著者はDMFデバイスおよびDMFハブの設計と開発への貢献のためにロナルドF.レンツィとマイケルS. BARTSCHに感謝します。この研究は完全にサンディア国立研究所で研究所監督研究開発プログラムによってサポートされていました。サンディア国立研究所は、契約DE-AC04-94AL85000下エネルギー省の国家核安全保障管理局の米国商務省のためサンディア社、ロッキード·マーティン社の完全子会社が管理·運営、マルチプログラム研究室です。

この一般的なプロトコルは、研究の多種多様なプラットフォームのアプリケーションをサポートするように設計されているが、この報告書に記載の代表研究に固有の側面は括弧内に分離されている図2プロトコルを使用して行う代表的な研究を示しています。プロトコルでは、すべての &quot;指示する&quot;コマンドはあらかじめ決められたスクリプトの使用を介して自動化されていることに注意してください。ステップ2、ステップ1（ステップ1.7と1.8の間に、例えば ）と並行して行うことができることにも注意して、そのステップ2.1は、多くの場合、バイパスされ（パターン化/コーティングされたDMFデバイスプレートを洗浄して再使用することができるので、ステップ5.2を参照のこと） 。 1。細胞灌流チェンバースを組み立て、移入<ol><li>コー​​ト滅菌（オートクレーブ）マイクロキャピラリー（フューズドシリカ長さ15cm;; 679μmの外径、534μmの番号）の内面無菌フィブロネクチン溶液（50μgの/ mlの30μlの）を描画するためにシリンジポンプを指示することによってフィブロネクチンと、それぞれに解決策を撤回するシリンジポンプを指示すると、マイクロキャピラリー乾燥（6時間室温（RT）を）せ、（37℃2℃時間）インキュベートし、マイクロキャピラリー。 </li><li> CapTite継手（6灌流チャンバー、8ポートシリンジポンプ）を使用して、ポリカーボネートの管（500μmの外径、100μmのID）とマルチポートシリンジポンプにチューブに各フィブロネクチンでコーティングされたマイクロキャピラリー（灌流チャンバー）を接続します。 </li><li>テープを用いて、所望の温度（37℃）に設定したヒートブロックに各灌流チャンバーの本体を固定する。 </li><li>新鮮な増殖培地（RPMI-1640、10％FBS、500単位/ mlペニシリン、500μgの中に懸濁させ、リザーバ内の灌流チャンバーの開放端（microcentifugeチューブまたはマイクロタイタープレートウェル）を含有する細胞（P388D.1マウスマクロファージ）浸し/所望の濃度（10 6細胞/ ml）でのストレプトマイシン）。 </li><li>十分な愛が続く各灌流チャンバーに細胞（10μlを、10 4細胞）を描画するためにシリンジポンプを指示ヒートブロックに固定部分に液体プラグ（約4.5センチ）を移動するには、r。 </li><li>細胞は灌流チャンバーのフィブロネクチンでコーティングされた内部表面（ - 2時間37℃で1）に準拠することができます。一方、セルリザーバからの新鮮な成長培地を含む新しいタンクに潅流チャンバの開放端を転送する。 </li><li>遵守期間の後、無駄に新鮮な培地（10μL）を撤回し、付着した以上の新しい液体プラグ（新鮮な培地）を配置するために十分な空気に従うように液体プラグ（馴化培地と付着していない細胞）を送信するためにシリンジポンプを指示細胞。 </li><li>細胞集団は、（ - 〜1,000細胞/チャンバーの集団を生成したマイクロスケールの文化の24時間16）が十分に平衡化と拡張になるまで定期的に（毎週2時間）で培地交換（ すなわち繰り返しステップ1.7）を実施し続けています。 </li></ol> 2。 DMFデバイスを製造し、ハブ·アーキテクチャを組み立てる<ol><l私は&gt;前述14,29、四十から六インジウムスズ酸化物（ITO）、フォトリソグラフィとエッチングにより電極（滴作動のドライバ）、及びパリレン-Cおよび50の5μmのとコートとパターンDMFデバイスの底板を説明テフロン-AFのNM。テフロン（登録商標）-AFでパターン化されていないITOガラス基板は、上述したように、コーティングによりDMF装置の天板を形成する。 </li><li> 、この間隔は、作動電極サイズ（2.5ミリメートル2）と組み合わせた液滴量（2.5μLを定義し、金属圧縮フレームを使用して、プレート間に400μmの間隔を維持する凹部を有するポリマーキャスト14,30にDMFのプレートを固定する電極当たり ）。 DMFアセンブリは単純な手段24によって達成することができることに注意してください。 </li><li>その同族作動電極の端まで延長し、各位置決めをDMFプレート間の空間に、 - ;インサートテフロンコーティングされた転送マイクロキャピラリー（4.0センチメートル長い3.5 OD、100μmの番号360ミクロン）。 </li><li>反対ENに各転送マイクロキャピラリー接辞CapTite金具のD。 </li><li> ITO電極に電圧を供給するための電気コネクタをかみ合わせます。電極の活性化配列は、所定のスクリプトを実行しているコンピュータ制御された電子インターフェースの使用を介して自動化されている。 </li><li>オプション：液滴の追跡を容易にするために電荷結合素子（CCD）カメラ（DCR-HC96（ソニー、日本））を備えた顕微鏡の並進ステージ（SZ-6145TR（オリンパス、日本））上に組み立てられたハブDMFを移動する31 </li></ol> 3。 DMFハブに灌流チェンバースを接続し、細胞集団を刺激<ol><li>培地リザーバから灌流チャンバーの開放端を取り外します（ステップ1.6を​​参照）、（ステップ2.4​​を参照）をDMFハブの転送マイクロキャピラリーの両端に貼付CapTiteフィッティングに挿入してください。 </li><li>無駄に灌流チャンバー内の液体プラグ（馴化培地）を送信するためにシリンジポンプに指示します。 </li><li>インストオンボードリザーバから（ 大腸菌 0.1％プルロニックF127 w / vの新鮮な培地では100μg/ mlで生体粒子）刺激を描き、微小毛細血管各転送に適切な大きさの液滴（10μL）を提供するDMFハブをruct 。オンボード·リザーバは、円筒状の区画（1.5×1.5センチずつ）で構成され、配管を介してDMFハブに接続されている。 </li><li>刺激が転送マイクロキャピラリーに差し込む描き、灌流チャンバーで細胞集団上のプラグを配置するために十分な空気に従うことをシリンジポンプに指示します。 </li><li>刺激（ - 4時間37℃1℃）で細胞をインキュベートする。オプション：定期的に新鮮な刺激のために為替（ つまりリピートステップ3.2から3.4）。 </li></ol> 4。刺激を終了し、解析のための細胞ライセートを回復<ol><li>オプション：ポスト刺激期間が必要な場合は、（ すなわち繰り返しステップ3.2新鮮培地に対して刺激含有液プラグを交換する- 3.4、）刺激のために新鮮な培地を代入し、インキュベートし、必要に応じて交換することを続けています。 </li><li>洗浄バッファー（プレリュード直接溶解モジュールバッファーB）（ すなわち繰り返しステップ3.2から3.4まで、刺激のための洗浄バッファーを代入）のExchange灌流チャンバー内の液栓、となど（5分）が必要とインキュベートする。 </li><li>為替溶解溶液（プレリュード直接溶解モジュールバッファー0.1％プルロニックF127 w / v）のための液体プラグ（バッファを洗う）（ つまりリピートステップ3.2から3.4まで、刺激のために溶解液を置換）、および必要に応じてインキュベート（5分） 。 </li><li> DMFハブに液体プラグ（細胞溶解物）を送信するためにシリンジポンプに指示します。 </li><li> DMFハブを分解し、DMFデバイス（これは液滴混合になることができますように、横向きプレートを傾けないように注意して使用して）の天板を取り外し、オフプラットフォーム分析用ピペットマンや注射器を使用して溶解物（遺伝子発現プロファイリングを収集定量PCR経由）。 </li></ol> 5。用クリーンプラットフォームハードウェア再利用<ol><li> 、RTで10分間、10％の漂白剤（水中v / v）で、転写マイクロキャピラリーとCapTite継手を浸す脱イオン水で十分にすすぎ、窒素ガスを用いたドライ。 </li><li> RTで10分間、10％の漂白剤でDMFデバイスプレートを浸し、イソプロパノールでよくすすぎ、脱イオン水に続いて、ドライ用いて窒素ガスを10分間160℃で焼成した。 </li><li>シリンジポンプのポリカーボネートチューブ、およびDMFデバイスのポリマーキャストと圧縮フレームは、洗浄することなく再利用することができる。灌流チャンバーマイクロキャピラリーは、各実験の後に破棄されます。 </li></ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+minimally+disturbed+synchronous+cultures+of+hematopoietic+cells.">Production of minimally disturbed synchronous cultures of hematopoietic cells.</a> BioTechniques. 32, 1098-1105 (2002).</li><li>Huang, X., Dai, W., Darzynkiewicz, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enforced+adhesion+of+hematopoietic+cells+to+culture+dish+induces+endomitosis+and+polyploidy.">Enforced adhesion of hematopoietic cells to culture dish induces endomitosis and polyploidy.</a> Cell Cycle. 4, 801-805 (2005).</li><li>Raje, 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=Charged+nylon+membrane+substrate+for+convenient+and+versatile+high+resolution+microscopic+analysis+of+Escherichia+coli+&amp;+mammalian+cells+in+suspension+culture.">Charged nylon membrane substrate for convenient and versatile high resolution microscopic analysis of Escherichia coli &amp; mammalian cells in suspension culture.</a> Cytotechnology. 51, 111-117 (2006).</li><li>Chatterjee, D., Hetayothin, B., Wheeler, A. R., King, D. J., Garrell, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet-based+microfluidics+with+nonaqueous+solvents+and+solutions.">Droplet-based microfluidics with nonaqueous solvents and solutions.</a> Lab Chip. 6, 199-206 (2006).</li><li>Perroud, T. D., 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=Microfluidic-based+cell+sorting+of+Francisella+tularensis+infected+macrophages+using+optical+forces.">Microfluidic-based cell sorting of Francisella tularensis infected macrophages using optical forces.</a> Analytical Chemistry. 80, 6365-6372 (2008).</li><li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+A+revolutionary+tool+for+transcriptomics.">RNA-Seq: A revolutionary tool for transcriptomics.</a> Nature Reviews Genetics. 10, 57-63 (2009).</li><li>Malone, J. H., Oliver, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarrays,+deep+sequencing+and+the+true+measure+of+the+transcriptome.">Microarrays, deep sequencing and the true measure of the transcriptome.</a> BMC Biology. 9, (2011).</li><li>Wu, 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=Microfluidically-unified+cell+culture,+sample+preparation,+imaging+and+flow+cytometry+for+measurement+of+cell+signaling+pathways+with+single+cell+resolution.">Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution.</a> Lab on a Chip. 12, 2823-2831 (2012).</li><li>Srivastava, N., 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=Fully+integrated+microfluidic+platform+enabling+automated+phosphoprofiling+of+macrophage+response.">Fully integrated microfluidic platform enabling automated phosphoprofiling of macrophage response.</a> Analytical Chemistry. 81, 3261-3269 (2009).</li><li>Herr, A. 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=Microfluidic+immunoassays+as+rapid+saliva-based+clinical+diagnostics.">Microfluidic immunoassays as rapid saliva-based clinical diagnostics.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 5268-5273 (2007).</li></ol>

著者は、彼らが競合する経済的利益を持っていないことを宣言します。

私たちは、マイクロスケールの細胞培養と刺激実験のためのシンプルで汎用性の高い自動化されたプラットフォームを開発しました。文化サイズがさらに小さい直径のマイクロキャピラリーを使用することにより減少させることができる、プラットフォームは、私たちは小さな文化ボリュームと細胞集団- （ - 20μLと100 2,000細胞、室ごとに1）で動作するようになります。これらのスケ?...

（in vitroでの解析では）文化の中で維持された細胞の研究では、複雑な生物学的システムと人間の健康を支配する基本的な原理と分子メカニズムに貴重な洞察を提供してきました。ペトリ皿とマイクロタイタープレートを利用して分析するための文化、刺激し、細胞の収集のための従来の方法は、ミリリットル規模な文化量の細胞（≥10 5）（≥0.1ミリリットル）の大規模な集団の研究のために設計されていた。しかし、細胞の限られた量が使用可能な多くのインスタンスが（ 例えば、初代細胞）、または細胞の小集団（ 例えば 、集団全体のセル間のばらつきを低減する）ことが望ましい、または必要な試薬を得ることが困難であるまたは高価（ 例えば精製細胞分泌因子）。このような問題が正常に培養規模を縮小することによって対処することができ、これは、 すべての消費を低減するという利点を有している試薬は、in vitroでの解析1,2 にするために必要。残念なことに、従来の装置および方法は、マイクロスケールの文化や3-11はあまりにも複雑で、ほとんどの研究室で日常的使用のために特化され、現在入手可能なマイクロ流体ベースの自動化されたシステムの正確で再現性のある操作をサポートしていません。 マイクロスケールのボリュームで（1 - 20μL） - 本稿では、自動化された文化、刺激し、細胞の小集団（2,000細胞100）の回復のためのシンプルで汎用性の高い技術プラットフォームの組み立てと使用を説明します。フィブロネクチンでコーティングされたマイクロキャピラリーが（ &quot;細胞灌流チャンバー&quot;モジュール）マイクロスケール文化の確立、維持、刺激のためのサイトとして機能のセット、およびデジタルマイクロフルイディクス（DMF：プラットフォーム·アーキテクチャ（ 図1）は、設計でモジュール化され&quot;転送&quot;マイクロキャピラリー（ &quot;中央ハブ&quot;モジュール）14,15路線細胞を装備）12,13デバイス灌流チャンバーへとからと試薬。 DMFは、ユーザが個々に、同時に複数の液滴に対応し、デバイスハードウェアを変更することなく操作（ すなわち、再構成試料処理列）を変更または再注文することを可能にする。その驚異的な柔軟性は、細胞培養16,17、酵素アッセイ18,19、20,21イムノアッセイ、DNA分析22,23、タンパク質処理、24,25など、幅広い用途でのキーテクノロジーとしての最近の出現で明らかであるおよび臨床検体処理。26,27地球中心ハブは、DMFデバイスに固有の柔軟性を利用し、さらに特化した周辺装置の操作のサブセット（ 例えば、細胞培養物）を実施する機会を提供微小インタフェースを追加することによってそれを増強するむしろDMFデバイス自体よりもモジュール。このように処理列車の区画もPLAの設計を簡素化TFORMアーキテクチャ（ すべての処理手順を実行することができるDMFデバイスを構築する必要がない）と新しい機能としての進化を促進するには（単に必要に応じて新たな周辺モジュールを統合）が必要です。から、交通機関へ、そして灌流チャンバー内で高精度な注射器で生成された圧力変化によって供給され、中央ハブ内の細胞と試薬の輸送はDMF装置13,28内の電極の逐次的活性化によって生成される力をエレクトロウェッティングにより駆動されるポンプ。これらの流体運動のすべては、単純な電子インターフェースを介して制御され、予め決められたスクリプトを使用して自動化されている。 代表的な例として、細菌によるチャレンジ（ 図2）により誘発された免疫細胞の転写応答の研究のためのプラットフォームの使用を示す。プラットフォーム上でこれらの実験を行うことは、私たちは、細胞の数が少ない（experimen あたり 〜1,000で動作するように有効にTAL条件）、実験·ツー·実験の変動、節約試薬、再直接ハンズオン労力を最小限に抑えます。それが与えることの利点だけでなく、そのアクセシビリティと汎用性を考えると、このプラットフォームでは、研究室や様々なアプリケーションでの使用を見つけて、限られた量で提供された細胞と刺激の分析を容易にする、特に有用であることが分かるはずです。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Prelude Direct Lysis Module</td>
 <td>NuGEN</td>
 <td>1400-24</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypan Blue (0.4% w/v)</td>
 <td>GIBCO</td>
 <td>15250-061</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell Stripper</td>
 <td>Cellgro</td>
 <td>25-056-C1</td>
 <td></td>
 </tr>
 <tr>
 <td>Ovation PicoSL WTA</td>
 <td>NuGEN</td>
 <td>3310-048</td>
 <td></td>
 </tr>
 <tr>
 <td>Agencourt RNAClean XP</td>
 <td>Beckman Coulter Genomics</td>
 <td>A63987</td>
 <td></td>
 </tr>
 <tr>
 <td>pHrodo BioParticles</td>
 <td>Invitrogen</td>
 <td>P35361</td>
 <td></td>
 </tr>
 <tr>
 <td>CCL4 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00443111_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>CCL5 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm01302428_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>PTGS2 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00478374_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>TNF TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00443258_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>GAPDH TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm99999915_g1</td>
 <td></td>
 </tr>
 <tr>
 <td>Pluronic F127</td>
 <td>Sigma Chemical</td>
 <td>2594628</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluorinert FC-40</td>
 <td>Sigma Chemical</td>
 <td>51142-49-5</td>
 <td></td>
 </tr>
 <tr>
 <td>Parylene C dimer</td>
 <td>Specialty Coating Systems</td>
 <td>28804-46-8</td>
 <td></td>
 </tr>
 <tr>
 <td>Teflon-AF</td>
 <td>DuPont</td>
 <td>AF1600</td>
 <td></td>
 </tr>
 <tr>
 <td>Polyimide tape</td>
 <td>ULINE</td>
 <td>S-11928</td>
 <td></td>
 </tr>
 <tr>
 <td>Indium tin oxide (ITO) coated glass substrates </td>
 <td>Delta Technologies </td>
 <td>CB-40IN-1107</td>
 <td></td>
 </tr>
 <tr>
 <td>DMF hub Teflon-coated fused-silica microcapillaries</td>
 <td>Polymicro Technologies</td>
 <td>TSU100375</td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber microcapillaries</td>
 <td>Polymicro Technologies</td>
 <td>TSP530700</td>
 <td></td>
 </tr>
 <tr>
 <td>Tubing and microcapillary fittings</td>
 <td>Sandia National Laboratories</td>
 <td>&nbsp;</td>
 <td></td>
 </tr>
 <tr>
 <td>Polycarbonate tubing</td>
 <td>Paradigm Optics</td>
 <td>CTPC100-500-5</td>
 <td></td>
 </tr>
 <tr>
 <td>8-port precision syringe pump equipped with 30 mm (500 &mu;l capacity) syringes</td>
 <td>Hamilton Company</td>
 <td>54848-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Parylene-C vapor deposition instrument</td>
 <td>Specialty Coating Systems</td>
 <td>PDS 2010 Labcoter 2</td>
 <td></td>
 </tr>
 <tr>
 <td>High-voltage function generator</td>
 <td>Trek</td>
 <td>615A-1 615-3</td>
 <td></td>
 </tr>
 <tr>
 <td>MVX10 microscope</td>
 <td>Olympus</td>
 <td></td>
 <td>Optional (facilitates tracking of droplets on DMF hub)</td>
 </tr>
 <tr>
 <td>QIClick digital CCD camera</td>
 <td>QImaging</td>
 <td>QIClick-F-CLR-12</td>
 <td>Optional (facilitates tracking of droplets on DMF hub)</td>
 </tr>
</tbody>
</table>

a versatile automated platform for micro-scale cell stimulation experiments

培養中の細胞（in vitroでの解析で ）の研究では、複雑な生物学的システムに重要な洞察を提供してきました。 in vitroでの解析でするための従来の方法と装置はよくミリリットル規模な量の細胞（≥10 5）（≥0.1ミリリットル）の多数の研究に適しています。しかしながら、関心および/またはその培養物、刺激、または処理に必要な試薬の細胞の消費を減らすために、培養規模を縮小することが必要または望ましいである多くの例がある。残念なことに、従来のアプローチでは、マイクロスケールの文化を正確かつ再現性のある操作をサポートしていないと、現在利用可能なマイクロ流体ベースの自動化されたシステムは、あまりにも複雑で、ほとんどの研究室で日常的使用のために特化されます。マイクロスケール体積 - この問題に対処するために、我々は自動化された培養物、刺激し、細胞の小集団（2,000セル100）の回収のためのシンプルかつ汎用性の高い技術プラットフォームを開発したS（1 - 20μL）。 &quot;転送&quot;マイクロキャピラリー（ &quot;中央ハブを装備し、デジタルマイクロフルイディクス（DMF）デバイス、プラットフォームは、マイクロスケール培養物を、確立され維持され、刺激され、その中フィブロネクチンでコーティングされたマイクロキャピラリー（&quot;細胞灌流チャンバー &quot;）、のセットで構成され&quot;）、および灌流チャンバーからのルート細胞および試薬れ、パワー灌流室と中央ハブの間の材料の輸送高精度シリンジポンプ、れると、材料の輸送を制御でき電子インターフェース、ある調整し、予め決められたスクリプトを介して自動化。例として、細菌によるチャレンジに基づい免疫細胞において誘発転写応答の研究を容易にするために、プラットフォームを使用した。プラットフォームを使用することにより、私たちは、細胞や試薬の消費量を減らす実験ツー実験の変動を最小限にするために有効で、再直接ハンズオン労働。それが与えることの利点だけでなく、そのアクセシビリティと汎用性、Oを考えるウルプラットフォームは、研究室やさまざまなアプリケーションでの使用を見出す、とだけ数量限定で提供された細胞と刺激の分析を容易にする、特に有用であることが分かるはずです。

自動化されたプラットフォームの証明として、我々は、免疫細胞の小集団は、マイクロスケール培養で増殖させた細菌でチャレンジし、炎症促進性応答（ 図2のプラットフォーム外分析のために溶解しされた研究を行うためにそれを使用し）。 6セル灌流チャンバーの各々は、成長培地10μlに再懸濁し、10 4免疫細胞（P388D.1ネズミマクロファージ）を?...

Watch this Scientific Journal Video about A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments at JoVE.com

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

我々は、マイクロスケール細胞刺激実験のために自動細胞培養と尋問プラットフォームを開発した。プラットフォームは、栽培と細胞の小集団を刺激し、分子解析のための溶解物の回収で、シンプルで汎用性の高い、かつ正確なコントロールを提供しています。プラットフォームはよく貴重な細胞および/または試薬を使用した研究に適しています。

マイクロスケール細胞刺激のための実験のための多目的自動化プラットフォーム

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

a-versatile-automated-platform-for-micro-scale-cell-stimulation

Research

JoVE Journal

Bioengineering

10.5K ビュー.  Sandia National Laboratories. 我々は、マイクロスケール細胞刺激実験のために自動細胞培養と尋問プラットフォームを開発した。プラットフォームは、栽培と細胞の小集団を刺激し、分子解析のための溶解物の回収で、シンプルで汎用性の高い、かつ正確なコントロールを提供しています。プラットフォームはよく貴重な細胞および/または試薬を使用した研究に適しています。

ビデオ: A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

器官型コラーゲンIアッセイ：3次元コンテキストでのセルの挙動を評価するための可鍛性プラットフォーム

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

生物工学

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
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GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.

GENPLAT：バイオマスの酵素の発見とカクテルの最適化のための自動化プラットフォーム

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

堅牢、マイクロ流体細胞内送達プラットフォームとしての細胞絞る

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

細胞および組織メカノでのアプリケーションのためのプラットフォームをストレッチ小説

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

ハイスループットシングルセルの単離および培養のためのマイクロ流体プラットフォーム

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

階層化アルギン酸構築：異種細胞集団の共培養のためのプラットフォーム

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

細胞の分化やトラクション軍の相関的分析のためのハイスループット細胞マイクロアレイプラットフォーム

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

パターニングタンパク質および細胞の多彩な方法

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

セミフレキシブル高分子を研究する多目的ツールとして DNA ナノチューブ

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

長期無細胞遺伝子発現の伝導のための多層マイクロ流体プラットフォーム

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...