 2の加湿雰囲気でインキュベーター中で2時間放置します。前の一次細胞を播種し、過剰なゼラチン溶液を除去します。 </li><li>無菌的に新鮮な犬の死体（ 図1A）から関心の血管（複数可）（ 例えば、大動脈、大静脈、静脈門脈）を取り外します。血管系の解剖学を維持興味のある容器の両端にストレートピンセットを置くことによって。内皮細胞の損傷を防止するために鉗子で組織の不必要な操作を避けてください。 <ol><li>手術用ハサミで両端にクランプされた容器をカットし、死体から取り外します。氷上のハンクス平衡塩溶液（HBSS）で輸送。 注：約5cmの血管の長さは、この手順のために好ましいが、1cmに測定するものはまた、培養に十分な細胞を提供します。 </li></ol></li><li>氷冷HBSSで満たされたペトリ皿に血管を転送します。手術用はさみを使用して、容器の外部から任意の付着組織と脂肪を除去し、（ 図1B）は無傷の容器自体を維持します。最適なビューのためのクランプまたは鉗子で脇に周囲の組織を引っ張る：容器は切断、すべての回ではっきりと見えることを確認してください。 <ol><li>合字と容器の任意の枝を閉じて（ 例えば 、ポリグラクチン3-0）、続いてそれらを削除手術用ハサミやメスで。 </li></ol></li><li>容器は、湾曲したハルステッドモスキート鉗子で終わる容器の他端に鉗子の先端をクランプし、その後撤回、それは完全に裏返し（ 図1C-G）になるまでゆっくりと容器を反転慎重を入力します。外側は今内皮細胞層で構成されています。いずれかの難易度は、容器を反転する際に遭遇した場合、摩擦を低減するために、再度HBSSに水没。 </li><li>それを完全に閉じるために、消化手順（ 図1H）への任意の非内皮血管組織の暴露を防止するために、容器の両端に巾着縫合糸を配置します。反転容器を操作するリガチャー端を使用してください。 </li><li>消化培養が後に行われた場合、前消化プロトコル（ステップ1.7）に反転容器を凍結保存。 <ol><li>クライオバイアルに反転容器を置き、凍結培地の細胞培養でいっぱい。凍結の共同を使用して、-80℃まで凍結ntainer。 -80℃で保存容器は1週間以内に使用される場合。長期保存の場合は、場所は-180℃で凍結バイアル。 ECの分離を行う場合は、水浴（37℃）で急速にクリオバイアルを解凍し、直ちに氷冷HBSS中に配置します。 1.7に示すように進んでください。 注：分離後のECの合計収率が原因で凍結プロセス中の生存能力の喪失に低くなることを考慮してください。 </li></ol></li><li> （ 図1I）50ミリリットルチューブに容器を移し、（解凍の際に又は残留凍結媒体）赤血球を除去するために、HBSS中でそれを2回すすいでください。 </li><li>断続的に穏やかで、37℃で1時間、イヌ内皮細胞増殖培地（CECGM）に30ミリリットルの溶液コラゲナーゼII型（0.15 U / ml）及びディスパーゼ（0.15 U / ml）で50ミリリットルチューブに容器をダイジェスト攪拌。 </li><li>チューブから容器を外し、250×gで5分間、細胞懸濁液を遠心してください。 </li><li> CECGM aの細胞ペレットを再懸濁しNDシード2mLの細胞懸濁液をウェルあたり（1 - 3のウェルは、容器のサイズに応じて）。培養物を空気中5％CO 2を含む加湿雰囲気中、37℃での細胞とは1週間に2回培地を変えます。 </li><li>通路70の密集度、細胞 - 80％に達しました。 <ol><li>死亡または非付着細胞を除去するために、予め温めたHBSSで細胞を洗浄。各ウェルに200μlの組換え細胞解離酵素（1X）を追加し、5分間、または全ての細胞が分離されるまで、バックインキュベーター内に置きます。 </li><li> 15mlチューブに細胞を移し、10％ウシ胎児血清（FCS）を含む培地10mlの（CEGM）を加えることによりトリプシン処理を停止させます。 250×gで5分間遠心分離します。ゼラチンプレコートプレートまたはフラスコ培養を続けます。 </li></ol></li></ol> 2.キャラクタリゼーション<ol><li>文化イヌ大動脈内皮細胞（CnAOECs）、顕微鏡で週二回、プライマリおよび市販の細胞株の形態を比較します。 注：T彼は、典型的な&gt; 70％の集密度に達したときに最も良く観察することができるパッチに電気部品のパターンを成長させます。 <ol><li> 0.5％w / vのゼラチンをプレコートしたT75フラスコ上CECGM文化CnAOECs。週に一度継代細胞密集度が70である - 80％。 <ol><li>継代のために、予め温めておいたHBSSで細胞を1回洗浄し、1 mlの組換え細胞解離酵素（1X）を追加します。 5分間、または全ての細胞が分離されるまで、バックインキュベーターでフラスコを置きます。 10％FCSを含む10mlの培地（CEGM）を添加することによってトリプシンを不活性化します。 、250×gで5分間、細胞懸濁液を遠心し、上清を捨て、1mlの培養培地中で細胞を再懸濁。 </li><li>細胞懸濁液の10μlのアリコートを取り、1希薄：0.4％トリパンブルーで1。自動細胞カウンターを用いて細胞を計数し、新しいT75フラスコに4.0×10 5生細胞をプレートアウト。 5％CO 2の加湿雰囲気中で37℃でフラスコや文化に予め温めておいたCECGMの10ミリリットルを追加します。 </li></ol></li></ol></li><li>培養CaPECsとCnAOECsの通路1からRNAを分離します。 <ol><li>試料調製試薬（SPR）の20μlを添加し、細胞を溶解するために1分間インキュベートし、ペレットとして、少なくとも1×10 3個の細胞を収集します。インキュベーション後、慎重に-70℃で全RNAとストアを含む細胞溶解物を収集します。 </li></ol></li><li>製造業者の説明書に従ってcDNA合成キット（ 例えば、iScript）を使用してcDNAにするmRNAを変換します。定量PCRを実行する準備ができてまで長期的に4°C一週間まで、または-20℃でで保存したcDNAを用いることができます。 </li><li>内皮細胞の起源を確認するために、内皮マーカーCD31の遺伝子発現を測定します。 MIQEのPRECISガイドライン10を使用して実験と定量化を行います。正規化のために、参照遺伝子GAPDH、RPS19、およびB2MG 11の発現を測定します。 <ol><li>ヌクレアーゼを含まない水で得られたcDNAの10倍希釈を準備します。 </li><li>準備します標準的なラインのためのプールされたcDNA試料から4倍希釈し、非鋳型対照としてヌクレアーゼを含まない水を使用しています。 qPCR反応のために50倍希釈合計に到達するために、サンプルを5倍希釈します。 4μlのcDNAおよびリバースとフォワードプライマー（ 表1）の20ピコモルと混合した蛍光体の6μLを使用して、384ウェルフォーマットで二重にピペット10μlの反応。 </li><li>変性のために95℃で10秒、アニーリングおよび伸長のためのTmで30秒間の39サイクル、続いてTaqポリメラーゼの活性化のため5分間、95℃にプログラムを設定します。各プライマーセットのTmを表1に示します。 </li><li>融解曲線を実行すると、1つの製品だけが増幅されていることを確認するためにあらゆるランの次に分析します。参照遺伝子の平均相対量を使用して試料の発現レベルを標準化し、反応効率が95％と105％の間である場合のΔCtを計算します。 </li></ol></li><li> AngIとECの機能性を評価ogenesisアッセイ。 <ol><li>予め冷却した血管新生のスライドの各ウェルに細胞外マトリックスの10μlを添加して、ウェルの表面を覆うようにピペットチップを用いて拡散。氷上で細胞外マトリックスを維持し、ゲル内への気泡の導入を避けます。 30分間、5％CO 2、37℃のインキュベーター中で水に浸したペーパータオルでペトリ皿にスライドを配置することによってゲルを固めます。 </li><li> 1.0×10 4、主なものを追加します。 50μlの内皮増殖培地ウェルあたりでのEC。 5％CO 2で37℃で6時間、スライドをインキュベートします。 </li><li> 6時間後、画像全体をよく含まれていることを確認し、20Xの倍率で写真を撮ります。 </li></ol></li></ol>

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The authors have no competing financial interests.

イヌのECに焦点を当てた研究ではCnAOECプライマリラインは犬3、12、13の内皮系統をモデル化するために使用されている。ヒトの研究では、HUVECの文化は、まだゴールドスタンダードと考えられています。明らかに、臍帯由来のECに単に焦点を当てすることは、心血管研究の会社制限があります。内皮細胞は、静脈の仕様を決定する特定の遺伝子の発現パターンを有します。出生後の血管...

イヌは、心血管疾患の研究のための大型動物モデルとして使用され、また先天性（遺伝的）血管異常1,2苦しむことができる。商業内皮細胞株は、しばしば、内皮細胞（EC）の機能を評価するために使用されるこれらの疾患を研究します。犬のための犬の大動脈由来（CnAOEC）利用可能な1の市販の内皮細胞株は、そこにあります。この細胞株は、主制御通常のEC 3-5として研究に使用されています。人間の心血管研究では、最も一般的に使用される内皮細胞株は、ヒト臍帯静脈内皮細胞（HUVEC）およびヒト臍帯動脈内皮細胞（HUAECs）は、それぞれ、ヒト臍帯静脈と動脈由来。 HUVECを、1980 6以降血管研究の黄金の標準として使用されてきました。これらは、内皮機能および疾患の適応を研究するための古典的なモデル系であると考えられます。異なる血管から単離された内皮細胞は、appearancで変化します遺伝的背景と微小環境7への曝露に起因する電子と機能。また、HUVECをとHUAECsは、臍帯からの条件に関して完全に模倣大人の血管が、彼らはにし、病気への応答さらされていない可能性があり、発達血管構造を導出しています。したがって、一般的には、心血管疾患にHUVECを中に見出された結果とHUAECsを翻訳することは不適切です。 大人ECの適応と行動を研究すると、関心の容器からの一次のECは、より直接的なアプローチとして使用されるべきです。これらの細胞を単離するために、いくつかの方法が報告されています。また、HUVECをするために使用され、広く記載された方法は、酵素消化液8と容器をフラッシュします。これは、多くの場合、平滑筋細胞および線維芽細胞9のような非電気部品の混入をもたらします。分離のためのもう一つの頻繁に使用される方法は、蛍光に続いみじん切り血管組織の酵素消化であります分化（CD）31 7、8。FACSソーティングの内皮細胞マーカーのクラスタとそれに続く細胞培養に基づいて選別（FACS）活性化細胞は、細胞を比較的大量に必要とし、したがって小血管からの内皮細胞の単離に適していません。したがって、我々は、高純度で、様々なイヌの血管から純粋な内皮細胞集団を単離するための新しい堅牢な方法を開発することを目的としました。新しい分離方法の効率を試験するために、我々は、単離され、大小異なるイヌの動脈と静脈からの純粋な犬の初代内皮細胞（CaPEC）の文化を、取得しました。この方法はまた、先天性細胞内または肝外門脈体循環シャント、犬2で共通の疾患などの罹患および/または異常な血管由来の内皮細胞の培養を可能にします。血管の大部分は整数のままための方法は、血管平滑筋細胞と同様の容器の追加の関連する細胞型の単離を可能にします手順の間に行動します。

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			<td height="21" style="height:21px;width:328px;">DMEM (1X) + GlutaMAX</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:211px;">31966-021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Hank&#39;s Balanced Salt Solution</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:211px;">14025-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Canine Endothelial Cells Growth Medium&#160;</td>
			<td style="width:164px;">Cell Applications</td>
			<td style="width:211px;">Cn211-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">CnAOECs</td>
			<td style="width:164px;">Cell Applications</td>
			<td>Cn304-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Fetal Calf Serum (FCS)&#160;</td>
			<td style="width:164px;">GE Healthcare</td>
			<td style="width:211px;">16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TrypLE Express</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:211px;">12604-013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">SPR</td>
			<td style="width:164px;">Bio-Rad</td>
			<td>170-8898</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">iScript synthesis kit</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:211px;">170-8891</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">SYBR green super mix</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:211px;">170-8886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Recovery Cell Freezing Medium</td>
			<td style="width:164px;">Gibco/Life Technologies</td>
			<td style="width:211px;">12648-010</td>
			<td>Keep on ice prior to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Freezing container, Nalgene Mr. Frosty</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:211px;">C1562</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Gelatin</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:211px;">G1890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Surgical scissors (Mayo or Metzenbaum)</td>
			<td style="width:164px;">B. Braun Medical</td>
			<td style="width:211px;">BC555R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mosquito forceps&#160;</td>
			<td style="width:164px;">B. Braun Medical</td>
			<td style="width:211px;">FB440R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Mosquito forceps curved</td>
			<td style="width:164px;">B. Braun Medical</td>
			<td style="width:211px;">FB441R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">polyglactin 3-0</td>
			<td style="width:164px;">Ethicon</td>
			<td style="width:211px;">VCP311H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Trypan blue</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:211px;">145-0013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Automated counting chamber</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:211px;">145-0102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Counting Slides, Dual Chamber</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:211px;">145-0011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Matrigel</td>
			<td style="width:164px;">BD Biosciences</td>
			<td style="width:211px;">BD356231</td>
			<td>Slowly thaw on ice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">&#181;-Slide Angiogenesis</td>
			<td style="width:164px;">Ibidi</td>
			<td style="width:211px;">81501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Endothelial Growth Medium</td>
			<td style="width:164px;">Lonza</td>
			<td style="width:211px;">CC-3156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">EGM-2 SingleQuot Kit&#160;</td>
			<td style="width:164px;">Lonza</td>
			<td style="width:211px;">CC-4176</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

別の血管が正常に記載の単離プロトコール（ 図2）に供しました。解剖や健康犬の大動脈、大静脈、静脈門脈、及び冠動脈反転することが可能であった（それぞれのイヌからの全ての船舶を、N = 4）。同じアプローチのECは2先天性門脈体循環シャント（肝外と肝内、n = 1のそれぞれ）から単離したと。大動脈を容易に反転されたが、胸部大動脈セグメント?...

Watch this Scientific Journal Video about Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins at JoVE.com

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

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-culture-primary-endothelial-cells-from-canine-arteries

Research

JoVE Journal

Medicine

11.2K ビュー.  Utrecht University. 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.

ビデオ: Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

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 ...

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

人間の前庭神経鞘腫の初代培養

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

ソリッド検体からの原発性卵巣癌細胞を得る方法

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

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 Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

マウス癌モデルからの循環腫瘍細胞の単離と伝播

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

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 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 ...

A Cell Culture Model of Resistance Arteries

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and signaling processes that can control vascular tone and blood pressure. As it is a projection from either the endothelial or smooth muscle cell, and due to its small size (on average, an area of ~1 &#181;m2), the MEJ is difficult to study in isolation. However, we have developed a cell culture model called the vascular cell co-culture (VCCC) that allows for in vitro MEJ formation, endothelial cell polarization, and dissection of signaling proteins and processes in the vascular wall of resistance arteries. The VCCC has a multitude of applications and can be adapted to suit different cell types. The model consists of two cell types grown on opposite sides of a filter with 0.4 &#181;m pores in which the in vitro MEJs can form. Here we describe how to create the VCCC via plating of cells and isolation of endothelial, MEJ, and smooth muscle fractions, which can then be used for protein isolation or activity assays. The filter with intact cell layers can be fixed, embedded, and sectioned for immunofluorescent analysis. Importantly, many of the discoveries from this model have been confirmed using intact resistance arteries, underscoring its physiological relevance.

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

抵抗血管の細胞培養モデル

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