この研究は健康の認可番号R01NS050452とR01EB009429総合研究所によって資金を供給された。カンチレバーチップの製造は、コーネル大学にあるナノ加工施設での共同研究者により外部で行われた。カンチレバーの製造工程で使用されるすべての機器はこの施設に位置していた。カンチレバー微細化と彼らの支援のためのマンディエッシュとジャン=マチューProtのに感謝します。カンチレバーの機能のビデオのアニメーションは、UCFでの合成リアリティ·ラボからチャールズ·ヒューズ、アレックスZeleninとエリック·インペリアーによって生成されました。

1カンチレバーチップ製造説明した製造工程を図示した細部は、 図1に示す。 <ol><li>オーブンでシリコン·オン·インシュレータ（SOI）ウェーハを配置し、それらを脱水し、20分間125℃で焼く。 </li><li>プラズマを用いて脱水したSOIウェハのハンドル層上に酸化ケイ素の1.5μmの厚さの層を堆積させる化学気相成長法（PECVD）ツールを強化。 </li><li>デバイス層を上に向けてスピンコーターチャック上のウエハを配置します。 、ウエハがセンタリングされていることを確認し1000回転/秒の加速度で60秒間、3000rpmでウェハの中心、そしてスピンでP20プライマーの2ミリリットルを分注する。 P20プライマーは、デバイス層にフォトレジストの接着を促進する。 </li><li>ウェハをスピンコータチャック上にある間に、ウェーハの中心にS1818フォトレジストを2mlを分配、およびo ​​1,000回転/秒の加速度で60秒間3,000 rpmでスピンフォトレジストの厚さ1.5μmの層をbtain。 </li><li>ホットプレート上でウェーハを置き、1分間115℃でソフトベークを行う。 </li><li>デバイス層上のフォトレジストにカンチレバーマスクからパターンを転写するためにフォトリソグラフィーコンタクトマスクアライナを使用してハードコンタクトUV露光を行う。 125 MJ / S1818フォトレジスト用cm 2の露光エネルギー値に基づいて露光時間を算出する。 </li><li>優しく前後にウェハを振りながら、1分間726 MIFフォトレジスト現像液にウェハを浸漬することにより、フォトレジストを開発する。 </li><li>脱イオン（DI）水を用いてウェーハをリンスし、それを乾燥、好ましくは、スピンリンス乾燥（SRD）ツールを使用して。 </li><li>アセトンに浸した綿棒を使用して（端から5mmまで）ウェーハのエッジからフォトレジストを除去します。 </li><li>フォトレジスト側を上にして、ディープ反応性イオンエッチング（DRIE）ツールで、ウェーハをロードし、デビを通じてパターニングされたシリコン層をエッチングするレシピを実行CE層。エッチストップとしての埋め込み酸化物層の機能なので、完全なエッチングを確実にするために必要であると予想されるよりも50％〜100％以上のサイクルでエッチングを行う。 </li><li>フォトレジストを除去するために20分間のホットフォトレジスト浴溶液にウェハを配置します。 DI水でウェーハを洗浄するためにクイックダンプリンサ（QDR）にウエハを転送します。フォトレジスト剥離後、ウェーハを洗浄し、乾燥さSRDツールでウェーハをロードします。期待どおりカンチレバーパターンが表示されていることを確認するために、顕微鏡下でウェハを検査します。 </li><li> PECVDツールを使用して、ウエハのデバイス層の上に非ドープ酸化シリコンの厚さ1μmの層を堆積。さらなる処理ステップ中にカンチレバーの保護層としてこの酸化物層は機能する。 </li><li>ウェハの裏面の処理を開始するために上に向け、ハンドル層をスピンコータチャックにウェハを配置します。 、ウエハがセンタリングされていることを確認し、ウェハの中央にP20プライマーの2ミリリットルを分配し、3000 rpmでスピン1,000回転/秒の加速度で60秒間。 </li><li>ウェハはスピンコーターチャックに入れたまま、ウエハの中心にSPR220-4.5フォトレジストの2ミリリットルを分配し、1000回転/秒の加速度で45秒間、2000rpmでスピンの6.5ミクロンの厚さの層を得るためフォトレジスト。 </li><li> 2分間115℃でソフトベークを実行するために近接ホットプレート上にウエハを配置します。 </li><li>前面/背面の位置合わせが可能なフォトリソグラフィコンタクトアライナーを使用して、前側カンチレバーパターンに裏面ウィンドウマスクを位置合わせし、ハンドル層上のフォトレジストに裏面ウィンドウマスクのパターンを転写するために、ハードコンタクトUV露光を行う。 SPR220-4.5フォトレジスト480 MJ / cm 2の露光エネルギー値に基づいて算出された露光時間を使用する。 </li><li> UV露光の後、ウェハは、次の処理ステップの前に30分間、暗い領域に残るう。 </li><li>ウエハを浸漬することによりフォトレジストを現像2分間726 MIFのフォトレジスト現像液を穏やかに前後にウェーハを振とうしながら。 </li><li>脱イオン（DI）水を用いてウェーハをリンスし、それを乾燥、好ましくは、スピンリンス乾燥（SRD）ツールを使用して。 </li><li> 12時間90℃のオーブンでウェーハを配置します。 </li><li>フッ素化ガスによるRIEシステムおよび酸化物をエッチングするためのレシピを用いてウエハの裏面に酸化シリコン層をエッチングする。ウェハの裏面上のパターン化された酸化物は、さらなる処理のためのエッチングマスクとして機能する。露出したウィンドウ内の酸化シリコンが完全にエッチングされることを保証するために、顕微鏡下でウェハを検査します。 </li><li>アセトン中に浸し、クリーンルーム綿棒を使用して（端から5mmまで）ウェーハのエッジ上にフォトレジストを除去します。 </li><li>とDRIEツールでウエハをロードし、エッチストップとしての埋め込み酸化膜の機能化を500μmの深さにパターン化されたハンドル層をエッチングするためにレシピを実行します。の過熱を防止するために、複数の実行中に、このエッチを分割シリコンの一貫性のないエッチングを引き起こすウェーハ。このエッチング工程は、完全にカンチレバー下のシリコンを除去します。 </li><li>カンチレバー下記の埋め込み酸化物層とカンチレバーの上に保護酸化物層を除去するために、25％希釈HFエッチング液を用いたウェットエッチング工程を行う。 注：この手順は、シリコンカンチレバーの底面を解放し、また、レーザーでカンチレバーをプローブするためのアクセスを提供するために下にあるウィンドウが開きます。 </li><li> DI水浴場のシリーズと窒素で丁寧に乾燥を用いてウェハを洗浄します。カンチレバーは、その拠点でのみサポートされているので、直接カンチレバーにDI水または不活性ガスの強制スプレーを使用しないでください。 </li><li>ハンドル層エッチング工程中に生成劈開ラインに沿ってウェハから各チップを切断する。 </li></ol> 2細胞培養<ol><li>以前に公開されている方法17に従って13Fカバースリップを準備します。 注：もし13F入り江rslipsが利用できない、95°以上の接触角を有する任意の疎水性表面を使用することができる。 </li><li>カンチレバーチップ、70％エタノール溶液中の13Fカバースリップを滅菌し、フローフード中で空気乾燥させ。 </li><li>標準12ウェルプレートの内側13Fカバースリップの上に個別のカンチレバーのチップを置きます。 </li><li>コー​​ト細胞型について最適化されたバイオポリマーまたは表面修飾を有するカンチレバーは、標準的な細胞培養プロトコルに従って使用されている。 </li><li>所望の濃度へのそれらの特異的な増殖培地中で細胞を再懸濁する。 注：このプロトコルは、カンチレバー窓から落下し、所望のカンチレバー表面に付着していない細胞のかなりの数になります。細胞調製は、したがって、標準的なカバースリップの準備のためよりも濃縮3~4なされるべきである。例えば、ラット骨格筋衛星細胞の播種は、典型的には500〜セル/平方ミリメートル15,16の播種密度を使用して行われる、および/平方mm 2000個の細胞でカンチレバー基板で使用されます。最適化実験は、適切な播種密度を確認するために、新しい細胞源を用いて実施されるべきである。 </li><li>媒体の気泡が完全にカンチレバー窓を覆う確保カンチレバーチップ表面上に細胞懸濁液200μlをピペット。媒体が窓から発散し、カンチレバーの上に静的な泡を形成しない場合は13Fのカバーガラスを交換し、メッキを再試行してください。 </li><li>インキュベーターにチップを含むプレートを移し、細胞が少なくとも1時間（好ましくは2〜3時間）のために接着させる。 </li><li>このメッキ期間の後、増殖培地1mlの文化を13Fカバースリップすることなく、きれいなだけでなく、チップを転送し、トップアップするために滅菌ピンセットを使用しています。 </li><li>インキュベーターにプレートを返します。 </li><li>カバーガラス上のインビトロ維持のための彼らの標準的なプロトコルに従って細胞を維持する。骨格筋細胞のために、細胞がコンフルエントになったら、筋管形成を誘導する培地組成のスイッチが必要になります。 </li></ol>カンチレバーのたわみ解析のためのハードウェアおよびソフトウェアのセットアップ3。 <ol><li>直立電気生理学顕微鏡のステージに加熱された培養皿を置きます。 </li><li>加熱された顕微鏡ステージに細胞が現在（+ 10mMのHEPES）中に懸濁さ給餌培地3mlを加える。 </li><li> 15mmの離間距離で加熱された培養皿の内側のステンレス鋼電極をマウントし、パルス発生器に接続し、変化する強度、周波数、及び波形の電界刺激パルスを生成することができる、システムは電場刺激を生成できるようにする細胞の適切なとき。 </li><li>ボルトヘリウムネオンレーザーは、顕微鏡台の下面に、XY並進ステージ上に取り付けられ、レーザビームは30°の角度で加熱した培養皿の底を通って導かれるように調整relativカンチレバーの面に電子。 </li><li>ボルト象限光検出器モジュール、顕微鏡ステージの下側に、XY並進ステージ上に取り付けられ、4象限の中央になるように反射されたレーザ光 ​​の土地の位置を調整してください。 図2は、設定されたハードウェアの概要を説明します記載されたプロトコルを実装するために必要な。 </li><li>カンチレバーを横切って走査するリニアアクチュエータを制御するためのソフトウェアプログラムを記述します。 図3に設けフローチャートを参照して、ソフトウェアプログラムを書くこのシステムで使用するためにプログラムされたグラフィカルインターフェイスは、 図4に設けられている。 </li></ol> 4。録音カンチレバーたわみデータ<ol><li>カンチレバー分析ハードウェアと関連ソフトウェアをオンにします。 </li><li>媒体への加熱されたステージのサーミスタを挿入し、それが37℃を読むことを待つ。 </li><li> cantileveと段階にカンチレバーチップを挿入ステージの右手側に向けrsは。 </li><li>顕微鏡の光源をオンにしてください。 </li><li>視野にカンチレバーのエッジをもたらし、カンチレバー1 NOTEの先端にレーザビームを位置決めするレーザー/光検出器制御ソフトウェアを使用するために顕微鏡の焦点を合わせる：カンチレバーがステージの右に向いていると仮定すると、カンチレバー1配列の左上に配置された1つであり、数字は左下に16にダウンを実行。カンチレバー17は、右上の位置にあり、右下の（ 図5A）で32に実行されます。 </li><li>を押して、録音ソフトウェア上で「再生」。 </li><li>位置の光検出器信号は光検出器を制御するステッピングモータを調整することにより、xとyの両方のフレームに「0」を読み出すようになっている。 </li><li>レーザー/光検出器制御ソフトウェアで設定カンチレバー1の位置（ 図4）。 </li><li>ステップ4.7を繰り返して、カンチレバー16の先端にレーザーを移動させ、cantilevを設定えー、レーザー/光検出器制御ソフトウェアの16ポジション。 </li><li>カンチレバー32は、手順4.7の先端にレーザーを移動させ、レーザー/光検出器制御ソフトウェアのカンチレバー32の位置を設定します。 </li><li>カンチレバー17は、手順4.7の先端にレーザーを移動させ、レーザー/光検出器制御ソフトウェアのカンチレバー17の位置を設定します。 </li><li>顕微鏡光源と実験室でのオーバーヘッドライトをオフにします。 </li><li>録音ソフトウェアを押して「記録」。 </li><li> 1Hzの周波数で40ミリ秒、5 Vのパルスに対するパルス発生器のハードウェアを設定し、マシンの電源を入れます。 注：必要に応じて、この時点では、特定の細胞ソースの最適化された刺激プロトコルを採用して、述べ設定は、ヒトとげっ歯類の細胞源12,13,15,16を使用して収集されたデータに基づくガイドラインである。 </li><li>レーザー/光検出器制御ソフトウェアを使用して、32カンチレバーアレイを横切って走査するためのハードウェアを設定し、それぞれの上に5秒間停止メール。 </li><li> 32カンチレバーのスキャンが完了すると、次に録音ソフトを停止し、データファイルを起動し、刺激装置をオフにしてください。 </li><li>収縮活動の証拠について、それぞれのカンチレバーから記録されたトレースを調べます。陽性反応をそれぞれのカンチレバーをメモしておきます。偏向は、ベースラインよりも少なくとも0.1 Vである場合に収縮がピークと定義される。 </li><li>レーザー/光検出器制御ソフトウェア上のスキャンプロトコルから任意の非応答性のカンチレバーを取り外します。 </li><li>アクティブカンチレバーは、細胞の自発収縮活動の測定値を得るために刺激することなく、再スキャンすることができる。 </li><li>培養された細胞の機能的出力への影響を観察するために、媒体への治療用化合物を添加した後、電気刺激または幅広い分野ずにスキャンを実行します。 </li><li>電気的に延長された期間とCONTRのスキャンレベルのために細胞を刺激することにより、疲労評価を実施<html> actilityは、それが特定のしきい値以下に低下するピーク力のためにかかる時間を測定した。 </li><li>運動ニューロンは筋肉との共培養で維持されている実験では、またはシナプス阻害剤（ 例えば 、D-ツボクラリン）と走査（グルタミン酸など）、神経刺激薬と運動ニューロン、筋管カンチレバー共培養の処理により、神経筋接合部の形成を測定自発的活動の増加と減少はそれぞれ16。 </li><li>計画された実験の必要性によって指示されるように、特定のスキャンを実行します。 </li></ol>カンチレバーたわみデータの解析5。 <ol><li>使用して、ストーニーの方程式は15細胞層または筋管（パスカル）、および（ナノニュートン）は、細胞の収縮力を直接測定における応力の読み出しに（ボルト）で生カンチレバー偏向データを変換する（詳細は下記）更新日： 広告/ 51866 / 51866eq1.jpg &quot;/&gt;式（1） <img alt="式2" fo:content-width="2in" width="300" src="/files/ftp_upload/51866/51866eq2.jpg" />式2 </li><li>ここで、δ=筋管によって生成カンチレバー先端のたわみや応力、筋管によって生成σC =ストレス、写真上のレーザー位置に電圧を関連するカンチレバー、C型検出器 =システム固有の係数の均一な厚膜と仮定すると全幅-detector、θ=カンチレバーの面に対するレーザおよび検出器の角度、E Siはシリコンの弾性率=、tのSiはSiの毒=ポアソン比V、T fは筋の厚さ 、カンチレバーの厚さ=シリコンカンチレバー先端から検出器へのレーザのL =片持ち梁の長さ、P =経路長、およびw <sub&gt; Siは、カンチレバーの幅を=。これらの式で使用される用語を説明するための概略を図6に提供される。 </li><li>ストレスとストーニーのの適用のために使用されたと仮定し、セルの断面積からの力の計算を等しくすることで、筋管が均一な膜である、筋管の力が式3につながる、フィルムの力に等しいと仮定すると方程式。 <img alt="式3" fo:content-width="1.5in" src="files/ftp_upload/51866/51866eq3.jpg" />式3 </li><li>機能的なデータ収集に続いて、免疫細胞化学分析のためにカンチレバーチップを修正するか、標準的な技術を使用してタンパク質またはDNA分析のために細胞を利用する。 </li><li>必要に応じて、代わりに、後の時点で機能的性能を再評価するために、分子解析のための準備のインキュベーターにセルを返します。 </li></ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-fiber+study+of+contractile+and+biochemical+properties+of+skeletal+muscles+in+streptozotocin-induced+diabetic+rats.">Single-fiber study of contractile and biochemical properties of skeletal muscles in streptozotocin-induced diabetic rats.</a> Diabetes. 43, 622-628 (1994).</li><li>Harber, M., Trappe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+muscle+fiber+contractile+properties+of+young+competitive+distance+runners.">Single muscle fiber contractile properties of young competitive distance runners.</a> Journal of applied physiology (Bethesda, Md: 1985). 105, 629-636 (2008).</li><li>Hvid, L. G., 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=Four+days+of+muscle+disuse+impairs+single+fiber+contractile+function+in+young+and+old+healthy+men.">Four days of muscle disuse impairs single fiber contractile function in young and old healthy men.</a> Experimental gerontology. 48, 154-161 (2013).</li><li>Edman, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+performance+of+striated+muscle.">Contractile performance of striated muscle.</a> Advances in experimental medicine and biology. 682, 7-40 (2010).</li><li>Krivickas, L. S., Walsh, R., Amato, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+muscle+fiber+contractile+properties+in+adults+with+muscular+dystrophy+treated+with+MYO-029.">Single muscle fiber contractile properties in adults with muscular dystrophy treated with MYO-029.</a> Muscle Nerve. 39, 3-9 (2009).</li><li>Sung, J. H., 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=Microfabricated+mammalian+organ+systems+and+their+integration+into+models+of+whole+animals+and+humans.">Microfabricated mammalian organ systems and their integration into models of whole animals and humans.</a> Lab on a chip. 13, 1201-1212 (2013).</li><li>Wilson, K., Molnar, P., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+functional+myotubes+with+a+Bio-MEMS+device+for+non-invasive+interrogation.">Integration of functional myotubes with a Bio-MEMS device for non-invasive interrogation.</a> Lab on a chip. 7, 920-922 (2007).</li><li>Wilson, K., Das, M., Wahl, K. J., Colton, R. J., Hickman, J. <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+contractile+stress+generated+by+cultured+rat+muscle+on+silicon+cantilevers+for+toxin+detection+and+muscle+performance+enhancement.">Measurement of contractile stress generated by cultured rat muscle on silicon cantilevers for toxin detection and muscle performance enhancement.</a> PLoS ONE. 5, (2010).</li><li>Binnig, G., Quate, C. F., Gerber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+Force+Microscope.">Atomic Force Microscope.</a> Physical Review Letters. 56, 930-933 (1986).</li><li>Pirozzi, K. L., Long, C. J., McAleer, C. W., Smith, A. S., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+embryonic+skeletal+muscle+myotube+physical+characteristics+with+contractile+force+generation+on+an+atomic+force+microscope-based+bio-microelectromechanical+systems+device.">Correlation of embryonic skeletal muscle myotube physical characteristics with contractile force generation on an atomic force microscope-based bio-microelectromechanical systems device.</a> Applied physics letters. 103, 83108 (2013).</li><li>Smith, A., Long, C., Pirozzi, K., Hickman, 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+functional+system+for+high-content+screening+of+neuromuscular+junctions+in+vitro.">A functional system for high-content screening of neuromuscular junctions in vitro.</a> Technology. 1, 37-48 (2013).</li><li>Stenger, D. A., 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=Coplanar+molecular+assemblies+of+amino-+and+perfluorinated+alkylsilanes:+characterization+and+geometric+definition+of+mammalian+cell+adhesion+and+growth.">Coplanar molecular assemblies of amino- and perfluorinated alkylsilanes: characterization and geometric definition of mammalian cell adhesion and growth.</a> Journal of the American Chemical Society. 114, 8435-8442 (1992).</li><li>Das, M., Rumsey, J. W., Bhargava, N., Stancescu, M., Hickman, J. 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+defined+long-term+in+vitro+tissue+engineered+model+of+neuromuscular+junctions.">A defined long-term in vitro tissue engineered model of neuromuscular junctions.</a> Biomaterials. 31, 4880-4888 (2010).</li><li>Rumsey, J. W., Das, M., Bhalkikar, A., Stancescu, M., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+the+mechanosensory+circuit+of+the+stretch+reflex+arc:+Sensory+neuron+innervation+of+intrafusal+muscle+fibers.">Tissue engineering the mechanosensory circuit of the stretch reflex arc: Sensory neuron innervation of intrafusal muscle fibers.</a> Biomaterials. 31, 8218-8227 (2010).</li><li>Das, M., Rumsey, J. W., Bhargava, N., Stancescu, M., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+tissue+engineering:+A+maturation+model+promoting+long-term+survival+of+myotubes,+structural+development+of+the+excitation-contraction+coupling+apparatus+and+neonatal+myosin+heavy+chain+expression.">Skeletal muscle tissue engineering: A maturation model promoting long-term survival of myotubes, structural development of the excitation-contraction coupling apparatus and neonatal myosin heavy chain expression.</a> Biomaterials. 30, 5392-5402 (2009).</li><li>Das, 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=Developing+a+novel+serum-free+cell+culture+model+of+skeletal+muscle+differentiation+by+systematically+studying+the+role+of+different+growth+factors+in+myotube+formation.">Developing a novel serum-free cell culture model of skeletal muscle differentiation by systematically studying the role of different growth factors in myotube formation.</a> In Vitro Cell Dev Biol Anim. 45, 378-387 (2009).</li><li>Stenger, D. A., Pike, C. J., Hickman, J. J., Cotman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+determinants+of+neuronal+survival+and+growth+on+self-assembled+monolayers+in+culture.">Surface determinants of neuronal survival and growth on self-assembled monolayers in culture.</a> Brain research. 630, 136-147 (1993).</li><li>Hickman, 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=Rational+pattern+design+for+in+vitro+cellular+networks+using+surface+photochemistry.">Rational pattern design for in vitro cellular networks using surface photochemistry.</a> Journal of Vacuum Scienc., &amp; Technology A: Vacuum, Surfaces, and Films. 12, 607-616 (1994).</li><li>Das, M., Molnar, P., Devaraj, H., Poeta, M., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+and+morphological+characterization+of+rat+embryonic+motor+neurons+in+a+defined+system.">Electrophysiological and morphological characterization of rat embryonic motor neurons in a defined system.</a> Biotechnology progress. 19, 1756-1761 (2003).</li><li>Rumsey, J. W., 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=Node+of+Ranvier+formation+on+motor+neurons+in+vitro.">Node of Ranvier formation on motor neurons in vitro.</a> Biomaterials. 30, 3567-3572 (2009).</li><li>Murugan, R., Molnar, P., Rao, K. P., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterial+Surface+patterning+of+self+assembled+monolayers+for+controlling+neuronal+cell+behavior.">Biomaterial Surface patterning of self assembled monolayers for controlling neuronal cell behavior.</a> International journal of biomedical engineering and technology. 2, 104-134 (2009).</li><li>Guo, X., Johe, K., Molnar, P., Davis, H., Hickman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+human+fetal+spinal+cord+stem+cell+line,+NSI-566RSC,+and+its+induction+to+functional+motoneurons.">Characterization of a human fetal spinal cord stem cell line, NSI-566RSC, and its induction to functional motoneurons.</a> Journal of Tissue Engineering and Regenerative Medicine. 4, 181-193 (2010).</li><li>Varghese, K., 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=A+new+target+for+amyloid+beta+toxicity+validated+by+standard+and+high-throughput+electrophysiology.">A new target for amyloid beta toxicity validated by standard and high-throughput electrophysiology.</a> PLoS ONE. 5, e8643 (2010).</li><li>Natarajan, A., 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=Patterned+cardiomyocytes+on+microelectrode+arrays+as+a+functional,+high+information+content+drug+screening+platform.">Patterned cardiomyocytes on microelectrode arrays as a functional, high information content drug screening platform.</a> Biomaterials. 32, 4267-4274 (2011).</li><li>Davis, H., 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=Rat+Cortical+Oligodendrocyte-Embryonic+Motoneuron+Co-Culture:+An+Axon-Oligodendrocyte+Interaction+Model.">Rat Cortical Oligodendrocyte-Embryonic Motoneuron Co-Culture: An Axon-Oligodendrocyte Interaction Model.</a> Journal of biomaterials and tissue. 2, 206-214 (2012).</li><li>Natarajan, A., DeMarse, T., Molnar, P., Hickman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+In+Vitro+Feed-Forward+Networks.">Engineered In Vitro Feed-Forward Networks.</a> J Biotechnol Biomater. 3, 2 (2013).</li></ol>

The authors have nothing to disclose.

細胞収縮の証拠マイクロカンチレバーを分析する重要なステップは、顕微鏡ステージ内のカンチレバーチップの配置、配列内のコーナーカンチレバーの先端がレーザーと光検出器のその後のアラインメントである。これが正確に行われない場合、ソフトウェアは、潜在的にデータ収集中に偽陰性の蓄積につながる、アレイ内の残りのカンチレバーの位置を推定することができません。オペレ?...

The in vitro culture of muscle cells from both human and rodent sources has been possible for decades1,2. However, while standard coverslip preparations are useful for biochemical assessment, they do not facilitate analysis of the cell&#8217;s primary functional output (contractility), and therefore are of somewhat limited value as a means to assess cellular maturation and performance. In order to maximize the amount of data obtainable from such in vitro cultures, it is necessary to advance the development of systems capable of housing such cells in configurations that permit the real-time assessment of their functional performance. The establishment of a multitude of three dimensional muscle models has made some progress toward fulfilling this need, and such systems have been used in a number of publications as a means to assess the contractile capacity of cultured muscle cells in vitro3-5. While such systems are invaluable for tissue modeling and reconstruction studies, they are limited in their applicability for studies of single cell responses. In such cases where single fiber studies are necessary, complex and labor intensive ex vivo methodologies remain the only option6-10. Furthermore, current movement toward the development of complex, multi-organ platforms for drug development and screening protocols requires the establishment of systems which are non-invasive, easily scalable and which integrate readily with supporting cells and tissue models11. 
Microscale cantilevers offer a simple method for assessing the functional contractile capacity of single cells/small populations of cells12,13. The technique is based on modified Atomic Force Microscopy (AFM) technology14, and uses a laser and photo-detector system to measure microscale cantilever deflection in response to cultured myotube contractile activity. Modified Stoney&#8217;s equations are then used to calculate stress in the myotube, and the force exerted by the myotube in order to generate the observed substrate deflection15. A scanning program has been written which enables simultaneous assessment of multiplexed cantilever arrays, offering potential moderate to high through-put applications for drug toxicity/efficacy studies15,16. Such technology may prove invaluable in the development of functional, pre-clinical assays for predicting drug efficacy in vivo. Furthermore, fabrication of cantilever chips in silicon does not impede post analysis processing of cells for standard biomolecular assays such as immunostaining, western blotting and PCR. 
This manuscript provides detailed instructions on the fabrication and preparation of microscale silicon cantilevers, the hardware and software set-up, and the operating guidelines for assessing the functionality of contractile cells cultured on these chips. Standard cell culture techniques can be implemented for plating and maintenance of cells on these surfaces, hence any contractile cell type for which reliable culture parameters exist should be able to integrate with this device with ease. The relatively simple 2D culture parameters utilized in this system makes integration of other cell models or addition of cell types that can interact with muscle (such as innervating neurons) straight-forward, greatly increasing the applicability of this model in the development of more complex functional in vitro assays and multi-organ models of mammalian systems.

<table border="0" cellpadding="0" cellspacing="0" width="690">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:299px;">Name of material/ equipment</td>
			<td style="width:128px;">Company</td>
			<td style="width:108px;">Catalog number</td>
			<td style="width:156px;">Comments/ Description</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Primary rat muscle growth medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Neurobasal medium</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">21103-049&#160;</td>
			<td dir="LTR" style="width:156px;">N/A</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">B27 (50x)</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">17504044</td>
			<td dir="LTR" style="width:156px;">1x</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Glutamax (100x)</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">35050061</td>
			<td dir="LTR" style="width:156px;">1x</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">G5 supplement</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">17503-012&#160;</td>
			<td dir="LTR" style="width:156px;">1x</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Glial-Derived Neurotrophic Factor</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRG400B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Brain-Derived Neurotrophic Factor</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRB600B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Ciliary Neurotrophic Factor</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRC400A</td>
			<td dir="LTR" style="width:156px;">40 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Neurotrophin-3</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRN500B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Neurotrophin-4</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRN501B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Acidic Fibroblast Growth Factor</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">13241-013&#160;</td>
			<td dir="LTR" style="width:156px;">25 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Vascular Endothelial Growth Factor</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">P2654</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Cardiotrophin-1</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRC700B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Heparin Sulphate</td>
			<td dir="LTR" style="width:128px;">Sigma</td>
			<td dir="LTR" style="width:108px;">D9809&#160;</td>
			<td dir="LTR" style="width:156px;">100 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Leukemia Inhibitory Factor</td>
			<td dir="LTR" style="width:128px;">Sigma</td>
			<td dir="LTR" style="width:108px;">L5158&#160;</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Vitronectin</td>
			<td dir="LTR" style="width:128px;">Sigma</td>
			<td dir="LTR" style="width:108px;">V0132</td>
			<td dir="LTR" style="width:156px;">100 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;"></td>
			<td dir="LTR" style="width:128px;"></td>
			<td dir="LTR" style="width:108px;"></td>
			<td dir="LTR" style="width:156px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Primary rat muscle differentiation medium</td>
			<td dir="LTR" style="width:128px;"></td>
			<td dir="LTR" style="width:108px;"></td>
			<td dir="LTR" style="width:156px;"></td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">NB Activ 4</td>
			<td dir="LTR" style="width:128px;">Brain Bits LLC</td>
			<td dir="LTR" style="width:108px;">NB4-500</td>
			<td dir="LTR" style="width:156px;">N/A</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;"></td>
			<td dir="LTR" style="width:128px;"></td>
			<td dir="LTR" style="width:108px;"></td>
			<td dir="LTR" style="width:156px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Class 2 red diode laser</td>
			<td>Newport</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Photo-detector</td>
			<td>Noah Industries</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Model 2100 Pulse stimulator</td>
			<td>A-M systems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Multiclamp 700B Digitizer</td>
			<td>Axon Instruments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Patch clamp microscope and stage</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Delta T4 culture dish controller</td>
			<td>Bioptechs</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Axoscope software</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">LabVIEW software</td>
			<td>National Instruments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">37oC, 5% CO2 incubator</td>
			<td>NAPCO</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Class 2 microbiological flow hood</td>
			<td>Labconco</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pipettes and tips</td>
			<td>Eppendorf</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

utilization of microscale silicon cantilevers to assess cellular contractile function in vitro

インビトロアッセイにおける複数の予測と生物学的に関連の開発は、播種した細胞の機能評価を容易にする多目的な細胞培養系の進歩に基づく。そのため、マイクロカンチレバー技術は、曲げ収縮誘発性基板の評価を通じて、骨格筋、心筋、および平滑筋細胞などの細胞型の範囲の収縮機能を測定するためにどのプラットフォームを提供する。多重化されたカンチレバーアレイのアプリケーションは、薬物の有効性および毒性、疾患表現型および進行、ならびに神経筋および他の細胞 - 細胞相互作用を評価するためのハイスループットプロトコルに適度な開発する手段を提供する。この原稿は、この目的のために信頼性の高いカンチレバーアレイを製造するための詳細、およびこれらの表面上に成功裏に培養細胞に必要なメソッドを提供する。さらなる説明は、機能的肛門を実行するのに必要なステップに設けられている新規なレーザーおよび光検出器システムを使用してそのようなアレイ上で維持収縮性細胞型のysis。ハイライトの精度と、このシステム、ならびにこのような技術を適用することができる研究の広い範囲を使用して可能な収縮機能の分析の再現可能な性質を提供した代表的なデータ。このシステムの成功広く採用は、組織のパフォーマンス、疾患の発症および新規治療的処置に対する応答のより正確な予測をもたらす、インビトロで迅速、低コストで機能的研究を実行するための手段を研究者に提供することができる。

カンチレバー上の収縮性細胞の成功した文化は、標準的な細胞培養技術（ 図5）を利用して、比較的簡単な手順です。収縮する細胞を支持するカンチレバーの割合は、細胞型を調べ、特定の培養技術が採用さに依存して変化する。ラット後肢由来の初代胚細胞を使用して、収縮活動を調べカンチレバーの12％で検出された（n = 4）であった。記載レーザーと光検出器システムを使?...

Watch this Scientific Journal Video about Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro at JoVE.com

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

このプロトコルは、 インビトロで筋細胞の収縮性を測定するための柔軟な培養表面としてマイクロスケールのシリコンカンチレバーの使用を記載している。細胞収縮を測定記録し、 インビトロで収縮機能を測定するための非侵襲的かつスケーラブルなシステムを提供し、力の読み取り値に変換することができるカンチレバー屈曲を引き起こす。

携帯収縮機能を評価するために、マイクロスケールのシリコンカンチレバーの活用<em&gt;体外</em

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

utilization-microscale-silicon-cantilevers-to-assess-cellular

Research

JoVE Journal

Bioengineering

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

10.1K ビュー.  University of Central Florida. このプロトコルは、 インビトロで筋細胞の収縮性を測定するための柔軟な培養表面としてマイクロスケールのシリコンカンチレバーの使用を記載している。細胞収縮を測定記録し、 インビトロで収縮機能を測定するための非侵襲的かつスケーラブルなシステムを提供し、力の読み取り値に変換することができるカンチレバー屈曲を引き起こ...

ビデオ: Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

強化されたマイクロおよびナノ操作のためのプラズモニックとフォトニック結晶ナノ構造の利用

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

生物工学

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

DNA損傷の検出のための半人工分子機械とその使用のin vitroでの組立に

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

パラメータ化拡張期充満フォーマリズムを経由Transmitral流れの運動学的モデルベースの​​解析によるグローバル拡張期機能の定量化

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

マイクロスケールシュリーレン法によるマイクロ流体デバイス内の混合不均一性の分析

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

再構成ヒト角膜上皮様（RHCE）組織モデルを使用して眼刺激性化学物質の有害性の同定のための眼刺激性試験（EIT）

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

エタノールへのリグノセルロースの生物変換のための堅牢なペントース発酵酵母の進化のための技術

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Measuring and Modeling Contractile Drying in Human Stratum Corneum

Stratum corneum (SC) is the most superficial skin layer. Its contact with the external environment means that this tissue layer is subjected to both cleansing agents and daily variations in ambient moisture; both of which can alter the water content of the tissue. Reductions in water content from severe barrier dysfunction or low humidity environments can alter SC stiffness and cause a build-up of drying stresses. In extreme conditions, these factors can cause mechanical rupture of the tissue. We have established a high throughput method of quantifying dynamic changes in the mechanical properties of SC upon drying. This technique can be employed to quantify changes in the drying behavior and mechanical properties of SC with cosmetic cleanser and moisturizer treatments. This is achieved by measuring dynamic variations in spatially resolved in-plane drying displacements of circular tissue samples adhered to an elastomer substrate. In-plane radial displacements acquired during drying are azimuthally averaged and fitted with a profile based on a linear elastic contractility model. Dynamic changes in drying stress and SC elastic modulus can then be extracted from the fitted model profiles.

This article describes a method of quantifying the dynamic drying behavior and mechanical properties of stratum corneum by measuring spatially resolved in-plane drying displacements of circular tissue samples adhered to an elastomer substrate. This technique can be used to measure how different chemical treatments alter drying and tissue mechanical properties.

計測とモデリング収縮乾燥ヒト角質層で

Stratum corneum (SC) is the most superficial skin layer. Its contact with the external environment means that this tissue layer is subjected to both ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

アン<em&gt;インビトロ</emマウス椎間板の&gt;器官培養モデル

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

長期ナノ材料暴露後のインビトロ遺伝子毒性試験のための高度な3D肝モデル

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.
Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic&#160;recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.