この作品は、ドイツ学術振興による臨床研究（IZKF）ミュンスターのための学際センター（SEED 03/12、SB）、（DFG）によってサポートされていました、細胞·イン·モーション·エクセレンスのクラスター（EXC 1003 - CIM）、ミュンスター大学（SB及びSGM）とそれ以外クローネ-フレゼニウス-財団（SB及びSGMへ2012_A88）による。私たちは、優れたイラスト平家ブルムに感謝します。

全ての動物実験は、地方当局（ &quot;; Fachbereich 87、Tiergesundheit、Tierschutz、LandesamtfürNatur、環世界ウントVerbraucherschutz NRWレックリングハウゼン、ドイツ&quot;）によって承認されたと動物愛護のドイツの法律に基づいて実施しました。 マウス実験1.一般的なコメント<ol><li>それぞれの施設内動物管理使用委員会のガイドラインに従ってマウスを使用して、すべての実験を行う。 </li><li>病原体を含まない条件下でマウスを維持し、食物および水を自由にアクセスできるようにすることを可能。 </li><li>生物学的特性は、年齢や性別に応じて変化することができますので、実験群における年齢および性別をマッチさせたマウスを使用してください。 </li><li>可能な限り動物実験を減らす洗練または交換してみてください。 </li></ol>パーコール勾配の調製<ol><li> 1ミリリットル10倍のPBS、1ミリリットルFCSおよび10ミリリットルのパーコールでの1X PBS 19ミリリットルを混ぜる。 </li><li>サントを行うガラス管（：0.2μmのフィルター孔の直径）でろ過をrile。その後、4℃でソリューションを保存します。 </li></ol>内皮細胞培地および細胞培養プレートのコーティングの調製<ol><li>内皮細胞培地。 <ol><li> 500mlの培地の調製：（約20％）を100mlのPDSを400ミリリットルDMEM培地（約80％）のミックスを0.25 mlのbFGF（20μg/ mlの、約0.05％）、0.5 mlのヘパリンを（100μl/ウェルmlであり、約0.1％）、0.5mlのpyrumycin（4 mg / mlの、約0.1％）。唯一の培養の最初の2日間において使用される細胞培養培地のためpyrumycin加える。 </li><li>ガラス管（：0.2μmのフィルター孔の直径）で無菌濾過を行う。その後、4℃でソリューションを保存します。 </li></ol></li><li>細胞培養プレートのコーティング： <ol><li>コーティング液1mlについては、500μlののddH 2 O、400μlのコラーゲンIV（0.4 mg / ml）で100μlのフィブロネクチン（0.1mg / mlの）の溶液を調製する。 </li><li> EACの全面を覆うコー​​ティング溶液を用いて細胞培養プレートの時間十分。 4℃で一晩プレートを保管してください。 </li></ol></li></ol>マウス脳微小血管内皮細胞（MBMEC）の4.絶縁<ol><li>頚椎脱臼により - （12週齢、C57BL / 6〜8）10成体マウスを生け贄に捧げる。その後5mlのPBSでマウスの脳や店舗を隔離（警告：！仕事無菌）。 </li><li>鉗子で脳茎、小脳や視床を削除します。慎重に滅菌吸取紙の上に脳を転がすことによって髄膜を取り外してください。その結果髄膜フリー前脳を収集します。 </li><li> DMEMを13.5ミリリットルで満たされた50ミリリットルファルコンチューブに脳を転送します。 </li><li>メディアが乳白色になるまで、10ミリリットルピペットで、最初の25ミリリットルピペットで、組織をミンチ。 </li><li>そして、軌道上で37℃で1時間インキュベートしたダルベッコ改変イーグル培地（DMEM）0.6 mlコラゲナーゼCLS2た（10mg / mlのDMEM中で）0.2mlのDNアーゼ（PBS中1mg / ml）との混合物で組織を消化振る180 rpmでR。 </li><li> DMEM 10 mlを加え、4℃で10分間、組織懸濁液を遠心1,000×gで細胞を添加した。 </li><li>上澄みを捨てる（警告：ペレットが失うものは簡単です！） </li><li>ミエリンを削除するには、25ミリリットルのBSA-DMEM（約25倍）（20％w / v）の25mlのピペット、および4℃で20分間千×gで遠心分離してペレットを再懸濁します。 </li><li>大径の割れたガラスパスツールピペットを用いて、上側ミエリン層（乳白色）を破棄し、その後、通常のパスツールピペットで、BSA層を捨てる。 </li><li> 9ミリリットルDMEM中でペレットを再懸濁し、1mlのコラゲナーゼ/ディスパーゼを0.1mlをDNA分解酵素（最終濃度を1mg / mlである）を追加します。 180 rpmでオービタルシェーカー上で37℃で1時間のためのソリューションのダイジェスト（警告を：再懸濁するためにピペットを使用していない - 細​​胞が失われる可能性があります）。 </li><li>消化中に、加速度、のWiで、4℃で1時間3000×gで超遠心機を用いた密度勾配を設定するためにパーコール液を遠心ブレーキthout。 </li><li>消化された細胞懸濁液にDMEMを10ミリリットルを追加します。遠心機で4℃で10分間千×gでサスペンション。その後、上清を破棄し、DMEMを2mlにペレットを再懸濁。 </li><li>加速とブレーキなしで4℃で10分間700×gでパーコール勾配遠心の上に慎重に再懸濁した細胞を追加します。注：細胞が間期におけるクラウドとして表示されます。 </li><li>約取る。長い滅菌針で細胞と間期の12ミリリットルのDMEM 5mlで新しい50ミリリットルファルコンに配置します。 </li><li> 4℃で10分間千×gで再び細胞を遠心します。その後（約使用。細胞培養プレートの媒体1単位cm 2面0.2 ml）を内皮細胞培地中でペレットを再懸濁する。 </li><li>コーティングされた細胞培養プレートからコーティング溶液を除去し（ ステップ3.2参照）と、2つの連続した洗浄工程において、滅菌PBSで各ウェルすすぐ。 </li><li>内皮に​​おける細胞培養を維持する細胞を37℃で培地および滅菌インキュベーター内で5％CO 2。 2〜3日ごとに培地を変更してください。 90％コンフルエンスで細胞を分割します。 </li></ol>

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The authors declare that they have no competing financial interests.

血液脳関門の機能障害は、多発性硬化症におけるBBBの破壊など広範囲に自己反応性免疫細胞によるCNS浸潤を促進し、オリゴデンドロサイトの出会いや破壊を可能にする様々な有害なCNS疾患の顕著な特徴である。虚血性脳卒中では、BBBの破壊および脳浮腫のその後の形成は、二次梗塞の成長と患者6の全生存期間に影響を与える重要な要因である。さらに、遠隔地BBBの変化によって焦点虚血性病変は、脳の広範な機能的変化を誘導することができる。しかし、根本的な分子メカニズムは不明な点が多い5。これとは対照的に、機能的BMECs高密度かつ排出トランスポーターの多様性は、そのような感染症のための脳の悪性腫瘍または抗生物質のための化学療法剤として有益な薬剤の濃度を低減します。そのため、血液脳バーをより深く理解生理学的および病態生理学的条件下でRIER機能は特に好ま方向21にバリア機能を調節することができる薬理学的薬剤の開発が求められている。 BBBのin vitroモデルにおける信頼性の高いは、BBBでの調節機構を研究するための不可欠なツールです。 多くの不死化された脳内皮細胞株を確立し、インビトロ BBBモデル22として用いられてきた。彼らはより速く成長し、いくつかの継代にわたって一定のBBB特性を保つように、これらの細胞株は、プライマリBMECsにわたって特定の利点を提供します。しかし、内皮細胞株は完全にin vivoの状況に実験結果の転写混ざりなどの重要なプロパティが変更された初代細胞を代替することはできません。例えば、マウス脳内皮細胞株bEnd.5高paracにつながる不連続に分布するタイトジャンクショ​​ンタンパク質の低いレベルのみを発現するellular透磁率23。のbEnd、別の頻繁に使用されるマウス脳内皮細胞株は、緻密で連続的に分布し、タイトジャンクショ​​ン、高内皮性、いくつかの排出トランスポーターと低傍細胞透過性を示している。しかし、のbEnd細胞層の主BMECsに比べ、一定の経と傍細胞基質24の浸透に関して本当の差別を欠いている。 初代細胞培養の調製では、混入細胞が有意に実験結果の妥当性を妨げる可能性があります。記載されたプロトコルでは、pyrumicin高純度を達成するために、内皮細胞のための選択剤として使用される。それにもかかわらず、細胞培養物の定期的な品質管理は、必然的に信頼性のある実験のために保証するために必要とされる。内皮細胞の典型的な形態学的特性（ 図1Aを参照）、transendothelia以外の品質管理のためのいくつかの方法があり、lの抵抗を測定する場合や、そのようなCD31内皮細胞マーカーの発現（ 図1Bを参照）、またはフォンウィルブランド因子（vWF）が評価されるかもしれない。 一匹のマウスの脳から単離された脳内皮細胞の数が比較的低く、複数の実験のための適切な細胞培養を設定することで、いくつかのマウスの脳をプールしなければならない。我々の経験では、少なくとも10匹のマウスは、それぞれの動物施設のリソースに応じて制限する要因かもしれない一つの実験のために使用される必要がある。バイアスや交絡因子を避けるために、同一の遺伝的および環境的背景を持つマウスのみをプールする必要があります。対照的に、ウシおよびブタの脳内皮細胞調製物は、一脳内の利用可能な脳内皮細胞の大多数を提供する。しかし、合併症のない飼育や住宅、利用可能なトランスジェニックマウスの広範かつ増え続けるレパートリーに加えて初代マウスBMECsは理想としてレンダリング血液脳関門の機能を研究するためのモデル。 BBB基礎となる分子機構の機能に関するいくつかの重要な知見は、二次元組織培養系における研究から来ている。最近では、三次元培養系は、内皮細胞が管腔と管状ネットワークを形成できるようにすることコラーゲンマトリックス内に配置されている図25に示すように、開発されてきた。これらの新規な細胞培養系は、 インビボで無傷の生物における血管のプロセスのより正確な再現を可能にする。このような周皮細胞およびアストロサイトなどNVU 3Dで細胞培養系のさらなる細胞の関与は、血液脳関門の機能を研究するin vitroでの革命をもたらすかもしれない。最初のモデルは、最近26を開発されてきた。 トラブルシューティングのためのいくつかの推奨事項があります。すべての無菌作業の最初は、内皮細胞培養物の品質に必須である。第二に、pyrumycinはセルのみのために追加する必要があります培養の最初の2日間に使用される培地は、長いアプリケーションが増加MBMEC細胞死および低品質のバリア機能につながるかもしれない。第三に、このプロトコルに適用される酵素は、製造業者の指示に従って使用して保存されるべきである。そうでなければそれらの適切な機能が損なわれる可能性があります。内皮細胞が付着しない場合はさらに、細胞培養プレートのコーティングが改正される可能性があります。フィブロネクチンおよびコラーゲンの濃度は、変更される場合があり、または他のマトリックスタンパク質は、コーティング溶液に添加してもよい。最後に、年齢、性別、季節および動物施設内の環境条件がMBMEC分離実験の比較可能性の品質に影響を与える可能性のある重要な要因である。これは、条件が独立した実験の間で同等であることを保証されるべきである。 記載されたプロトコルは、マウスBMECsを分離するための基本的な神経免疫法として考慮されるべきである。単離された細胞は、Uかもしれませんそのような移動アッセイ、遺伝子およびタンパク質発現の研究​​、電気生理学的評価または透過性実験としてBBB機能へのさらなる洞察を得るために、さまざまなアプリケーションのためのsed。上述したように、BMECsの三次元細胞培養系は、NVUの文脈におけるBBBの調節に関与する複雑な機構を研究するための新たな機会を提供するかもしれない。したがって、一次内皮細胞の単離は、将来的にBBB研究の分野で非常に貴重な技術のままである。

<html>高度に専門化血液脳関門（BBB）の一部である脳微小血管内皮細胞（BMEC）フォームの単層。 BMECsは基底膜に付着し、接合部タンパク質によって相互接続されている。一緒に周皮細胞、平滑筋細胞、星状細胞エンドフィート、それらは、いわゆる神経血管単位（NVU）1を構築循環する血液細胞と混合する。血液および中枢神経--系（CNS）との間に不透過性バリアの以前の概念に対して、NVUは脳血管およびCNS 2の間の流体、分子および細胞の転移を制御し、動的な非常に特異的で、安定化インタフェースです。 NVUの機能不全または調節不全は、開始および/ ​​または虚血性脳卒中、HIV-脳症、多発性硬化症、アルツハイマー病またはパーキンソン病など3-6 CNSの神経血管、感染性、炎症性または変性疾患、種々のに寄与し得る。 _content &quot;&gt; BMEC単層が緊密に接合部複合体により封止されているタイト（TJ）から構成され、接合（AJ）をアドヘレン7。高い電気抵抗とBBBの低い傍細胞透過性は、主にTJ蛋白質8に基づいています。のTJであるこのようzonulaoccludens（ZO）タンパク質などのアダプター分子によって内皮細胞の細胞骨格に連結されているクローディンとオクルディンファミリーの膜貫通タンパク質によって形成された複合体ZO1-3 4アドヘレン接合は、主に内在性膜タンパク質によって組み立てられる。カテニン8を介して細胞骨格にリンクされている血管内皮（VE）カドヘリン、。 BBBの密封は、血液およびCSFの間で基板および細胞の自由な交換を防ぐことができます。この規則の例外は、脂質媒介拡散9によってBBBを横切ることができる分子量&lt;400 Daの、親油性、小分子である。より大きいおよび/または通路例えば、グルコース、アミノ酸、ペプチド、タンパク質、および多くの薬剤のような親水性分子は、5つの主要なカテゴリーに分類することができる、高度に制御された経細胞輸送システム10に限定される：担体輸送、イオン輸送、能動排出輸送、受容体媒介輸送、およびカベオラ媒介輸送4。これらの輸送​​体系は、正確な信号の生成、形質導入および統合のために必要とされるCNSの恒常性を維持するのに役立ちます。また、BMECsも積極ectoenzymes、様々の発現により明確な分子の遷移を制御することができる。これらの酵素は阻害しまたはBBB 11の移行を可能にする特定の内因性および外因性の基質を細胞表面に局在し、変更されている。 CNSは、長い時間のための免疫特権臓器と考えられたのに対し、最近の知見は、免疫survのではなく、ダイナミックで厳密に調節システムを提案CNSのeillance。 BMECsは決定的に免疫細胞の遊走の調節に関与する。その表面リンパ球上のセレクチンの発現によって選択的に緩く内皮に付着するように誘導される。白血球上の特異的受容体との遭遇ケモカインの分泌は、式またはそのようなLFA-1などの白血球インテグリンのコンホメーション変化をもたらす（リンパ球機能関連抗原-1）およびVLA-4（非常に遅く抗原4）。インテグリンは、間に、またはBBB 12-14のBMECsを通して脳実質内への遊出を可能にするそれらの内皮counterreceptors、 例えば VCAM-I（血管細胞接着分子）、ICAM-I（細胞間接着分子）を結合することにより、強固な接着を媒介する。これらおよび他の知見は、免疫細胞の遊走の調節における内皮細胞自体の積極的な役割を強調する。 さらに、BMECs NVUの一部が脳血流liの調節に関与しているよう地元のニューロンの代謝要求にnked。星状細胞の刺激内皮細胞に血管平滑筋細胞の弛緩15に至る酸化窒素などの血管作動性物質を産生する。 開発において、ならびに成体の脳を示し平行パターニングおよび開発における規制1,4の多くの特性を共有し、血管新生および神経発生。内皮細胞は、これらのプロセス16,17に決定的に関与している。 要約すると、BMECsは、CNSの適切な発達と機能を保証するために不可欠な機能を提供する。 BBBの機能不全は、多くの重篤な神経疾患にリンクされています。しかし、ごく少数の標的は、特異的かつ効率的な治療の18脳血管系界面において同定されている。 in vitroモデルは、簡略化のために見よ機能と複雑な生理的システムの調節に関与するメカニズムを理解するために使用されているngの時間。分離は、この原稿と特定のマウスノックアウト-株の様々な特定のネズミBMECs のin vitro研究により記載されるように、新たな治療の道を開く生理学的および病態生理学的条件下で、BBB機能および調節のさらなる理解を提供するかもしれない。

<table border="0" cellpadding="0" cellspacing="0" width="808">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">bFGF</td>
			<td style="width:133px;">Peprotech</td>
			<td style="width:123px;">100-18B</td>
			<td style="width:331px;">Basic fibroblast growth factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">BSA</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">A9418</td>
			<td>Bovine serum albumine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Collagenase CLS2</td>
			<td style="width:133px;">Worthington</td>
			<td style="width:123px;">LS004176</td>
			<td>High relative level of protease activity</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Collagenase-dispase</td>
			<td style="width:133px;">Roche</td>
			<td align="right" style="width:123px;">10269638001</td>
			<td style="width:331px;">Collagenase from V. alginolyticus, Dispase from B. polymyxa</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Collagen IV</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">C0543</td>
			<td style="width:331px;">From Engelbreth-Holm-Swarm murine sarcoma basement membrane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">DMEM</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">D5030</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">DNAse</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">D4513</td>
			<td>Deoxyribonuclease I from bovine pancreas</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">FCS</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">F6178</td>
			<td>Fetal calf serum (also named fetal bovine serum)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Heparin</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">H3393</td>
			<td>Anticoagulant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">PDS</td>
			<td style="width:133px;">First Link UK Ltd.</td>
			<td style="width:123px;">60-00-850</td>
			<td>Plasma-derived serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Percoll</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">P1644</td>
			<td>Warm to room temperature before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Pyrumycin</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">P8833</td>
			<td>Should only be used in first 2 d of culture</td>
		</tr>
	</tbody>
</table>

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.

単離直後、マウスBMECsと混入細胞は、細胞培養培地（ 図1A、0日目）に浮遊集塊を形成する。彼らはそのような毒素の相対抵抗をもたらすP-糖タンパク質のような排出トランスポーターの高レベルで発現するのでpyrumycinによる治療は、マウスBMECsの選択につながる。混入細胞媒介性細胞傷害19 pyrumycinはるかに受けやすい。 MBMECsはコラーゲンIV /フィブロネクチンコーティングした細胞培養プレート（ 図1A、1日目と2日目）にアタッチし始めるのに対し、pyrumicin選択の過程で、混入細胞は、アポトーシスまたは壊死性細胞死を入力してください。 5日目までは、MBMECsはおよその密度に増殖する。 80から90パーセント。これらは、典型的な紡錘状の外観によって特徴付けられる。合流で、彼らは密集し、長手方向に整列し、非重複接触禁止細胞（ 図1A、5日目）の単層を形成する。バイ（; 図1B PECAM1）は、内皮細胞マーカーCD31によって実証されるようにpyrumicin選択、最大99％までMBMECの高純度が達成される。 MBMECsはしっかりとタイトジャンクショ​​ンタンパク質によって相互接続された密封された単分子層を形成する。培養8日後、内皮細胞層は、電気抵抗のための安定した値を構築する（20の間の典型的な値に到達する- - 30Ωのxセンチ2）7の後に、キャパシタンス（ 図1C）は、無細胞のような、この時点では機能的アッセイ。エバンスブルーまたはデキストランで例えば遊走実験3、20,21および透過性テストは、実施すべきである。 <img alt="図1" src="/files/ftp_upload/52204/52204fig1highres.jpg" width="500" /> 図1.形態学的およびネズミBMECsの機能特性。送信光マイクロによって見培養BMECsの（A）代表経時変化5日間のSCOPY。スケールバーはすべての画像に適用される。内皮細胞マーカーCD31のための（B）免疫蛍光染色を5日目にCD31（緑色）及びDAPI（青色）で、（C）細胞を5日間前培養した後のための自動化システムに移した生物物理学的特性（抵抗と容量）の分析。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52204/52204fig1highres.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Isolation of Primary Murine Brain Microvascular Endothelial Cells at JoVE.com

Isolation of Primary Murine Brain Microvascular Endothelial Cells

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-of-primary-murine-brain-microvascular-endothelial-cells

Nanyang Technological University

Research

JoVE Journal

Neuroscience

32.2K ビュー.  University of Münster. 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. 

ビデオ: Isolation of Primary Murine Brain Microvascular Endothelial Cells

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 and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

胎児マウス神経管から神経堤細胞の単離および培養

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

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 and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

胚性齧歯類の脳から単離、プライマリ脳ドーパミンニューロンの文化と長期メンテナンス

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost oligodendrocytes, myelin, axons, and neurons. Remyelination is an endogenous repair mechanism whereby new myelin is produced subsequent to proliferation, recruitment, and differentiation of oligodendrocyte precursor cells into myelin-forming oligodendrocytes, and is necessary to protect axons from further damage. Currently, all therapeutics for the treatment of multiple sclerosis target the aberrant immune component of the disease, which reduce inflammatory relapses but do not prevent progression to irreversible neurological decline. It is therefore imperative that remyelination-promoting strategies be developed which may delay disease progression and perhaps reverse neurological symptoms. Several animal models of demyelination exist, including experimental autoimmune encephalomyelitis and curprizone; however, there are limitations in their use for studying remyelination. A more robust approach is the focal injection of toxins into the central nervous system, including the detergent lysolecithin into the spinal cord white matter of rodents. In this protocol, we demonstrate that the surgical procedure involved in injecting lysolecithin into the ventral white matter of mice is fast, cost-effective, and requires no additional materials than those commercially available. This procedure is important not only for studying the normal events involved in the remyelination process, but also as a pre-clinical tool for screening candidate remyelination-promoting therapeutics.

Demyelinating diseases can be modeled in animals by focal application of lysolecithin into the CNS. A single injection of lysolecithin into mouse spinal cord produces a lesion that spontaneously repairs over time. The goal is to study factors involved in de- and remyelination, and to test agents for enhancing repair.

リゾレシチンの焦点注入による実験的脱髄およびマウス脊髄の再ミエリン化

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

2次元培養系を用いた主な大脳皮質細胞のイメージングをライブします。

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

プライマリ マウス骨格筋の微小血管内皮細胞の分離

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

シアログリカンの代謝標識による原発神経幹および前駆細胞の細胞表面マーカーの同定

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

ヒト幹細胞由来内皮細胞およびGABAergicニューロンの移動、化学誘引、共培養アッセイ

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

複数の中枢神経系領域からのマウスグリアの解剖と分離

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...