Name: microfluidic flow chambers using reconstituted blood to model hemostasis and platelet transfusion in vitro
Uploaded: 2016-03-19T14:00:00.000+00:00
Duration: 10 min 25 s
Description: Watch this Scientific Journal Video about Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro at JoVE.com

The authors have no acknowledgements.

このプロトコルは、ヒト試料の研究のための制度倫理指針に従っており、インフォームドコンセントは、関係する全てのドナーから得られました。ここに記載された実験の承認は、アントワープ大学病院の施設内倫理委員会から入手しました。 注：指定しない限り、温度表示は、常に室温です。 1.準備フローチャンバーのセットアップ<ol><li>レーン、チューブおよびピンの準備<ol><li>渦激しくコラーゲン懸濁液および50μg/ mlの最終濃度になるように、プロバイダにより供給された等張グルコース溶液で1/20に希釈します。 注：私たちは、主にI型線維の構成された、ウマ腱コラーゲンを使用します。ウマコラーゲンI型はしばしば「HORM「コラーゲンと呼ばれ、歴史的なだけでなく、生物学的な理由の両方のためのアッセイ9のこのタイプの黄金の標準です。ヒトIII型コラーゲンを使用することもできるが、目eはあまりよくコートをフィブリルおよび血小板応答は、強力ではありません。他のコーティング表面は、例えばフォン・ヴィレブランド因子（VWF）、フィブリノゲン、フィブロネクチン、ラミニン、ビトロネクチン、トロンボスポンジン-1、またはこれらの16の組み合わせのために、使用することができます。 </li><li>プロバイダーのコンテナから新しい使い捨てバイオチップを取ります。ここで使用されるバイオチップの寸法はミリメートル3で0.4Wのx 0.1Hのx 20Lです。 </li><li>ピペットの出口としてチップとマークの一端のマイクロ流体バイオチップのレーン（複数可）に0.8μlの。レーンが1.1.1で調製した塗布溶液を含有するコ​​ラーゲンと5/6日に満たされていることを確認します。気泡がないことを確認してください。 注：チャンネルは、部分的に（説明を参照）、チャネルの入口にコラーゲン線維の蓄積を回避するためにコーティングされています。 </li><li>加湿と密閉容器内の4時間または一晩4℃でインキュベートします。 </li><li> / v）のウシ血清アルブミンwは（ブロッキング緩衝液（1.0％をピペットでコーティングされたチャネルを遮断他端とマークで、D、0.1％（w / v）のグルコース、10mMの4-（2-ヒドロキシエチル）-1-ピペラジンエタンスルホン酸（HEPES）の緩衝化生理食塩水（/ v）の塩化ナトリウム、pH7.4のW、0.9％（HBS）で入口として。車線が完全に空気の泡を避けブロッキング緩衝液で満たされていることを確認します。 </li><li>同じサイズ（12センチ）でチューブをカットします。レーンあたり1を使用し、ピンで各チューブを接続します。例えば、二重に実行する二つの条件を比較した実験では、4チューブストレッチと4本のピンを用意する必要があります。 </li><li>シリンジと26 Gの針または添付コネクタを使用して蒸留水でチューブを洗浄します。 </li><li>ブロッキング緩衝液でチューブを飽和。 1時間の最小値のため、閉鎖し、加湿容器に保管してください。 </li></ol></li><li>ポンプとマニホールドの準備<ol><li>ポンプを洗浄し、蒸留水でマニホールド、気泡を除去。 </li><li>出口で固定10μlのチップを用いたバイオチップレーン（複数可）のうち、ブロッキング緩衝液を吸引します。の表面を清掃してください精密無料ワイプほこりや変性アルコールによるバイオチップは、プリントやほこりを除去します。 </li><li>自動化された顕微鏡ステージ上のバイオチップを修正しました。複数のレーンが1回の実行で同時に使用されている場合は、バイオチップコンセントに8レーンマニホールドスプリッタを接続します。 注：8レーンマニホールドスプリッタは、ポンプやバイオチップに接続されたハードウェアの一部（ 図S1）です。これは、添付のソフトウェアで操作者が定義することができるバイオチップ又はレーンの組み合わせ（8個）のレーンすべての動作が利用できることができます。 </li><li>バイオチップの入口でそれらを修正するためにチューブでピンを使用してください。 HBSで満たされた1.5ミリリットルコニカル試験管に（ピンなし）チューブのもう一方の端を置きます。バッファおよび不十分な付着コラーゲンをブロックし、残りを除去するために、ポンプを使用して、1ミリリットルのHBSですべてのチューブとその接続されたレーンをすすぎます。 </li></ol></li></ol>血液試料の調製<ol><li>コレクションとの分離健康なボランティアからの新鮮な全血。17 <ol><li>抗凝固剤としてエチレンジアミン四酢酸（EDTA）を含む真空チューブ内の血液の最初のミリリットルを収集し、排他的に自動血液分析器を用いて完全血球算定（CBC）のために、このサンプルを使用しています。 </li><li>避難チューブを用いて、適切な抗凝固剤で血液量を収集します。フィブリン形成が研究プロトコールおよびクエン酸ナトリウムの一部ではない場合がある場合、フローチャンバー実験のための標準的な抗凝固剤は、ヘパリンまたはヒルジンです。 注：ヘパリンは、結果に記載のすべての実験のための抗凝固剤として使用しました。 注：血液の量は、実行される実験の数に依存します。 3車線のため、約1管（7ミリリットル）。 </li><li>回転子保留中の血液の再構成にチューブを入れます。 注：このアッセイは、瀉血の3時間以内に完了する必要があります。 </li><li>多血小板血漿を準備するために250グラムで15分間遠心分離（PRP）。ゆるく充填されたペレットの乱れを防止するために遠心ブレークを使用しないでください。 <ol><li>複数のチューブが収集された場合には、単一の円錐形遠心分離管に血液をプールします。 注：遠心分離は、PRPの収量および差動セル「汚染」に応じて、よりゆっくりと以下の長い行うことができる好ましいです。 </li></ol></li><li>削除して、いくつかの血小板が充填された赤血球を得たPRPと軟膜を捨てます。 我々の手中に平均1μl当たり13±5×10 3パック赤血球画分中の血小板数がある（平均±SD、n個= 12）：注意してください。 </li></ol></li><li>血液再構成<ol><li> 5分および4ミリリットルあたり20秒、37℃で解凍血液型AB（アカゲザルD負）プラズマ。 </li><li>自動血液分析装置を用いて、2.1.5で製造した濃縮赤血球のヘマトクリットを決定します。 </li><li>血液バンクに血小板濃度を決定するには、濃縮血小板股関節を用意しましたtは、上記赤血球画分を再構成するために使用されます。 </li><li> 1ミリリットルのサンプルで40％のヘマトクリットおよび250×10 3血小板/μLをもたらすパックされた赤血球と血小板濃縮物の体積を計算します。 注：細胞の他の標的力価は、試験プロトコルに応じて、任意に設定することができます。 </li><li>転送がクリップピペットチップを使用し、1mlのサンプル容量に達するまで、血小板濃縮物を加える新しいチューブに赤血球と血漿を充填しました。 注：プラズマは血栓形成率に大きな影響を持っているので、研究の変数に応じて、血漿分画は、全ての再構成のサンプルで同じでなければなりません。例えば、異なるドナーから、または別の抗凝固剤で撮影を繰り返し凍結融解または血漿は、結果に影響を与えることができます。 </li><li>静かに反転させて再構成された血液を混ぜ、CBCを行います。 </li><li>血小板画分の量が置換された「ブランク」対照試料を調製し0.9％の同じ体積CBCを使用して再構成された血液中の内因性血小板（ すなわち、非血液バンク血小板）の濃度を決定するために水中で（M / v）の塩化ナトリウム。 </li></ol></li><li>ラベリング<ol><li>ピペットで1ミリリットル1μlの5 mMのカルセインAM（5μM最終濃度）を含有する試験管に血液を再構成しました。 注：他の細胞の色素14を使用することができます。 </li><li>転倒混和し。 </li><li>使用前に37℃で5分間インキュベートします。 </li></ol></li></ol> 3.灌流アッセイ<ol><li>レーンの底に付着したコラーゲン繊維上の目的に焦点を当てます。理想的には、この焦点を当て戦略の位相差や微分干渉コントラスト（DIC）の設定を使用します。デジタルで選択されたZ-位置を固定するための実験ソフトウェアで「選択されたタイル領域に設定されている現在のZ]を選択します。 </li><li>メートルの試験ソフトウエアでレーン（XY）に関心領域（ROI）を選択します実験中に記録されますicroscope。 注：ROIは任意に灌流レーン内で選択した任意の表面積することができます。そのように、これは比較的小さくても、その領域の可変流量プロファイルの副作用を回避するために、近い車線の入口及び出口に血栓形成を分析しないことが望ましいです。 ROIの表面積は、血小板の有意な数を含むか、または信号の平滑化を可能にするために血栓べきです。このプロトコルでは、ROIは2センチ車線の中央に0.62ミリメートル2の結果3等しいサイズのサイドバイサイド画像のデジタルでステッチ集合体です。 </li><li>自動化されたステージ上でバイオチップの隣に、これらを反転して位置によって穏やかにサンプルを混ぜます。 </li><li>再構成された血液サンプルを含む試験管内のバイオチップの入口に接続されたチューブを置きます。 </li><li> （希望として、または他のせん断応力）試験管にリンクされ、これらのチャネルを50ダイン/ cm 2のポンプを起動しますポンプのソフトウェアを使用して再構成した血液サンプルを含みます。 注：他のせん断応力を使用することができます。 </li><li>レコード画像顕微鏡の取得と実験ソフトウェアを使用してリアルタイムで5分ごとに15秒。 注：他の時系列が、実験のセットアップに応じて使用することができます。 注：私たちは、一般的に100倍の倍率（10倍対物レンズと10倍レンズ）を使用しますが、より高い（または低い）倍率が簡単に代替として使用することができます。 </li></ol> 4.ウォッシュアウト<ol><li>すべてのチューブの出口に取り付けられ、次亜塩素酸ナトリウム（漂白剤）、0.5％（v / v）を、水で最終​​的に0.1 MのNaOH、続いて蒸留水を用いてマルチチャンネルマニホールドまたはポンプに接続さを洗い流します。有害廃棄物のバイオチップの入口に固定チューブを捨てます。 </li></ol> 5.データ解析<ol><li>画像解析ソフトで血栓成長速度を決定します。次のコマンドはZEN2012に特異的です。 <ol&gt; <li>血小板の表面被覆率を決定するために、プラグインの画像解析を開きます 。 </li><li>正の信号と相関画素強度を定義するためのインタラクティブなタブを分析 、 すなわち付着血小板または付着血小板中の蛍光閾値を設定します。 </li><li>自動的に選択された閾値との間の信号を有する画素を含むもの「オブジェクト」の（μm2の中の）独立した表面領域を含むことになるスプレッドシートを生成するために、 表の作成 ​​に使用します。これは、各時点で行われます。 注：蛍光閾値を選んだしたら、解析ソフトウェアは自動的に基準を満たす視野に「オブジェクト」を検出します。これらのオブジェクトは、血栓、小さな血小板凝集または単一の血小板であり、画素数をカバーします。すべてのオブジェクトは、別々のスプレッドシートに記載されています。 </li><li> xml形式でこれらのスプレッドシートを保存し、スプレッドシートプログラムで開きますさらなる計算のために。 </li><li>合計して、選択したオブジェクトの合計表面積とは、測定フィールド（μmの2）の総面積で結果を分けます。これは、相対的な表面被覆率（％）を生じるであろう。毎回ポイントのためにそう。 </li><li>灌流時間の関数にこれらの表面被覆率をプロットし、線形回帰によって傾きを算出し、その特定の状態の血栓成長の速度論を得ました。 </li></ol></li></ol>

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggregation+of+blood+platelets+by+adenosine+diphosphate+and+its+reversal.">Aggregation of blood platelets by adenosine diphosphate and its reversal.</a> Nature. 194, 927-929 (1962).</li><li>Cattaneo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+the+Standardization+of+Light+Transmission+Aggregometry:+A+Consensus+of+the+Working+Party+from+the+Platelet+Physiology+Subcommittee+of+SSC/ISTH.">Recommendations for the Standardization of Light Transmission Aggregometry: A Consensus of the Working Party from the Platelet Physiology Subcommittee of SSC/ISTH.</a> J Thromb Haemost. 11, 1183-1189 (2013).</li><li>Deckmyn, H., Feys, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assays+for+quality+control+of+platelets+for+transfusion.">Assays for quality control of platelets for transfusion.</a> ISBT Sci Series. 8, 221-224 (2013).</li><li>Andre, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticoagulants+(thrombin+inhibitors)+and+aspirin+synergize+with+P2Y12+receptor+antagonism+in+thrombosis.">Anticoagulants (thrombin inhibitors) and aspirin synergize with P2Y12 receptor antagonism in thrombosis.</a> Circulation. 108, 2697-2703 (2003).</li><li>Casari, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=von+Willebrand+factor+mutation+promotes+thrombocytopathy+by+inhibiting+integrin+alphaIIbbeta3.">von Willebrand factor mutation promotes thrombocytopathy by inhibiting integrin alphaIIbbeta3.</a> J Clin Invest. 123, 5071-5081 (2013).</li><li>Gitz, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+platelet+survival+after+cold+storage+by+prevention+of+Glycoprotein+Ib&#945;+clustering+in+lipid+rafts.">Improved platelet survival after cold storage by prevention of Glycoprotein Ib&#945; clustering in lipid rafts.</a> Haematologica. , (2012).</li><li>Maurer-Spurej, E., Labrie, A., Brown, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Routine+Quality+Testing+of+Blood+Platelet+Transfusions+with+Dynamic+Light+Scattering.">Routine Quality Testing of Blood Platelet Transfusions with Dynamic Light Scattering.</a> Part Part Syst Char. 25, 99-104 (2008).</li><li>Van Kruchten, R., Cosemans, J. M., Heemskerk, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+whole+blood+thrombus+formation+using+parallel-plate+flow+chambers+-+a+practical+guide.">Measurement of whole blood thrombus formation using parallel-plate flow chambers - a practical guide.</a> Platelets. 23, 229-242 (2012).</li><li>Zwaginga, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-based+assays+for+global+assessment+of+hemostasis.+Part+2:+current+methods+and+considerations+for+the+future.">Flow-based assays for global assessment of hemostasis. Part 2: current methods and considerations for the future.</a> J Thromb Haemost. 4, 2716-2717 (2006).</li><li>Zwaginga, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-based+assays+for+global+assessment+of+hemostasis.+Part+1:+Biorheologic+considerations.">Flow-based assays for global assessment of hemostasis. Part 1: Biorheologic considerations.</a> J Thromb Haemost. 4, 2486-2487 (2006).</li></ol>

The authors have nothing to disclose.

マイクロ流体フローチャンバー実験は、血液の流れに血小板機能を調査するための優れたツールであり、実験的な文脈を変化させるインビトロでの止血を評価するために使用されます。貧しい施設間の標準化9にもかかわらず、我々は我々の研究室内実験変動が許容可能であることを示しています。これは確実に与えられた研究の範囲内（ペアリング）のサンプルを比較するこ?...

止血は制限された時空間コンテキスト1で細胞、タンパク質、イオンと組織の組み合わせと調整活動が必要です。制御されていない活動は、血液凝固に関連する疾患のスペクトルに出血や血栓症および罹患率や死亡率につながる可能性があります。マイクロ流体フローチャンバー実験は、 インビトロで止血を模倣困難な技術です。このアプローチは、血小板のための先導的な役割で止血に参加するプロセスの複雑な相互作用の調査を可能にします。 血管損傷後、血小板は血液の損失を防止するために、露出した内皮下マトリックス（糖）タンパク質に付​​着します。接着に続いて、血小板が最終的にフィブリンおよび事務所の結果によって安定化された血小板のネットワークの形成につながるシグナル伝達を自動およびパラクリンに応答して活性化し、骨材、血栓2を封止巻か。他のほとんどの血小板機能検査、experiとは異なり、フローチャンバーを持つメントは、アカウントへの血流の物理的パラメータ、したがって、参加した細胞や生体分子3,4上のレオロジーの影響を取ります。 チャンバー実験フロー止血（サブ）に影響の重要なパラメータを変化させることによって、止血および血栓症の中で画期的な洞察を生成した接着剤マトリックス、レオロジー及び流れプロファイル、細胞組成、毒素または薬物、イオン強度および多くの存在を含む処理されます。過去20年間では、大規模なサンプル容量（10〜ml）を必要とする低スループットフローチャンバー実験は、多くの場合、小さな平行板室からなり、制御された壁面せん断条件5で全血を灌流するための近代的な技術を含むマイクロ流体室に進化してきました。ハードウェアのセットアップを簡略化しており、以下（血液）のボリュームが必要とされる主な理由Microscalingが大幅に実験をよりアクセスし、versatiレンダリング、アッセイのスループットを増加していますル。例えば、小型の実験動物からの血液は、現在、動物を犠牲にすることなく使用することができます。遺伝的に改変されたマウスの血液サンプルは、このように止血を促進または阻害鍵分子の同定におよび新規の基本的な洞察6に支援しています。 専門の研究室は、多くの場合、まだソフトウェアによってblueprintedすることができリトグラフ金型に重合したポリジメチルシロキサン（PDMS）7からインスタンスのカスタムメイドのフローチャンバーを使用します。その結果、チャンバは、安価な使い捨てであり、容易に事後解析のために分解することができます。また、分岐点または鋭いターンを含む血管の基本的にはどのようなデザインは、コマンド上に構築することができます。この利点は、標準化は既にフローチャンバー実験と主要な問題であり、PDMSは、カスタムメイドの室がこれを支援していないので、その欠点です。この特定の問題、コーティング（条件）、蛍光プローブ、抗凝固剤、温度の上サンプリングと分析の間eratureと時間がすべての悪い8を標準化されています。これらの変数の標準化は、困難、それにもかかわらず、研究室間の結果の比較を可能にするために必要とされます。このトピックでは、生物レオロジー9,10に関する科学と標準化小委員会における血栓止血に関する国際社会の主要な対象です。 血小板濃縮物（PC）は、血小板減少症および/または出血を引き起こす様々な疾患に罹患している患者に輸血されています。しかし、PC中の血小板は、特に貯蔵時間11の関数で、脱感作することが知られており、劣化プロセスは、加齢および一般血小板貯蔵病変と呼ばに連結されました。時々 、このような血小板はかつて12輸血血液循環に復元することが主張されているが、これのための証拠は乏しいです。さらに、PCを構成する血小板の機能は、日常的にテストされていないため、このようなアッセイとの間の関係治療的または予防的有効性は13不明です。マイクロ流体フローチャンバーは、コレクションと発行間の操作のチェーンを最適化するために、PC内の血小板機能を調査するための手段を提供しています。それは我々が以前に14,15を公開しているし、ここに記載されているように、PCの直接（ペアリング）比較のための強力な研究ツールです。

<table><tbody><tr><td>BD vacutainer tube with EDTA&amp;nbsp;</td><td>Becton, Dickinson and Company</td><td>368856</td><td></td></tr><tr><td>BD vacutainer tube with Heparin</td><td>Becton, Dickinson and Company</td><td>368480</td><td></td></tr><tr><td>BD vacutainer tube with Sodium Citrate</td><td>Becton, Dickinson and Company</td><td>366575</td><td></td></tr><tr><td>Hirudin Blood tube</td><td>Roche</td><td>6675751 001</td><td></td></tr><tr><td>BD vacutainer Eclipse</td><td>Becton, Dickinson and Company</td><td>368650</td><td>Blood collection needle with preattached holder</td></tr><tr><td>Pipette tips 100-1,000</td><td>Greiner bio-one</td><td>740290</td><td></td></tr><tr><td>Pipette tips 2-200</td><td>Greiner bio-one</td><td>739280</td><td></td></tr><tr><td>Pipette tips 1-10</td><td>Eppendorf</td><td>A08928</td><td></td></tr><tr><td>Tube 5 ml</td><td>Simport</td><td>11691380</td><td></td></tr><tr><td>Conical tube 15 ml</td><td>Greiner bio-one</td><td>1888271</td><td></td></tr><tr><td>Conical tube 50 ml</td><td>Greiner bio-one</td><td>227261</td><td></td></tr><tr><td>10 ml Syringe</td><td>BD</td><td>309604</td><td></td></tr><tr><td>Precision wipes</td><td>Kimtech</td><td>5511</td><td></td></tr><tr><td>Vena8 Fluoro+ Biochips</td><td>Cellix</td><td>188V8CF-400-100-02P10</td><td>Named in &lt;strong&gt;Figure S1&lt;/strong&gt; A as &#039;Biochip&#039;</td></tr><tr><td>Vena8 Tubing</td><td>Cellix</td><td>TUBING-TYGON-B1IC-B1OC-ROLL 100F</td><td>Named in &lt;strong&gt;Figure S1&lt;/strong&gt; B as &#039;Disposable tubing&#039;</td></tr><tr><td>Vena8 Needles</td><td>Cellix</td><td>SS-P-B1IC-B1OC-PACK200</td><td>Named in &lt;strong&gt;Figure S1&lt;/strong&gt; B as &#039;Pin&#039;</td></tr><tr><td>Connectors for single inlet cables of biochips</td><td>Cellix</td><td>CONNECTORS-B1IC-PACK100</td><td></td></tr><tr><td>Multiflow8 connect</td><td>Cellix</td><td>MF8-CONNECT-BIC3-N-THROMBOSIS</td><td>Named in &lt;strong&gt;Figure S1&lt;/strong&gt; B as &#039;Reusable tubing&#039; and &#039;Splitter&#039;</td></tr><tr><td>Humidified box</td><td>Cellix</td><td>HUMID-BOX</td><td></td></tr><tr><td>Software microfluidic pump</td><td>Cellix</td><td>N/A</td><td>Venaflux Assay</td></tr><tr><td>Horm Collagen</td><td>Takeda/Nycomed</td><td>1130630</td><td>Native equine tendon collagen (type I)&lt;br/&gt; Isotonic glucose solution to dilute collagen is supplemented</td></tr><tr><td>HEPES buffered saline (HBS)</td><td>in house preparation</td><td>in house preparation</td><td>10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (0.9% (w/v) NaCl, pH 7.4)</td></tr><tr><td>Blocking buffer</td><td>in house preparation</td><td>in house preparation</td><td>1.0% (w/v) bovine serum albumin and 0.1% (w/v) glucose in HBS</td></tr><tr><td>Calcein AM</td><td>Molecular probes</td><td>C1430</td><td></td></tr><tr><td>Bleach 10%</td><td>in house preparation</td><td>in house preparation</td><td></td></tr><tr><td>0.1 M NaOH</td><td>in house preparation</td><td>in house preparation</td><td></td></tr><tr><td>Denaturated alcohol</td><td>Fiers</td><td>T0011.5</td><td></td></tr><tr><td>Mirus Evo Nanopump</td><td>Cellix</td><td>188-MIRUS-PUMP-EVO</td><td>with Multiflow8. Named in &lt;strong&gt;Figure S1&lt;/strong&gt; A as &#039;Pump&#039; and &#039;Manifold&#039;</td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Axio Observer Z1</td><td>equipped with a colibri-LED and high resolution CCD camera</td></tr><tr><td>Software microscope&amp;nbsp;</td><td>Zeiss</td><td>N/A</td><td>ZEN 2012</td></tr><tr><td>Hematology analyzer</td><td>Sysmex</td><td>N/A</td><td></td></tr><tr><td>Table Top Centrifuge</td><td>Eppendorf</td><td>521-0095</td><td></td></tr><tr><td>Platelet incubater</td><td>Helmer</td><td>PF-48i</td><td></td></tr><tr><td>Incubation water bath</td><td>GFL</td><td>1013</td><td></td></tr><tr><td>Pipette</td><td>Brand</td><td>A03429</td><td></td></tr><tr><td>Tube Roller</td><td>Ratek</td><td>BTR5-12V</td><td></td></tr><tr><td>Sterile docking device</td><td>Terumo BCT</td><td>TSCD</td><td></td></tr><tr><td>Tubing Sealer</td><td>Terumo BCT</td><td>AC-155</td><td></td></tr><tr><td>Vortex</td><td>VWR</td><td>58816-121</td><td></td></tr></tbody></table>

microfluidic flow chambers using reconstituted blood to model hemostasis and platelet transfusion in vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

アッセイ内変動を示すために、3つの同一の再構成された全血試料を、コラーゲン被覆された表面（ 図1）を介して同時に灌流しました。これは、8.7％の変動係数をもたらしました。この統計は、関連するサンプル間の信頼性の比較を可能にする許容可能なアッセイ内およびintralaboratory変化を示唆しています。 ...

Watch this Scientific Journal Video about Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro at JoVE.com

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

マイクロ流体チャンバーモデル止血や血小板輸血に再構成された血液を使用しました<em&gt;インビトロ</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

microfluidic-flow-chambers-using-reconstituted-blood-to-model

Research

JoVE Journal

Bioengineering

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

9.3K ビュー.  Belgium Red Cross-Flanders. Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro. 

ビデオ: Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

腫瘍微小環境を再作成するためのマイクロ流体デバイス<em&gt;体外で</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

生物工学

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

誘電泳動と層流を用いたマルチチャネルマイクロ流体デバイスの分離ビーズと細胞

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

層流のせん断応力下の内皮前駆細胞を評価するために、平行平板フローチャンバーと定常流回路

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

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 ...

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-&#945; (TNF-&#945;), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

の体系的な分析<em&gt;体外</emマルチウェルプレートマイクロ流体システムを使用して&gt;セルローリング

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

血管細胞リモデリングの研究への応用生理学的および病理学的血流のインビトロモデル

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

血小板輸血や止血のためのマイクロ流体フローチャンバーモデルは、リアルタイムでの血小板沈着およびフィブリン形成を測定します

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

全血-内皮相互作用と血栓動態をリアルタイムで研究するインビトロ微小流体疾患モデル

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...

免疫・感染学

An In Vitro Hemodynamic Loop Model to Investigate the Hemocytocompatibility and Host Cell Activation of Vascular Medical Devices

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive conjugates was compared to uncoated PVC tubes, latex tubes, and a stent for intravascular application that was placed inside the PVC tubes. Evaluation of hemocompatibility was done using an in vitro hemodynamic loop model that is recommended by the ISO standard 10993-4. The tubes were cut into segments of identical length and closed to form loops avoiding any gap at the splice, then filled with human blood and rotated in a water bath at 37 &#176;C for 3 hours. Thereafter, the blood inside the tubes was collected for the analysis of whole blood cell count, hemolysis (free plasma hemoglobin), complement system (sC5b-9), coagulation system (fibrinopeptide A), and leukocyte activation (polymorphonuclear elastase, tumor necrosis factor and interleukin-6). Host cell activation was determined for platelet activation, leukocyte integrin status and monocyte platelet aggregates using flow cytometry. The effect of inaccurate loop closure was examined with x-ray microtomography and scanning electron microscopy, that showed thrombus formation at the splice. Latex tubes showed the strongest activation of both plasma and cellular components of the blood, indicating a poor hemocompatibility, followed by the stent group and uncoated PVC tubes. The coated PVC tubes did not show a significant decrease in platelet activation status, but showed an increased in complement and coagulation cascade compared to uncoated PVC tubes. The loop model itself did not lead to the activation of cells or soluble factors, and the hemolysis level was low. Therefore, the presented in vitro hemodynamic loop model avoids excessive activation of blood components by mechanical forces and serves as a method to investigate in vitro interactions between donor blood and vascular medical devices.

Presented here is a protocol for a standardized in vitro hemodynamic loop model. This model allows to test the hemocompatibility of perfusion tubes or vascular stents to be in accordance with ISO (International Organization for Standardization) standard 10993-4.

血管医療機器の血球適合性と宿主細胞活性化を調べるインビトロ血球ループモデル

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive ...

医学

<meta charset="utf8"></head><body>外傷および輸血における血小板機能を評価するためのプラットフォーム

Microfluidics in Assessing Platelet Function

Microfluidics incorporate physiologically relevant substrates and flows that mimic the vasculature and are, therefore, a valuable tool for studying aspects of thrombosis and hemostasis. At high-shear environments simulating arterial flow, a microfluidic assay facilitates the study of platelet function, as platelet-rich thrombi form in a localized stenotic region of a flow channel. Utilizing devices that allow for small sample volume can additionally aid in evaluating platelet function under flow from volume-limited patient samples or animal models. Studying trauma patient samples or samples following platelet product transfusion may aid in directing therapeutic strategies for patient populations in which platelet function is critical. Effects of platelet inhibition via pharmacological agents can also be studied in this model. The objective of this protocol is to establish a microfluidic platform that incorporates physiologic flow, biological surfaces, and relevant hemostatic mechanisms to assess platelet function with implications for the study of trauma induced coagulopathy and transfusion medicine.

<meta charset="utf8"></head><body>血小板機能の評価におけるマイクロ流体工学

Platelet function under flow can be assessed and simulated hemostatic resuscitation can be modeled using a microfluidic device, which has applications in trauma and transfusion medicine.

血小板機能の評価におけるマイクロ流体工学

Microfluidics incorporate physiologically relevant substrates and flows that mimic the vasculature and are, therefore, a valuable tool for studying ...

マイクロ流体チャンバーモデル止血や血小板輸血に再構成された血液を使用しました

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腫瘍微小環境を再作成するためのマイクロ流体デバイス