この作品は、統合失調症研究のための国民の同盟とうつ病（NARSAD）若手研究者賞と米国立衛生研究所のAVにR01NS073635を助成金によってサポートされていました。

1。試薬および溶液の調製の<ol><li> 35mm培養皿のコーティング：コラーゲン1型溶液を、20mM酢酸（〜100mgのタンパク質/バイアル）中の水溶液として供給される。滅菌組織培養グレードの水を用いて0.01％の使用濃度にコラーゲン溶液の適切な容量に希釈する。 2〜8℃で、室温（RT）又は37℃で3-4時間、コラーゲン溶液1ml、または一晩でコートディッシュコー​​トディッシュから余分な溶液を除去し、それを一晩乾燥させます。メディアを追加する前に、組織培養グレードの水またはPBSでお皿を洗浄します。被覆皿1ヶ月間4℃で保存することができる。 </li><li>完全ダルベッコ改変イーグル培地（DMEM）を準備します。 （;吸油量1 mlの1×濃度）を、10％FBSおよび抗生物質 - 抗真菌溶液でDMEM（500ml）に補う。完全DMEM 2ヶ月間4℃で保存することができる。 </li><li>アリコートをDNアーゼI（0.001 mg / ml）を有するDMEM（50 ml）を調製し、（Dと称されるMEM-DNアーゼI）。 </li><li>コラゲナーゼおよびディスパーゼ（DMEM-collagenase/dispaseと呼ぶ）（1 mg / ml）を含むDMEMの別のアリコート（10ml）に調製する。 </li><li> EGF（5μg）を、ECGS（100mg）および抗生物質 - 抗真菌溶液5μlを加えた内皮細胞培養培地（ECCM、500ミリリットル）を、準備します。 ECCM 2ヶ月間4℃で保存することができる。 </li><li>使用前にすべてのメディアを予熱し。 </li><li> 0.5％BSAおよび2mM EDTAを含むPBS、pHが7.2：精製緩衝液を準備します。冷緩衝を維持（2〜8℃）。 </li></ol> 2。終脳の胚および解剖の除去実験動物を用いて、全ての実験は、マクリーン病院の動物のケアと使用委員会によって承認され、実験動物の管理と使用に関するNI​​Hガイドラインに準拠している。 <ol><li>時限妊娠CD1マウス（胎生15日目）を使用します。膣栓の発見の日は、胎生0（E0）と考えられている。 CD1ダムは一般的に大規模なリットルを生産するため、10月12日胚Sは妊娠ダムから期待されている。 </li><li>子宮切開のために、ケタミン（100 mg / kgの）/キシラジン（5 mg / kg）を腹腔内注射で妊娠マウスを麻酔。マウス約20〜30 gで、10ミリリットル/キログラムケタミン/キシラジン麻酔液の0.3ミリリットルを提供し、その痛みや苦痛は麻酔薬の腹腔内注射時に最小限であることを確認してください。注射のために手の拘束を使用してください。 </li><li>足指のピンチで深麻酔をチェックしてください。深く麻酔したマウスから胚を取り出します。母親から除去すると、すぐに各胚を首を切る。氷冷PBS中の滅菌ペトリ皿に胚ヘッドを配置します。 </li><li> 2013版：麻酔薬の過剰投与（130 mg / kgのペントバルビタール、IP）、動物の安楽死のためのAVMAガイドラインと一致して安楽死させる方法、とすぐに安楽死により子宮摘出に従ってください。 </li><li>実体顕微鏡下で、細かいマイクロチップはさみや細かい鉗子で頭から脳を削除します。注：高品質の解剖イオンが正常胚の脳から軟膜膜を除去することが不可欠である。これは実施によって達成される。細かい鉗子とマイクロチップのはさみはまた、良好な解剖を取得するための重要なツールです。 </li><li>軟膜膜を剥がすために、脳やマイクロチップのはさみをしっかりグリップを得るために鉗子を使用してください。中脳と後脳を取り出して、終脳を分離します。氷の中の2ミリリットルPBSで35mm培養皿にすべての胚から軟膜フリー終脳を転送します。 </li></ol> 3。細胞単離<ol><li>メスの刃との1〜2mmの断片に終脳をミンチし、完全DMEM 2ミリリットルを含む15ミリリットルファルコンチューブ内の組織を収集します。 </li><li>塊が消え、乳白色の懸濁液が達成されるまで、1ミリリットルピペットで静かに徹底的に組織を解離する。 </li><li>遠心分離機は、室温で5分間800〜1,000×gで細胞を解離し。 </li><li>慎重にゆっくりとCEの関連付けを解除したDMEM-DNアーゼIの上清を再懸濁ペレットを削除LLS再度RTで5分間、800〜1,000×gでで1 mlピペット及び遠心分離機を用いて。 </li><li>暖かい完全DMEMにペレットを再懸濁。無菌70μmのナイロンメッシュを通してフィルタセル。フィルタリング細胞を収集し、氷中に保管してください。 </li><li>細胞部分がメッシュ上に保持され、RTで1〜2分間DMEM-collagenase/dispase（2ml）でさらに消化集める。室温で5分間800〜1,000×gの遠心分離によって、DMEM-DNアーゼIにセル3回洗浄します。ステップ3.5で収集フィルタリング細胞とこれらの細胞をプール。完全DMEMで一度全細胞を洗浄します。細胞数および磁気標識ステップを決定するために進んでください。注：これは、磁気標識の前に単一細胞懸濁液を得ることが重要である。 </li></ol> 4。磁気標識<ol><li>このプロトコルは、10 7個の細胞の磁気標識を示す。 10 7以上の細胞数が低いため、同じ試薬量および10 7を超える細胞数のために使うと、試薬容量と総容量のスケールアップこのため（ 例えば、2×10 7全細胞のために、すべての示された試薬の二倍のボリュームを使用します）。氷の中のすべての試薬および細胞を維持する。 </li><li>血球計数器で細胞数を決定します。 </li><li>室温で10分間300×gで遠心細胞懸濁​​液。完全に上清を吸引し。 </li><li> CD31マイクロビーズを10μlに続く細胞ペレットに精製緩衝液の90μl/107セルを追加。製造業者によって提供されるマイクロビーズ、抗マウスCD31のモノクローナル抗体をコンジュゲートされる。 </li><li>よく混ぜ、冷蔵庫で15分間インキュベートする。注：磁気標識の間より高い温度および/またはより長いインキュベーション時間は、非特異的結合をもたらし得る。 </li><li> 10分間300×gで精製緩衝遠心1-2エルフテックス7細胞を追加することで細胞を洗浄。上清を除去し、精製緩衝液500μl中に細胞を再懸濁。磁気分離または精製ステップに進みます。 </li></ol> 5。 PUrification <ol><li> MACSセパレーターの磁場中でMSカラムに配置します。 </li><li>精製緩衝液500μlでカラムを洗浄します。 </li><li>カラムに細胞懸濁液を適用します。高い細胞数が列を詰まらせる可能性があるMSカラムに適切な数の細胞をロードします。注意：各MSカラムは、2×10 7セルの最大数を収容することができ、より高い細胞数を、より多くの列を使用しています。 </li><li>収集および非標識細胞を含むフロースルーを捨てる。 </li><li>精製緩衝液500μlで3回カラムを洗浄。洗浄工程の間に、列のリザーバは、その後の精製緩衝液の一定分量を追加する前に空であることを確認してください。 </li><li> MACS分離器から列を削除し、15ミリリットルファルコンチューブの上に置きます。 </li><li>プランジ​​ャは、MSカラムと共に供給される。 MSのカラムにピペット精製緩衝液の1ミリリットル、すぐにしっかりと列にプランジャーを押すことにより、磁気的に標識した細胞を洗い流す。 </li><li>プレートCEL35mm培養皿上のlsは、コラーゲンタイプ1でプレコートし、95％O 2および5％CO 2、37℃に設定したインキュベーター中ECCM中の細胞を成長させる。 </li><li> 4日ごとに培地を変更します。料理がコンフルエントのとき10〜12日後に実験やサブカルチャーのためのPVECsを使用してください。 </li></ol> 6。 PV ECのサブカルチャー<ol><li> ECCMを取り外し、滅菌PBS、1〜2 mlのPVECSの単層をすすぎます。 </li><li>穏やかに細胞単層を覆うように、0.25％トリプシン-EDTA溶液1.5mlを加える。 </li><li>インキュベーターに料理を返します。 5-7分以内に、細胞は、皿の表面から剥離します。 </li><li>細胞が剥離した後、直ちにトリプシンインヒビター等量のを追加し、数回粉砕する。 </li><li>無菌15ミリリットルファルコンチューブに細胞を移す。 5分間500×gでチューブを遠心分離する。 </li><li>上清を除去し、予め温めECCMの1ミリリットル中にペレットを再懸濁します。 </li><li>文化、IMを拡大するために細胞を播種老化または他のアッセイ。 </li></ol>

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利害の対立が宣言されていません。

PVEC年代は、より生理学的に関連する神経血管の相互作用に焦点を当てた研究のための他の組織源からの大人の脳内皮細胞とのECよりもあり、また治療の可能性を持っている。 PVECの準備のためには、死んだ細胞は、CD31マイクロビーズに非特異的に結合することができるので、優れた実行可能性を達成するために、高速動作するように郭清を伴う重要な始まりです。単一細胞懸濁液は、従来の...

血管新生、神経発生および神経細胞移動は、中枢神経系（CNS）の開発、修復および再生における重要な事象である。いくつかのエレガントな研究は、内皮細胞が可溶性因子の放出を介して直接接触することによりニューロンの増殖およびその逆を刺激することが示されている。我々はそれが好奇心これらの研究1-3の大部分であることが見出さ神経前駆細胞/神経幹細胞は、胚の脳から単離されている間、それらは成人の脳、他の成体組織源から、または内皮細胞株と内皮細胞と共培養される。これは、部分的には、胚の脳から内皮細胞の純粋な集団を単離し、培養に関連する技術的な問題が原因である可能性があります。しかし、血管形成、神経発生、および神経細胞の移行は、より堅牢な胚の脳の正常な成人の脳に比べて桁違いに生じる同時イベントです。 embryoniの脳室周囲血管網Cの前脳（終脳）は、大脳基底核の原基内にある容器から発信され、胎生11（E11）4,5で終脳背側腹側からの整然とした勾配の形で開発しています。脳室周囲血管網の血管のこの神経叢は起源、解剖学的位置、成長パターン、および発生調節4,5に基づき、軟膜血管とは異なります。脳室周囲の血管新生勾配の伝播方向は、終脳の横断神経遺伝勾配と一致します。終脳内では、脳室周囲の血管新生勾配及び移行のGABAニューロンの勾配は接線方向にも空間的に6に重なる。タイミングに関しては、血管新生勾配は、約日までに神経遺伝勾配及びGABAニューロン勾配の進んでいる。したがって、内皮細胞は、脳室周囲空間的および時間的によく終脳の神経発生をサポートするために重要な手がかりを提供するように配置され、ニューロン移動4,6。したがって、神経前駆細胞及び/又はニューロンと共培養実験において、胚性脳室周囲の内皮細胞の使用は、神経血管の相互作用を研究し、神経変性疾患または虚血性/外傷性脳損傷の治療のための新規な手段を開発するためのより良好なモデルを提供するであろう。 我々は、上皮細胞の混入を制限することなく、分子的および脳室周囲血管網4,6（軟膜のECと区別するPVECs称される）の内皮細胞からの機能的に区別される軟膜血管内皮細胞を分離するだけでなく、軟膜膜を除去することの重要性を強調。ここでは、私たちが日常的にPVECsの豊かで純粋な収量を得るために、我々の研究室で使用する方法について説明します。これらの内皮細胞は、単一の時限妊娠マウスから単離された胚性前脳から調製される。これらは、膨張継代培養し、将来の使用のために正常に凍結することができる。

<table><tbody><tr><td>DNase I</td><td>Sigma</td><td>D-4527</td><td>&amp;nbsp;</td></tr><tr><td>Collagen, Type 1 solution from rat tail&amp;nbsp;</td><td>Sigma</td><td>C3867</td><td>&amp;nbsp;</td></tr><tr><td>DPBS</td><td>Quality Biologicals</td><td>114057-131</td><td>&amp;nbsp;</td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>M4055</td><td>&amp;nbsp;</td></tr><tr><td>BSA</td><td>Sigma</td><td>A2058</td><td>&amp;nbsp;</td></tr><tr><td>MS column</td><td>Miltenyi Biotech</td><td>130-042-201</td><td>&amp;nbsp;</td></tr><tr><td>CD31 microbeads</td><td>Miltenyi Biotech</td><td>130-097-418</td><td>&amp;nbsp;</td></tr><tr><td>MACS separator</td><td>Miltenyi Biotech</td><td>130-042-102</td><td>&amp;nbsp;</td></tr><tr><td>MACS multi stand</td><td>Miltenyi Biotech</td><td>130-042-303</td><td>&amp;nbsp;</td></tr><tr><td>Cell strainer 70 &amp;micro;m</td><td>BD Bioscience</td><td>352350</td><td>&amp;nbsp;</td></tr><tr><td>Antibiotic and antimycotic solution</td><td>Sigma</td><td>A5955</td><td>&amp;nbsp;</td></tr><tr><td>FBS</td><td>Sigma</td><td>F4135</td><td>&amp;nbsp;</td></tr><tr><td>Collagenase/Dispase</td><td>Roche</td><td>10269638001</td><td>&amp;nbsp;</td></tr><tr><td>DMEM&amp;nbsp;</td><td>Lonza</td><td>12-604F</td><td>&amp;nbsp;</td></tr><tr><td>35 mm Culture dish</td><td>BD Bioscience</td><td>353001</td><td>&amp;nbsp;</td></tr><tr><td>15 ml Falcon tube&amp;nbsp;</td><td>BD Bioscience</td><td>352097</td><td>&amp;nbsp;</td></tr><tr><td>50 ml Falcon tube</td><td>BD Bioscience</td><td>352098</td><td>&amp;nbsp;</td></tr><tr><td>ECCM kit</td><td>BD Bioscience</td><td>355054</td><td>Kit Includes Endothelial cell growth supplement, EGF, and Soybean Trypsin Inhibitor</td></tr><tr><td>Endothelial Cell Growth Supplement (ECGS)</td><td>BD Bioscience</td><td>354006</td><td>&amp;nbsp;</td></tr><tr><td>RBECGM</td><td>Cell Applications</td><td>R819-500</td><td>&amp;nbsp;</td></tr><tr><td>DMEM F12&amp;nbsp;</td><td>Life Technologies</td><td>10565-018</td><td>&amp;nbsp;</td></tr><tr><td>GlutaMAX</td><td>Life Technologies</td><td>305050-061</td><td>&amp;nbsp;</td></tr><tr><td>Tissue culture grade water</td><td>Life Technologies</td><td>15230162</td><td>&amp;nbsp;</td></tr><tr><td>0.25% Trypsin</td><td>Life Technologies</td><td>15050</td><td>&amp;nbsp;</td></tr><tr><td>Soybean trypsin inhibitor</td><td>BD Bioscience</td><td>5425</td><td>&amp;nbsp;</td></tr><tr><td>Matrigel</td><td>BD Bioscience</td><td>354234</td><td>&amp;nbsp;</td></tr><tr><td>Qtracker 655 Cell Labeling Kit</td><td>Life Technologies</td><td>Q25021</td><td>&amp;nbsp;</td></tr><tr><td>CellLight Plasma Membrane-RFP, BacMam 2.0</td><td>Life Technologies</td><td>C10608</td><td>&amp;nbsp;</td></tr><tr><td>Biotinylated Isolectin B4 antibody</td><td>Sigma</td><td>L2140</td><td>&amp;nbsp;</td></tr><tr><td>Anti-Von Willebrand factor</td><td>Sigma</td><td>F3520</td><td>&amp;nbsp;</td></tr><tr><td>Anti-CD31/PECAM-1</td><td>BD Pharmingen</td><td>550274</td><td>&amp;nbsp;</td></tr><tr><td>VECTASHIELD Hardset Mounting Media with DAPI</td><td>Vector Laboratories</td><td>H-1500</td><td>&amp;nbsp;</td></tr><tr><td>Ketamine</td><td>Butler Schein Animal Health Supply</td><td>44028</td><td>&amp;nbsp;</td></tr><tr><td>Xylazine</td><td>Lloyd Laboratories</td><td>1009</td><td>&amp;nbsp;</td></tr><tr><td>Stereomicroscope</td><td>Motic</td><td>SMZ-168</td><td>&amp;nbsp;</td></tr><tr><td>Hemocytometer</td><td>Fisher Scientific</td><td>267110</td><td>&amp;nbsp;</td></tr><tr><td>Inverted microscope</td><td>Olympus CK-40</td><td>CK-40</td><td>&amp;nbsp;</td></tr><tr><td>Fluorescent microscope</td><td>Olympus FSX-100</td><td>FSX-100</td><td>&amp;nbsp;</td></tr><tr><td>Fine forceps</td><td>Roboz Surgical Instrument</td><td>7 inox</td><td>&amp;nbsp;</td></tr><tr><td>Fine microtip scissors</td><td>Roboz Surgical Instrument</td><td>RS5611</td><td>&amp;nbsp;</td></tr></tbody></table>

isolation and culture of endothelial cells from the embryonic forebrain

胚の脳内皮細胞は、血管新生および神経血管の発達との相互作用の研究における重要なツールとして機能することができる。胚前脳、軟膜および脳室周囲の2血管網は、空間的に独特であり、異なる起源と成長パターンを持っている。軟膜および脳室周囲血管網から​​の内皮細胞は、固有の遺伝子発現プロフィールおよび機能を有している。ここでは、胚前脳（終脳）の脳室周囲血管網（PVECs）からの内皮細胞の純粋な集団の単離、文化、および検証のためのステップバイステップのプロトコルを提示する。このアプローチでは、胎齢15日のマウスから得た軟膜膜を欠く終脳は、みじん切りされたコラゲナーゼ/ディスパーゼを用いて消化し、単一細胞懸濁液中に機械的に分散した。 PVECsは強い磁場を使用してマイクロビーズに結合させanti-CD-31/PECAM-1抗体で陽性選択を用いて細胞懸濁液から精製される分離方法。コンフルエントとなるまで、さらに継代培養細胞は、精製された内皮細胞培養培地中のコラーゲン1コーティングした培養皿上で培養される。位相差光学顕微鏡、蛍光顕微鏡で可視化されるようPVECsは、このプロトコルの展示石畳と紡錘形の表現型を用いて得られる。 PVEC培養の純度は、内皮細胞のマーカーで設立されました。私たちの手では、この方法では、確実にかつ一貫してPVECsの純粋な集団が得られます。このプロトコルは、前脳の血管新生への機械論的な洞察を得るPVEC相互作用、および神経細胞の種類とのクロストークを理解し、治療的血管形成のための途方もない可能性を秘めていることを目的とした研究の利益になる。

1-12日目からPVECsの表現型の特徴は、位相差光学顕微鏡検査（ 図2）によって示されている。細胞分裂（ 図2A）の1日目を表示形態学特性上皿に付着した細胞。 5-8日の間、石畳からPVECs遷移は、内皮細胞およびそのin vivoの状態（ 図2Bおよび2C）により類似の典型的な形の形態をスピンドルます。 12日目までにPVEC文化が完全な合流点（ <stro...

Watch this Scientific Journal Video about Isolation and Culture of Endothelial Cells from the Embryonic Forebrain at JoVE.com

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

このビデオでは、10〜12日以内に、胚前脳からの内皮細胞の純粋培養を調製するための簡単​​で信頼性の高い戦略を示し、脳血管新生のさまざまな側面に焦点を当てた研究のために有用であろう。

胚前脳からの内皮細胞の単離および培養

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

isolation-culture-endothelial-cells-from-embryonic

Research

JoVE Journal

Neuroscience

15.3K ビュー.  Harvard Medical School. このビデオでは、10〜12日以内に、胚前脳からの内皮細胞の純粋培養を調製するための簡単​​で信頼性の高い戦略を示し、脳血管新生のさまざまな側面に焦点を当てた研究のために有用であろう。 

ビデオ: Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

ひと臍帯血由来の血管内皮前駆細胞の分離

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

発生生物学

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

として不死化マウス脳微小血管内皮細胞株の生成<em&gt; in vitroで</em&gt;血液脳関門モデル

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

神経科学

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

初代マウス脳微小血管内皮細胞の単離

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

新生児マウスからの肺血管内皮細胞の単離と培養

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

生物学

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

胚心室内皮細胞の単離

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

マウス大動脈内皮細胞の単離および初代培養

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

マウスの冠動脈内皮細胞の単離

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

A Triple Culture Cell System Modeling the Human Blood-Brain Barrier

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls and restrict access to the brain parenchyma. However, with the development of nanotechnologies, large panels of new nanomaterials were developed to improve drug delivery, highlighting the need for reliable in vitro microsystems to predict brain penetration in the frame of preclinical assays. Here is a straightforward method to set up a microphysiological system to model the BBB using solely human cells. In its configuration, the model consists of a triple culture including brain-like endothelial cells (BLECs), pericytes, and astrocytes, the three main BBB cellular actors necessary to induce and regulate the BBB properties in a more physiological manner without the requirement of tightening compounds. The model developed in a 12-well plate format, ready after 6 days of triple culture, is characterized in physical properties, gene, and protein expressions and used for polymeric nanogel transport measurement. The model can be used for an extensive range of experiments in healthy and pathological conditions and represents a valuable tool for preclinical assessments of molecule and particle transport, as well as inter-and intracellular trafficking.

This protocol describes a method to establish a human blood-brain barrier (BBB) in vitro model. The endothelial cells and pericytes are seeded on each side of an insert filter (blood compartment), and astrocytes are seeded in the bottom well (brain compartment). The model characterized was used for nanoparticles transport experiments.

ヒト血液脳関門をモデル化した三重培養細胞系

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls ...

Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow increases with decreasing diameter from arteries to arterioles that ultimately feed into capillaries. Due to their smaller size and location in the parenchyma, arterioles have been relatively understudied and with less reproducibility in findings than surface pial arteries. Regardless, arteriolar endothelial cell structure and function&#8212;integral to the physiology and etiology of chronic degenerative diseases&#8212;requires extensive investigation. In particular, emerging evidence demonstrates that compromised endothelial function precedes and exacerbates cognitive impairment and dementia.
In the parenchymal microcirculation, endothelial K+ channel function is the most robust stimulus to finely control the spread of vasodilation to promote increases in blood flow to areas of neuronal activity. This paper illustrates a refined method for freshly isolating intact and electrically coupled endothelial &#34;tubes&#34; (diameter, ~25 &#181;m) from mouse brain parenchymal arterioles. Arteriolar endothelial tubes are secured during physiological conditions (37 &#176;C, pH 7.4) to resolve experimental variables that encompass K+ channel function and their regulation, including intracellular Ca2+ dynamics, changes in membrane potential, and membrane lipid regulation. A distinct technical advantage versus arterial endothelium is the enhanced morphological resolution of cell and organelle (e.g., mitochondria) dimensions, which expands the usefulness of this technique. Healthy cerebral perfusion throughout life entails robust endothelial function in parenchymal arterioles, directly linking blood flow to the fueling of neuronal and glial activity throughout precise anatomical regions of the brain. Thus, it is expected that this method will significantly advance the general knowledge of vascular physiology and neuroscience concerning the healthy and diseased brain.

Intensive preparation of intact mouse cerebral endothelial &#34;tubes&#34; from cerebral parenchymal arterioles is illustrated for studying cerebral blood flow regulation. Further, we demonstrate the experimental strengths of this endothelial study model for fluorescence imaging and electrophysiology measurement of key cellular signaling pathways, including changes in intracellular [Ca2+] and membrane potential.

マウス脳実質の動脈内皮の単離と機能解析

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow ...

Endothelial Cell Culture from Rat Brain Microvessels: A Procedure to Isolate and Culture Endothelial Cells from Microvessels of Rat Brain

Source: Molino, Y. et al. <a href="https://www.jove.com/v/51278/setting-up-an-vitro-model-rat-blood-brain-barrier-bbb-focus-on-bbb">Setting-up an&#160;In Vitro&#160;Model of Rat Blood-Brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport.</a> J. Vis. Exp.&#160;(2014)
This video presents the isolation of microvessels from a rat brain using a combination of density gradient centrifugation and enzymatic digestion mehods. The isolated microvessels can be cultured in a culture flask pre-coated with collagen and fibronectin matrix to obtain endothelial cell colonies.

ラット脳微小血管からの内皮細胞培養:ラット脳の微小血管から内皮細胞を単離および培養する手順

Source: Molino, Y. et al. Setting-up an&#160;In Vitro&#160;Model of Rat Blood-Brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated ...