著者は、テクニカル サポート、エリザベート ヘレナ ブルーン、サラ クリスティン ・ クリステンセン、ニルス ・ m ・ クリステンセンを認識したいし、ルンドベック財団許可番号 R155 2013 14113。

利害の衝突を報告しません。

精製と pBEC の増殖
浄化のプロシージャの間には、重要な手順は、髄膜の迅速かつ効果的な除去・分離精製収率、純度およびモデルの適切な設置が重要です白と灰色の物質に含まれます。提示された体外BBB モデルの pBECs を使用して、我々 は改善し、孤立した灰白質、サイズ選択的微小血管、コラゲナーゼで消化の隔離のためのフィルタ リングの機械的均質化に基づ?...

血液-脳関門の細胞の構造と機能
循環、中央神経系 (CNS) のインターフェイスで BBB は適切な機能と神経系の保護のために不可欠である中枢神経系微小環境の homoeostatic コントロールのキー規制サイトとして機能します。BBB のサイトは血管内腔内皮細胞です。脳の毛細血管の内皮細胞を形成する複雑な細胞間のタイトな接合と特定の流入と流出のトランスポーターの強く偏光式パターン血液と脳1の間非常に特定の分子輸送の確保します。タイト結合錯体の構造コンポーネント オクルディンとクローディン家族、精巣毛細血管(ZO) 蛋白質、cingulin からの蛋白質を含めるし、接合部接着分子 (ジャム) を関連付けられています。クローディン 5 は傍細胞接合部制限で特に重要です。この特徴的な BBB 内皮細胞表現型の誘導そして維持を一緒に脳血管内皮細胞フォームは基底膜ニューロンとアストロ サイト ペリサイト周囲の細胞とのダイナミックな相互作用を含む、神経血管ユニット (NVU)2,3。これらの相互作用に関与するメカニズムがまだ完全に理解されない、しかし、短期的に BBB 透過性の調節ができ、長期 BBB 機能4を誘導する細胞間化学信号の交換が含まれます。アストロ サイトは特に脳血管内皮細胞の表現型に貢献する知られているし、グリア由来神経栄養因子 (細胞内 cAMP に影響を与える)5、塩基性繊維芽細胞成長因子6、規制などのソースは、ヒドロコルチゾン7、および変換増殖因子 β (TGF-β)8。しかし、TGF-β の効果は議論9をされています。
生体内での in Vitro BBB から
BBB 生物学に関する貴重な情報を提供するin vivo研究継続します。ただし、細胞モデルは追加の洞察力を提供し、健康および病気の BBB の詳細な分子と機能的な側面を理解するための便利なツールを構成できます。BBB の成分と細胞のタイプ間の複雑な相互作用が生体外モデルを完全に達成することは困難、以来があった、脳血管内皮細胞の最初の精製とモノラル文化にこれらのアプリケーション10,11,12、浄化手順と BBB の成長条件の広範な開発培養細胞モデル、体内バリアに大きい類似に 。一般的に使用される生体外でBBB モデルは、齧歯動物、ブタ、ウシ由来の初代培養細胞および不死化細胞株に基づいています。各モデルには、さまざまな長所と欠点があります。比較とモデルの選択、BBB 構造タンパク質受容体、酵素、トランスポーターの発現など検証マーカーが現在確立されたモデル1の概要を作成する使用されます。
プロトコルの目的
BBB の 1 つの重要な特徴は気密バリア性と高い TEER まだ利用可能なモデルの数が多いが反映しても体内のレベルです。いくつかの研究所から開発・最適化への取り組みを取り入れ、このプロトコルの目的は、プライマリ プライマリ pBECs MC ACM の有無、NCC に基づく BBB モデル体外高 TEER を確立する方法を提示するにはラットや豚由来のアストロ サイトは。応用手順およびモデルの確立汚染細胞を排除し、pBECs の BBB の表現型への分化を改善する努力があります。この作品は低傍細胞透過性とタイトな接合部の主要なタンパク質、トランスポーター、および受容体の機能発現を良いと信頼性の高い、高 TEER モデルの確立になりました。ただし、アストロ サイトは脳血管内皮細胞の表現型に貢献の要因、文化の 3 つの異なる条件は脳血管内皮細胞の 3 つの異なった表現型を表します。NCC モデルは創、運輸研究と細胞内輸送に関与する特定の専門にされたメカニズムの研究だけでなく、細胞間相互作用の調査のため特に有益で BBB 機能の最大の表現が、有利であります。
プロトコルの起源と歴史
ここで説明した pBEC モデルは、ルイーズ ・ モーガン博士と同僚は、成功以前ウシ脳血管内皮細胞モデル13に基づく方法でエーザイ研究所 (ロンドン) で開発された豚のモデルに基づいています。セル準備の元のメソッドは、純度を改善するために二次培養の手順に続いて、微小血管をキャッチするメッシュのナイロンを使用して 2 段ろ過が。メソッドの以前の開発、最適な BBB の表現型とバリアの堅さは ACM を含む補足の媒体の成長によって達成されました。メソッドにさらに変更は、マンチェスター英国14,15教授 (名) rothwell さんのラボで r. スキナーによって作られました。メソッドは、アボットの研究室、KCL ロンドン、どこ Patabendige では、大幅に簡単にアストロ サイトまたは ACM の使用の回避によって、ピューロマイシンをペリサイトなど汚染細胞を排除する準備をした。 によって採用されました。初論文 MC モデルに、生体内でBBB、効果的なタイトな接合、膜輸送系受容体を介したトランスサイトーシス16,17などのいくつかの重要な機能が保持されることを確認しました。,18,19,20. S. ユソフ後再び培養アストロ サイトをテストし、これはアボット ラボ21で現在使用されている最寄りのバリアント、TEER、大幅それを発見します。モデルが正常にされている今簡素化灰色を含む問題抽出、1 つだけメッシュろ過ステップ、および単一のフィルター コーティングの手順を使用して、m. ニールセンに転送ラボ オーフス、さらに変更がされているの導入 (そのプロトコルに関して)コラーゲンとフィブロネクチンを組み合わせること。豚アストロ サイト (このプロトコル) の分離の適用手順は、・ トムセンら22で説明したオールボーで t. Moos 研究室で開発したプロトコルに基づいていた。TEER とロンドンとオーフスで生成されたモデルの他のプロパティは、モデル ラボ間で転送が容易に、注意深い観察と方法の手順の合理化によく反応するという考えに信任を貸す、似ています。確かに、s. ユソフはローカル条件およびティッシュ ソースにさらに調整を関与している熱帯の国 (マレーシア)23に、今 MC モデルを確立しています。
別の方法や現在の利点は、モデルを確立
PBECs ウシおよび齧歯動物の起源の脳血管内皮細胞に比べると、次の分離24生体内でBBB の表現の損失の低い率を持っていることの利点を提供します。さらに、pBECs、bEND.5 と bEND.3 (50 Ω cm2) など細胞の単分子膜のよく報告されるレベルと比べて MC (800 Ω cm2)16で育てられたときも比較的タイトな内皮障壁を形成できます。25,26,原発性脳腫瘍と cerebEND (500 Ω cm2)29,31,32cEND (300-800 Ω cm2)28,29,30, 27(100-300 Ω cm2) マウスの血管内皮細胞ss ="xref"> 33,34,35,36とラット (100-300 Ω cm2)37,38。しかし、TEER は浄化と文化のプロシージャの依存関係を示しています。ほとんどの場合、ACM の追加やアストロ サイトとの共培養内皮層1の気密性の内皮細胞で増加差別化効果に示します。それにもかかわらず、培養条件を最適化する努力、牛ベースのモデルのみを示している TEER 値豚ベース モデル (800 Ω cm2 MC、最大 2500 Ω cm2の培養アストロ サイトの平均) に匹敵する13 ,,3940,41,42,43,44,45。ウシ脳に基づくモデルとして内皮細胞は大きな変動、研究所14,45,46,47,48, 内と間を示しています。再現性は問題になる可能性があります。ここで報告 pBEC モデルで貢献の研究所は変動の少ない、両方の研究所の間で非常によく似た TEER と傍細胞透過性の値を達成しています。したがって、それはここで紹介した方法を使用して低い変動と堅牢なモデルを確立する他の研究所の可能なはず。タイトな内皮細胞層を形成するに加え pBECs を持つモデルがタイト結合蛋白質、機能 BBB トランスポーター、受容体、酵素、及び実証適性研究15の範囲の表現によって検証されて以前,16,17,19,20,22,49,50,51,52,53,54,55,56,57,58,59。 さらに、pBECs の共培養に未発表のトランスクリプトーム データは BBB トランスポーターと受容体 (未発表結果、ニールセンら) の予想されるプロファイルを示しています。
豚の病気の進行を反映60、好ましい特徴の他の確立されたモデルよりも高い程度に人間の生物学とゲノム、解剖学、生理学、豚ベースの BBB モデルがそれ以上の利点、製薬業界。ブタ脳肉業界の共通の副産物であると血管内皮細胞、動物実験のために必要な 1 つのブタの脳から高純度精製収率を提供するの数を最小限に抑える脳の簡単にアクセスできるソースそれら構成します。浄化の一次電池の耕作はやや時間がかかるとモデルの設定の標準化のための専門知識が必要ですが、一次電池は最も信頼性の高い BBB モデルを生成します。不死化細胞株はバリアの堅さ、トランスポーター発現プロファイルと微小環境規制が生体内で実験結果61,を反映していないなど重要なプロパティとして、代用をすることはできません。62体外モデルは、生細胞の高解像度のイメージング、目標を使用してサンプリングまたは観察された細胞に近いアプローチを許可することで細胞内のプロセスの可視化を実現の利点を提供。高倍率と優れた光学的品質63。これは生きている動物の 2 光子顕微鏡の使用のためのケースではありません。さらに、体外モデルは、タグ付きタンパク質の可視化や、人身売買の調査を許可するセルを使って能力を提供します。
モデルの応用
BBB の機能は固定されていないと生理学と病理学の両方で動的に変調することができます。神経変性を含む多くの神経学的な病気で炎症・感染症、混乱、BBB の透過性亢進には、64,65,66,67 が観察されます。.減らし、病気の進行や後の損傷を防ぐために、BBB の変調の基礎となる分子メカニズムの同定と解析、主要な重要です。このコンテキストで信頼性の高い体外モデルは、製薬業界での高い需要と、さらに中枢神経系薬の BBB 透過性を予測する上で重要な役割を果たします。透過スクリーンとして任意の生体外モデル制限傍細胞経路、生理学的に現実的な細胞アーキテクチャおよび機能発現トランスポーター メカニズム68が表示されます。以前研究16,17,57, と傍細胞透過性とここで TJ と AJ タンパク質の発現を示した本モデルこれらのすべての条件を満たして、BBB の範囲に適しています両方の通常の生理学と病理学を研究。提示の浄化と栽培方法の強みには、シンプルさの組み合わせ、結果堅牢で信頼性の高い高 TEER体外BBB モデルで再現性とアストロ サイトを含める機能に影響を与えるがあります。この目的は、ブタのアストロ サイトとラットの起源が同様の方法22pBECs の BBB の表現型を拡張するために示されています。

<table><tbody><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>F1141</td><td></td></tr><tr><td>Collagen IV</td><td>Sigma-Aldrich</td><td>C5533</td><td></td></tr><tr><td>Poly-L-lysine</td><td>Sigma-Aldrich</td><td>P1524</td><td></td></tr><tr><td>DMEM/F-12</td><td>Lonza</td><td>BE12-719F</td><td></td></tr><tr><td>DMEM/Low Glucose</td><td>Sigma-Aldrich</td><td>D6046</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco Invitrogen</td><td>15140</td><td></td></tr><tr><td>Plasma derived serum (PDS)</td><td>First Link UK Ltd.</td><td>60-00-89</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco Invitrogen</td><td>10-270-106</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Gibco Invitrogen</td><td>15090-046</td><td></td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H3393</td><td></td></tr><tr><td>Puromycin</td><td>Sigma-Aldrich</td><td>P8833</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H4001</td><td></td></tr><tr><td>8-CPT-cAMP</td><td>Biolog</td><td>C010</td><td></td></tr><tr><td>RO 20-1724</td><td>Sigma-Aldrich</td><td>B8279</td><td></td></tr><tr><td>Gentamicin Sulfate</td><td>Lonza</td><td>17-518Z</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>34896</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>EtOH</td><td>VWR</td><td>20,824,296</td><td>Mix the 70 % solution from the 96 % EtOH</td></tr><tr><td>DNAse 1</td><td>Sigma-Aldrich</td><td>D4513</td><td></td></tr><tr><td>Collagenase CLS2</td><td>Sigma-Aldrich</td><td>C6885</td><td></td></tr><tr><td>ddH&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>Made with Elga System</td></tr><tr><td>T75 flasks</td><td>Thermo Scientific</td><td>156499</td><td></td></tr><tr><td>Costar Transwell inserts (Cell permeable membrane inserts)</td><td>Costar</td><td>CLS3401</td><td>12-well plate, 12 mm diameter, 0.4 &amp;mu;m polycarbonate membrane</td></tr><tr><td>15 ml centrifuge tubes</td><td>Cellstar</td><td>188271</td><td></td></tr><tr><td>50 ml centrifuge tubes</td><td>Cellstar</td><td>227261</td><td></td></tr><tr><td>Petri dishes</td><td>Thermo Scientific</td><td>150350</td><td></td></tr><tr><td>Cryo vials</td><td>Thermo Scientific</td><td>377224</td><td></td></tr><tr><td>500 ml bottle</td><td>Thermo Scientific</td><td>159910/159920</td><td></td></tr><tr><td>Scalpels</td><td>Swann-Morten</td><td>REF0211</td><td>Type 24</td></tr><tr><td>Tissue homogenizer</td><td>Sigma</td><td>D9188</td><td></td></tr><tr><td>140 &amp;mu;m filters</td><td>MERCK</td><td>NY4H04700</td><td></td></tr><tr><td>40 &amp;mu;m filters</td><td>Corning</td><td>431750</td><td></td></tr><tr><td>EndOhm chamber system</td><td>World Precision Instruments</td><td>ENDOHM-12</td><td>EndOhm chamber for 12mm Culture Cups</td></tr><tr><td>EVOM2 electrode system</td><td>World Precision Instruments</td><td>300523+STX100C</td><td>TEER measurement system with rigid STX-100C electrode pair</td></tr><tr><td>Long needle</td><td>Sigma</td><td></td><td>Attach to a syringe</td></tr><tr><td>Fine-tip curved forceps</td><td>KLS Martin</td><td>12-409-12-07</td><td></td></tr><tr><td>Broad tip forceps</td><td>VWR</td><td>82027-390</td><td></td></tr><tr><td>Filter holder</td><td>MERCK Milipore</td><td>Swinnex-47</td><td></td></tr><tr><td>50 ml syringe</td><td>Braun</td><td>4617509F</td><td></td></tr><tr><td>10 ml syringe</td><td>Terumo</td><td>SSt20ESI</td><td></td></tr><tr><td>Anti-Occludin antibody</td><td>Abcam</td><td>ab31721</td><td>1:100</td></tr><tr><td>Anti-p120 Catenin antibody</td><td>BD Transduction laboratories</td><td>610133</td><td>1:200</td></tr><tr><td>Anti-ZO-1 antibody</td><td>Invitrogen</td><td>61-7300</td><td>1:200</td></tr><tr><td>Anti-Claudin 5 antibody</td><td>Sigma-Aldrich</td><td>SAB4502981</td><td>1:100</td></tr><tr><td>Donkey anti rabbit IgG conjugated with Alexa Flour 568</td><td>Thermo Scientific</td><td>A10042</td><td>1:500</td></tr><tr><td>Donkey anti mouse IgG conjugated with Alexa Flour 488</td><td>Thermo Scientific</td><td>A21202</td><td>1:500</td></tr><tr><td>Sucrose</td><td>Perkin Elmer</td><td>NEC100X250UC</td><td>0.15&amp;micro;l/ml final working conc</td></tr><tr><td>Lucifer Yellow</td><td>Sigma</td><td>L0144</td><td>10 &amp;micro;g/ml final working conc</td></tr></tbody></table>

improved method for the establishment of an in vitro blood-brain barrier model based on porcine brain endothelial cells

このプロトコルの目的提示浄化と pBECs のと体外血液脳関門 (BBB) モデル モノカルチャー (MC)、アストロ サイト調整培 (ACM) と MC で pBECs に基づく栽培に最適化されたプロシージャと非接触ブタのアストロ サイトとの共培養 (NCC) またはラットの起源。pBECs は隔離され、国内豚 5-6 ヶ月歳の脳皮質から毛細血管のフラグメントから培養されました。これらのフラグメントは、髄膜、分離および灰白質、ろ過、酵素消化、遠心分離の均質化を慎重に除去して精製しました。汚染細胞をさらになくすためには、キャピラリーの断片がピューロマイシン含有培養した培地。60 ~ 95% 合流 pBECs キャピラリーのフラグメントから成長したに継代し、透過性の膜フィルターが挿入され、モデルに設立されました。次の差別化要因と扱われた細胞バリア堅さと BBB pBECs の特徴的な表現型を増加する: 膜側の透過物 8-CPT-キャンプ (ここで省略のキャンプ)、ヒドロコルチゾン、ホスホジエステラーゼ阻害薬、RO-20-1724(RO)。手順は、9-11 日の期間にわたって実施された、アストロ サイトが 2-8 週間前もってを培養した NCC モデルを確立するとき。プロトコルに記載されている手順の遵守が厳しく制限されている傍細胞透過性の内皮層の確立を許可している、NCC (TEER) 1249 ± 80 Ω cmの平均内皮下電気抵抗を示すモデル2と傍細胞透過性 (Pアプリ) ルシファー 0.90 10-6 ± 0.13 10-6 cm 秒-1の黄色の (± SEM を意味する n = 55)。この pBEC 表現型のさらなる評価のタイトな接合部蛋白質クローディン 5、良い表現を示した ZO 1、オクルディンと単接合蛋白 p120 カテニン。健康および病気の BBB の研究の範囲の提示されたモデルを使用ことができますそして、厳しい傍細胞透過性このモデル輸送と細胞内輸送の研究に適しています。

BBB のIn Vitroモデルの確立
提示された、最適化された方法で pBECs の栽培やアストロ サイト (図 1) と MC 有無 ACM または NCC 透過膜挿入システムの確立実施した 9-11 日間 (図 2)。内皮細胞の選択、精製されたキャピラリー フラグメントの初期の文化は、ピューロマイシン最大 5 ?...

Watch this Scientific Journal Video about Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells at JoVE.com

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

体外血液脳関門ブタ脳血管内皮細胞に基づくモデルの確立のための改良法

プロトコルの目的は、プライマリ ブタ脳血管内皮細胞 (pBECs) に基づく体外血液脳関門 (BBB) モデルの確立のための最適化手順を提示することです。モデルを示しています高再現性、高気密性、輸送および創薬における細胞内輸送の研究に適しています。

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

improved-method-for-establishment-an-vitro-blood-brain-barrier-model

Research

JoVE Journal

Medicine

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

14.7K ビュー.  Aarhus University. プロトコルの目的は、プライマリ ブタ脳血管内皮細胞 (pBECs) に基づく体外血液脳関門 (BBB) モデルの確立のための最適化手順を提示することです。モデルを示しています高再現性、高気密性、輸送および創薬における細胞内輸送の研究に適しています。

ビデオ: Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

人間の細胞に基づく、血液脳関門の接触モデルの作製のための改善された方法

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

生物工学

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

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

設定アップ<em&gt;体外</emラット血液脳関門の&gt;モデル（BBB）：BBB不透過性および受容体媒介輸送に注力

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

医学

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

ヒトiPSCベースの血液脳バリアチップの生成

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

発生生物学

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 ...

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is characterized by an interruption in blood supply to the brain leading to cell death and cognitive dysfunction. During and after ischemic stroke, blood-brain barrier (BBB) dysfunction facilitates injury progression and contributes to poor patient recovery. Current BBB models primarily include endothelial monocultures and double co-cultures with either astrocytes or pericytes.
Such models lack the ability to fully imitate a dynamic brain microenvironment, which is essential for cell-to-cell communication. Additionally, commonly used BBB models often contain immortalized human endothelial cells or animal-derived (rodent, porcine, or bovine) cell cultures that pose translational limitations. This paper describes a novel well-insert-based BBB model containing only primary human cells (brain microvascular endothelial cells, astrocytes, and brain vascular pericytes) enabling the investigation of ischemic brain injury in vitro. 
The effects of oxygen-glucose deprivation (OGD) on barrier integrity were assessed by passive permeability, transendothelial electrical resistance (TEER) measurements,and direct visualization of hypoxic cells. The presented protocol offers a distinct advantage inmimicking the intercellular environment of the BBB in vivo, serving as a more realistic in vitro BBB model for developing new therapeutic strategies in the setting of ischemic brain injury.

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Here, we describe the method for establishing a triple cell culture model of the blood-brain barrier based on primary human brain microvascular endothelial cells, astrocytes, and pericytes. This multicellular model is suitable for studies of neurovascular unit dysfunction during ischemic stroke in vitro or for the screening of drug candidates.

虚血性脳卒中をin vitroで研究するためのヒト血液脳関門のトリプル初代細胞培養モデル

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is ...

生化学

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

アン<em&gt;インビトロ</em&gt;インピーダンス分光法を用いた血液脳関門のモデル：T細胞 - 内皮細胞の相互作用に焦点を当て

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

免疫・感染学

Analyzing the Permeability of the Blood-Brain Barrier by Microbial Traversal through Microvascular Endothelial Cells

The human blood-brain barrier (BBB) is characterized by a very low permeability for biomolecules in order to protect and regulate the metabolism of the brain. The BBB is mainly formed out of endothelial cells embedded in collagen IV and fibronectin-rich basement membranes. Several pathologies result from dysfunction of the BBB followed by microbial traversal, causing diseases such as meningitis. In order to test the effect of multiple parameters, including different drugs and anesthetics, on the permeability of the BBB we established a novel human cell culture model mimicking the BBB with human brain microvascular endothelial cells. The endothelial cells are grown on collagen IV and fibronectin-coated filter units until confluence and can then be treated with different compounds of interest. In order to demonstrate a microbial traversal, the upper chamber with the apical surface of the endothelial cells is inoculated with bacteria. After an incubation period, samples of the lower chamber are plated on agar plates and the obtained colonies are counted, whereby the number of colonies correlate with the permeability of the BBB. Endogenous cellular factors can be analyzed in this experimental set-up in order to elucidate basic cellular mechanisms of the endothelial cells contributing to the BBB. In addition, this platform allows performing a screen for compounds that might affect the permeability of the endothelial cells. Finally, bacterial traversal can be studied and linked to different pathologies, such as meningitis. It might be possible to extend the model and analyze the pathways of the bacteria through the BBB. In this article, we provide a detailed protocol of the described method to investigate the permeability of the BBB.

The human blood-brain barrier selectively prevents penetration of hydrophilic molecules and pathogens into the brain. Several pathologies, including meningitis and postoperative delirium, are associated with an increased permeability of the blood-brain barrier. Here, we describe an endothelial cell culture model to test the barrier permeability by microbial traversal.

微小血管内皮細胞を介した微生物トラバーサルによる血液脳関門の透過性の解析

The human blood-brain barrier (BBB) is characterized by a very low permeability for biomolecules in order to protect and regulate the metabolism of the ...

ヒト多能性幹細胞由来の3次元血液脳関門モデル

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting the brain from pathogens. The inherent barrier properties of the BBB pose a challenge for therapeutic drug delivery into the CNS to treat neurological diseases. Impaired BBB function has been related to neurological disease. Cerebral amyloid angiopathy (CAA), the deposition of amyloid in the cerebral vasculature leading to a compromised BBB, is a co-morbidity in most cases of Alzheimer&#39;s disease (AD), suggesting that BBB dysfunction or breakdown may be involved in neurodegeneration. Due to limited access to human BBB tissue, the mechanisms that contribute to proper BBB function and BBB degeneration remain unknown. To address these limitations, we have developed a human pluripotent stem cell-derived BBB (iBBB) by incorporating endothelial cells, pericytes, and astrocytes in a 3D matrix. The iBBB self-assembles to recapitulate the anatomy and cellular interactions present in the BBB. Seeding iBBBs with amyloid captures key aspects of CAA. Additionally, the iBBB offers a flexible platform to modulate genetic and environmental factors implicated in cerebrovascular disease and neurodegeneration, to investigate how genetics and lifestyle affect disease risk. Finally, the iBBB can be used for drug screening and medicinal chemistry studies to optimize therapeutic&#160;delivery to the CNS. In this protocol, we describe the differentiation of the three types of cells (endothelial cells, pericytes, and astrocytes) arising from human pluripotent stem cells, how to assemble the differentiated cells into the iBBB, and how to model CAA in vitro using exogenous amyloid. This model overcomes the challenge of studying live human brain tissue with a system that has both biological fidelity and experimental flexibility, and enables the interrogation of the human BBB and its role in neurodegeneration.

著者スポットライト:In vitro血液脳関門モデルを使用したアルツハイマー病の調査に向けた個別化アプローチ 

The blood-brain barrier (BBB) has a crucial role in sustaining a stable and healthy brain environment. BBB dysfunction is associated with many neurological diseases. We have developed a 3D, stem-cell-derived model of the BBB to investigate cerebrovascular pathology, BBB integrity, and how the BBB is altered by genetics and disease.

神経疾患のモデル化と治療標的化のための血液脳関門の in vitro 再構築

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting ...

体外血液脳関門ブタ脳血管内皮細胞に基づくモデルの確立のための改良法

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人間の細胞に基づく、血液脳関門の接触モデルの作製のための改善された方法

として不死化マウス脳微小血管内皮細胞株の生成

設定アップ

ヒトiPSCベースの血液脳バリアチップの生成

初代マウス脳微小血管内皮細胞の単離

虚血性脳卒中をin vitroで研究するためのヒト血液脳関門のトリプル初代細胞培養モデル

アン

微小血管内皮細胞を介した微生物トラバーサルによる血液脳関門の透過性の解析

神経疾患のモデル化と治療標的化のための血液脳関門の in vitro 再構築

神経疾患のモデル化と治療標的化のための血液脳関門の in vitro 再構築