我々は、T.ハント、M.ローゼンブルース、そして彼らのアドバイスや有用な議論のためのラムラボに感謝します。私たちは、ジョージア工科大学の電子工学とナノテクノロジーのためのG.スピナーと研究所からの支援を認める。この仕事のための財政支援は、NIHの助成金K08-HL093360、UCSF REAC賞、NIHのナノメディシン開発センター賞PN2EY018244、アトランタの子供の医療の内皮細胞生物学のためのセンターからの資金によって提供されました。

1。内皮マイクロデバイスの作製<ol><li>外側のマスクベンダにマイクロ流体デバイスのコンピュータ支援設計（CAD）図面を提出することによりフォトマスクを作成します。使用されるマスクは、ソーダライムガラス上にクロム層から構成されていた。このケースでは、微小流体チャネル幅は30μmであった。 </li><li> 15分間ピラニア（硫酸と過酸化水素の10:1比）ベアシリコンウェーハを洗浄し、30秒間フッ化水素酸に浸漬。約10秒間脱イオン（DI）水で洗い流してください。 </li><li> 30μmの高さにウエハ上にスピンコーターを用いて、スピンマイクロケムSU-8 2025レジスト。 SU-8 2025、3000 rpmの回転速度が推奨されます。 SU-8の他の粘度は、この高さを実現しますどのご利用いただけます。 SU-8を使用するための完全な手順は、ご利用いただけます<a href="http://www.microchem.com" target="_blank">www.microchem.com</a> </li><li> 95℃のホットプレート上でSU-8でウェハを置きます過剰な溶媒を駆動するために5分。 注意：オーブンはホットプレートとは異なるウェハを乾燥し、推奨されていません。 </li><li>ウエハ上に所望の機能の形状でマスクを配置し、紫外光（160 MJ / cm 2に公開 マスクアライナ（カールカールサス、MA-6））365 nmで測定した。このクロスは、フォトレジストをリンクします。 </li><li>さらにSU-8の重合を促進するためにさらに5分間95℃でホットプレート上でバックウェハを置きます。 </li><li>非架橋SU-8を削除するには、4分間PGMEA（プロピレングリコールメチルエーテルアセテート）から主に構成されるSU-8 Developerでウェハを浸します。 </li><li> 10秒に100％イソプロピルアルコール（IPA）と新しく開発されたウェーハを洗浄します。ウェハは、次いで溶媒が数分間、クリーンドラフト内でウェハから蒸発させることにより加圧された窒素を使用するか、または乾燥させることができる。 </li><li>テープのペトリ皿にパターニングSU-8のドライウェハーのエッジは、事前に運動を発散。 </li><li>ウェーハの完全なコーティングを確保するためにピペット、ペトリ皿の上部カバー、およびスワールウエハを用いてウェーハにSigmacote 1 mLを適用し、カバーし、すべての溶媒が蒸発するまでウェハを数分間乾燥させて削除します。 </li></ol> 2。 PDMS（ポリジメチルシロキサン）の準備<ol><li> 10:1の比（W / W）でPDMSポリマーと硬化剤を混合し、真空デシケーターを用いた気泡を除去します。脱PDMSに必要な時間の長さは、使用可能な真空システムの強さによって異なりますが、通常は数分から1時間の範囲である。なし以前に注ぎPDMSと6インチのペトリ皿には、ポリマーの60 mLの全量をお勧めします。 </li><li> 、ウエハ上に厚さ約5mmの混合物を注ぐ。また、厚さ約1mmのPDMSの薄いシートを作成するためにフラットボトム皿に注ぐ。 60一晩オーブンで℃で硬化します。 </li><li>ナイフやメスを用いて、Cの周りに切り取るPDMSデバイスをuredとウェハからそれを削除します。さらに、デバイスよりも若干大きくPDMSの薄いシートを切った。 </li><li> 1.0ミリメートルの穴パンチを使用してPDMSデバイスの入口と出口の穴を作成します。これは、どちらかのピンの副、ハリスユニコア、または同様の装置を用いて達成することができる。 </li><li>デバイスの表面とスコッチテープを使用してシートをクリーニングします。 </li><li>プラズマクリーナーを使用して、30秒間酸素プラズマにPDMSデバイスの表面とPDMSシートを公開します。酸素プラズマは、物理的に接触結合PDMSの露出面に反応種を作成します。 </li><li>順番に、PBS中のヒト血漿から50μg/ mlのフィブロネクチンで埋め平滑末端ポイント針と注射器に接続されている大規模なチューブの小さな長さ（数センチ）に小さいチューブを接続します。チューブと針が摩擦フィットがチューブの間に作成されるようにサイズ調整されています。小さなPDMSマイクロデバイスの入口にチューブを挿入pplyシリンジに圧力がフィブロネクチン溶液を完全にチャネルを埋めるために、出口ポートに小型の100μLのドロップを作成するには、チャネルがぬれたままいることを確認する。 40から60分間37℃でデバイスをインキュベートします。 </li><li>鈍ポイント針にPBSを充填した新鮮なシリンジを接続します。 PBSでデバイスをリンスするために注射器に圧力を適用します。 </li></ol> 3。内皮細胞とマイクロ流体デバイスを播種<ol><li> 8パーセントデキストランと内皮細胞増殖培地中でヒト臍帯静脈内皮細胞（HUVEC）の1,000,000細胞/ mLを準備します。 50万〜2,000,000細胞/ mLの範囲が正常に使用されています。セルローディング培地にデキストランの添加は、フィブロネクチンでコーティングされたマイクロ流体システムを入力すると、内皮細胞の移動速度を減少させ、流体の粘度を増加させます。これは、順番に、細胞がマイクロデバイス内に正常に付着し、文化れる可能性が増加します。 </li><li> ℃ステップ2.7のように新しいシリンジとチューブをONNECT。しかし、長いチューブ（長さ約1メートル）である。 </li><li>このシステムのパフォーマンスを最適化するために、それは全体の灌流システム内の任意のリークや気泡を防ぐために、この時点で非常に重要です。ソリューションやメディアの中の気泡の有無のいずれかのリーク電流は流れを変更し、内皮細胞の正常な播種を防ぐことができます。したがって、チューブのタイトフィッティングは、任意の接続間の漏れをなくすことが不可欠である。細胞は、マイクロデバイスに導入されており、全体の灌流システムは、気泡の導入を避けるために、流体にチューブを接続する前に細胞溶液でプライミングする必要があります前に、注射器、および/またはチューブ内の気泡を除去する必要があります。 </li><li>シリンジポンプを用いて、Cおよび5％CO 2、37℃で2時間1.23μL/ minの体積流量でPDMSデバイスに細胞懸濁液を注入する。シリンジポンプにいたとき、最適な結果を達成しました<html> PDMSデバイスと同じ高さ。入口には、インキュベーター内で巻いすることができます長いチューブは、細胞やメディアが適切にデバイスに接触する前に温めていることを保証します。本システムでは、1.23μL/ minの体積流量は、全血を使用してこれらのチャネルで〜1ダイン/ cm 2の壁面せん断応力に対応する最小のマイクロ、で〜1 mm / sの途中速度を概算します。 </li><li>同じシリンジポンプ、長いチューブを使用して、1.23μL/ minの割合で2から8日間新鮮な増殖培地を灌流。成功したデバイスは、内皮細胞の単層は24-48時間以内のデバイスの内側に成長しているでしょう。以前の実験では、コンフルエントな単層が適切にデバイス14を通して細胞間接合部のVE-カドヘリンを発現していることが示されている。 </li><li>実験のためのシステムへの血液や細胞懸濁液を注入します。 </li></ol> 4。代表的な結果その後_content &quot;&gt;このプロトコルを使用して、標準的なリソグラフィの微細加工技術は生理学的に微小血管（ 図1A）のsizescaleを模倣するマイクロ流体チャネルを生成するために必要な金型を作成するために使用されています。最適化された灌流法を用いて、内皮細胞種とconfluently文化細胞播種の24〜48時間以内マイクロ流体システムの全体の内面（ 図1B）は、マイクロ流体システムは、透明であるため、全体のマイクロデバイスは、イメージングとデータ収集のために明視野/蛍光顕微鏡ステージ上に配置することができます。 我々のシステムは、その後sickled赤血球と異常な白血球と血管内皮接着の増加剛性が微小血管の閉塞に寄与するような鎌状赤血球症などの生物物理学的特性を、変更を伴う血液疾患を研究するために適用することができます。臨床的に承認された薬、ヒドロキシウレアは、症状が、そのdを改善する微小血管の流れのirect効果は不明である。我々のアッセイは、アカウントセルの剛性と密着性の両方にかかり、ヒドロキシウレアが大幅に鎌状赤血球症（ 図2）の流れを改善することを示しています。 このシステムは理想的な血液細胞は、微小血管系でお互いと内皮細胞と相互作用する任意の血液学的プロセスを研究するために適しているとして、鎌状赤血球症は、微小血管·オン·チップ用のアプリケーションの一例にすぎません。より基本的なアプリケーションは、白血球生物学と他の多くの間造血幹細胞生物学を含めながら、他の臨床的に関連するアプリケーションでは、炎症性疾患、敗血症/肺傷害、血栓microangiopathies、マラリア、がんの転移が含まれています。 <img src="/files/ftp_upload/3958/3958fig1.jpg" alt="図1"/> 図1。）初期PDMSマイクロ流体デバイスを内皮前。ここでは、マイクロデバイスは、ウィットを注入されH食品は、システムのsizescaleと全体のデザインを説明するために着色。 B）明視野顕微鏡は、マイクロ流体システムが完全にここで説明したプロトコルを使用して、細胞播種の48時間以内にendothelializedであることを示している。 <img src="/files/ftp_upload/3958/3958fig2.jpg" alt="図2"/> 図2）。マイクロと微小血管·オン·チップは、ほとんどの鎌状赤血球血管閉塞イベントのサイト、後毛細血管細静脈（30μm）のサイズを概算します。ヒドロキシウレアを受けて鎌状赤血球症患者から、ヒドロキシを受けていない患者から全血は、2つの異なる微小血管系オンチップデバイスを介して流される。 endothelializedマイクロ内の相対的な容易にヒドロキシウレアの流れを受けて鎌状赤血球症患者からのB）全血。 C）同じ血行動態の条件下では、ヒドロキシウレアを受けていない鎌状赤血球症患者から全血は、しかし、microchと​​はるかに遅い流れを示すannel閉塞。一番下のチャンネルが完全に流れがないと妨害や他のendothelializedマイクロチャネル内の流速は、ヒドロキシウレアの条件よりも有意に低くなっています。すべての3つの画像中のスケールバーは30μmである。

<ol>
	<li>Mezzano, D., Quiroga, T., Pereira, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Level+of+Laboratory+Testing+Required+for+Diagnosis+or+Exclusion+of+a+Platelet+Function+Disorder+Using+Platelet+Aggregation+and+Secretion+Assays.">The Level of Laboratory Testing Required for Diagnosis or Exclusion of a Platelet Function Disorder Using Platelet Aggregation and Secretion Assays.</a> Semin. Thromb. Hemost. 35, 242-254 (2009).</li><li>Young, E. W. K., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+microfluidic+cell+culture+in+controlled+microenvironments.">Fundamentals of microfluidic cell culture in controlled microenvironments.</a> Chemical Society Reviews. 39, 1036-1048 (2010).</li><li>Young, E. W. K., Simmons, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macro-+microscale+fluid+flow+systems+for+endothelial+cell+biology.">Macro- microscale fluid flow systems for endothelial cell biology.</a> Lab on a Chip. 10, 143-160 (2010).</li><li>Higgins, J. M., Eddington, D. T., Bhatia, S. N., Mahadevan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sickle+cell+vasoocclusion+and+rescue+in+a+microfluidic+device.">Sickle cell vasoocclusion and rescue in a microfluidic device.</a> Proceedings of the National Academy of Sciences. 104, 20496-20500 (2007).</li><li>Rosano, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+physiologically+realistic+in+vitro+model+of+microvascular+networks.">A physiologically realistic in vitro model of microvascular networks.</a> Biomedical Microdevices. 11, 1051-1057 (2009).</li><li>Meer, A. D. v. a. n. d. e. r., Poot, A. A., Duits, M. H. G., Feijen, J., Vermes, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+Technology+in+Vascular+Research.">Microfluidic Technology in Vascular Research.</a> Journal of Biomedicine and Biotechnology. 2009, (2009).</li><li>Karunarathne, W., Ku, C. -. J., Spence, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dual+nature+of+extracellular+ATP+as+a+concentration-dependent+platelet+P2X1+agonist+and+antagonist.">The dual nature of extracellular ATP as a concentration-dependent platelet P2X1 agonist and antagonist.</a> Integrative Biology. 1, 655-663 (2009).</li><li>Kotz, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+microfluidics+for+neutrophil+genomics+and+proteomics.">Clinical microfluidics for neutrophil genomics and proteomics.</a> Nat. Med. 16, 1042-1047 (2010).</li><li>Rosenbluth, M. J., Lam, W. A., Fletcher, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+cell+mechanics+in+hematologic+diseases+with+microfluidic+biophysical+flow+cytometry.">Analyzing cell mechanics in hematologic diseases with microfluidic biophysical flow cytometry.</a> Lab on a Chip. 8, 1062-1070 (2008).</li><li>Shelby, J. P., White, J., Ganesan, K., Rathod, P. K., Chiu, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+model+for+single-cell+capillary+obstruction+by+Plasmodium+falciparum-infected+erythrocytes.">A microfluidic model for single-cell capillary obstruction by Plasmodium falciparum-infected erythrocytes.</a> Proceedings of the National Academy of Sciences. 100, 14618-14622 (1073).</li><li>Borenstein, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+endothelialized+microvascular+networks+with+circular+cross-sections+in+a+tissue+culture+substrate.">Functional endothelialized microvascular networks with circular cross-sections in a tissue culture substrate.</a> Biomedical Microdevices. 12, 71-79 (2010).</li><li>Nesbitt, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+shear+gradient-dependent+platelet+aggregation+mechanism+drives+thrombus+formation.">A shear gradient-dependent platelet aggregation mechanism drives thrombus formation.</a> Nat. Med. 15, 665-673 (2009).</li><li>Prabhakarpandian, B., Shen, M. -. C., Pant, K., Kiani, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+for+modeling+cell-cell+and+particle-cell+interactions+in+the+microvasculature.">Microfluidic devices for modeling cell-cell and particle-cell interactions in the microvasculature.</a> Microvascular Research. 82, 210-220 (2011).</li><li>Tsai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+modeling+of+the+microvascular+occlusion+and+thrombosis+that+occur+in+hematologic+diseases+using+microfluidic+technology.">In vitro modeling of the microvascular occlusion and thrombosis that occur in hematologic diseases using microfluidic technology.</a> The Journal of Clinical Investigation. , (2011).</li><li>Green, D. A., Murphy, W. G., Uttley, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haemolytic+uraemic+syndrome:+prognostic+factors.">Haemolytic uraemic syndrome: prognostic factors.</a> Clinical &#38; Laboratory Haematology. 22, 11-14 (2000).</li></ol>

利害の衝突が宣言されません。

私たちのendothelializedマイクロデバイス·システムは、in vivoの実験ではと組み合わせて使用するときに最も適していると、その還元のアプローチは、人間と動物モデルで観察された血液学的プロセスの生物物理学的メカニズムの解明に役立つことがあります。さらに、我々のシステムには限界がないわけではない。例えば、我々の流体チャネルは、クロスセクションの正方形です?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>鈍ポイント針</td><td> OKインターナショナル</td><td> 920050-TE </td><td>精度TE針20ゲージ×1/2 &quot;、ピンク</td></tr><tr><td>デキストラン</td><td> Sigma-Aldrich社</td><td> 31392 </td><td></td></tr><tr><td>フィブロネクチン</td><td> Sigma-Aldrich社</td><td> F0895 </td><td></td></tr><tr><td>穴パンチャー（ピン万力） </td><td>技術革新</td><td></td><td></td></tr><tr><td>ヒト臍帯内皮細胞（HUVEC） </td><td>ロンザ</td><td> CC-2519 </td><td></td></tr><tr><td>プラズマクリーナー</td><td>プラズマ</td><td> PDC-326 </td><td></td></tr><tr><td>ポリジメチルシロキサン（PDMS） </td><td>フィッシャー·サイエンティフィック</td><td> NC9285739 </td><td> Sylgard 184シリコーンエラストマーKIT </td></tr><tr><td> Sigmacote </td><td> Sigma-Aldrich社</td><td> SL2 </td><td></td></tr><tr><td> SU-8 2025 </td><td>マイクロケム</td><td> Y111069 </td><td></td></tr><tr><td> SU-8開発</td><td>マイクロケム</td><td> Y020100 </td><td></td></tr><tr><td>注射器ポンプ</td><td>ハーバード装置</td><td> 70から3008 </td><td> PHD-ULTRA </td></tr><tr><td>チューブ（大きい） </td><td>コー​​ル·パーマーインストゥルメント·カンパニー</td><td> 06418から02 </td><td> Tygonregマイクロボアチューブ、0.020 &quot;内径x 0.060&quot; OD </td></tr><tr><td>チューブ（小さい） </td><td>コー​​ル·パーマーインストゥルメント·カンパニー</td><td> 06417から11 </td><td> PTFEマイクロボアチューブ、0.012 &quot;内径x 0.030&quot; OD </td></tr></tbody></table>

endothelialized microfluidics for studying microvascular interactions in hematologic diseases

微細加工技術の進歩により、マイクロ·nanoscales 1,2で生物学的および生化学的な実験を実施するための安価で再現性のあるマイクロ流体システムの生産を有効にしている。さらに、マイクロフルイディクスは、具体的にするため、簡単に動的な流体の環境及び生物学的条件3-6を制御する能力を、定量的に血液と血管のプロセスを分析するために使用されています。このように、研究者は最近では血液細胞の変形能、血液細胞の凝集、微小血管の血流、血液細胞血管内皮細胞の相互作用6-13勉強するマイクロ流体システムを使用しています。しかし、これらのマイクロ流体システムのいずれかの培養内皮細胞を含めたり、大きかったしませんでした微小血管の病理学的プロセスに関連するsizescaleより。培養内皮細胞を用いたマイクロ流体プラットフォームを正確に反復する携帯電話、物理的、およびhemodynその微小循環のアミック環境は微小血管を伴う血液疾患の根本的な生物物理学的病態の理解を促進するために必要です。 ここでは、血液疾患で発生する病理学的生物物理学の微小血管の相互作用を研究するために、標準的な内皮細胞培養技術と組み合わせて、シンプルな単一のマスクの微細加工プロセスを用いて、微小血管の in vitroモデルで &quot;endothelialized&quot;を作成する方法を報告します。この &quot;微小血管·オン·チップ&quot;はしっかりと生物学的ならびに生物物理学的条件を制御し、標準的なシリンジポンプと明視野/蛍光顕微鏡を用いて操作された堅牢なアッセイを用いて研究を提供しています。このような微小循環血行状態、内皮細胞の種類、血液細胞の種類（s）と濃度（S）、薬物/阻害濃度等、などのパラメータはすべて容易に制御することができます。このように、私たちのマイクロシステムを提供します定量的に微小血管の流れが原因で、細胞接着、凝集し、変形能は、既存のアッセイでは使用できない機能に変化が損なわれている病気のプロセスを調査する方法。

Watch this Scientific Journal Video about Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases at JoVE.com

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

微小サイズのチャネル（&lt;30μm）を持つマイクロ流体デバイスの全体の内部の三次元表面全体に文化内皮細胞単層をに方法が記載されています。この<em&gt; in vitroで</em&gt;微小血管モデルは、血液細胞、内皮細胞、血液疾患における可溶性因子との間の生物物理学的相互作用の研究を可能にします。

血液疾患における血管相互作用を研究するためのEndothelializedマイクロフルイディクス

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

endothelialized-microfluidics-for-studying-microvascular-interactions

Research

JoVE Journal

Bioengineering

15.8K ビュー.  Emory University School of Medicine. 微小サイズのチャネル（

ビデオ: Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

タンパク質の折り畳みを研究するためのマイクロ流体ミキサー

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

生物工学

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

使用プラ​​センタアクロス異物およびナノ材料の輸送速度の決定<em&gt; ex vivoで</em&gt;ヒト胎盤灌流モデル

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

マルチ深円形の断面内皮マイクロチャンネル·オン·ア·チップの開発のための手順

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

における損傷応答のライブイメージングや研究のためのマイクロ流体チップを使用<em&gt;ショウジョウバエ</em&gt;幼虫

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

連続フローMicrospotterを使用して変更表面に細胞のサブマージ印刷

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

サンドイッチ状の微小環境は、Cell /材料の相互作用を活用します

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

光ファイバを用いて、液滴マイクロフルイディクスのための多色蛍光検出

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

組織工学のための血管新生単位としてマウスの脂肪組織由来の微小血管フラグメントの単離

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

神経細胞のコミュニケーションと軸索の信号伝搬特性を研究するためマイクロ電極アレイとのインタ フェース

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

脳癌と神経変性疾患を研究するためのヒト神経オルガノイド

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...