Name: expanding nanopatterned substrates using stitch technique for nanotopographical modulation of cell behavior
Uploaded: 2016-12-08T14:00:00.000+00:00
Duration: 9 min 6 s
Description: Watch this Scientific Journal Video about Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior at JoVE.com

This work was partly supported by NSF CBET 1227766, NSF CBET 1511759, and Byars-Tarnay Endowment. We gratefully acknowledge use of the West Virginia University Shared Research Facilities which are supported, in part, by NSF EPS-1003907.

EBLモールドからPDMS金型の1レプリケーション<ol><li>シリコン型29を製作<ol><li>スピンコートは、200μlのポリメチルメタクリレート（PMMA）、1分間2,500 rpmで1×1cmのシリコン（Si）基板上に溶液が薄膜を形成します。 </li><li> 2分間180℃でSi基板上にPMMA膜を焼きます。 </li><li> 300μC/ cm 2の面積の用量で集束電子ビームを用いて、PMMAフィルム上に設計されたナノパターンを書きます。 </li><li> 80秒間現像液中でのPMMAナノパターンを開発します。 </li><li> 10 kVで、0.5 mAで放出電流及び0.5オングストローム/秒の蒸着速度の出力電圧で電子ビーム蒸発器を使用して、厚さ50nmのニッケル層とPMMAのナノパターンを堆積させます。 </li><li> 80℃で20分間20ミリリットルリムーバーでPMMA部分を持ち上げ。 </li><li>反応性イオンエッチング（RIE）Si基板にナノパターンを所望の深さのシリコンモールドを取得します。 注：のTetRのガス混合物afluoromethane（CF 4）、150 W、400 WとRIE電力の誘導結合プラズマ（ICP）電源投入時/酸素（O 2）（90％/ 10％）は、560ナノメートルの深さにSi基板をエッチングするために使用されます。 </li></ol></li><li> SilanizeのSiモールド<ol><li> 100ミリメートルPSペトリ皿にカバーガラスとSiモールドを入れて、換気フード内に配置ガラスデシケーターでそれらを転送します。 </li><li>カバースリップ上に10μlの1H、1H、2H、2H-Perfluorooctyltrichlorosilaneをドロップします。 注意：1H、1H、2H、2H-Perfluorooctyltrichlorosilaneは皮膚腐食性および重篤な眼の損傷を引き起こす可能性があります。適切な個人保護具（PPE）を着用してください。 </li><li>部分的にペトリ皿をカバーしています。 </li><li>シリコン金型のシラン化を完了するために、ドラフト内で5時間、真空下でデシケーターを保管してください。 </li></ol></li><li> PDMSプレポリマーを準備<ol><li>使い捨ての計量ボートに10gのPDMS樹脂1.05グラムの硬化剤を秤量します。 </li><li>プラスチック製のスプーンを使って徹底的にPDMSプレポリマーを混ぜます。 </li><li>透明な混合物が観察されるまで約20分間真空下でプラスチック製デシケーター中でPDMSプレポリマーを脱気。 </li></ol></li><li> PDMS金型を複製します<ol><li> 60ミリメートルペトリ皿にシラン化したSiモールドを置きます。 </li><li>ペトリ皿中のSiモールドにPDMSプレポリマーを注ぎます。 </li><li>すべての気泡が消えるまで約10分間プラスチック製デシケーターと脱ガスにペトリ皿を置きます。 </li><li>ホットプレートにペトリ皿を移し、4時間70℃でPDMSプレポリマーを硬化させます。 </li><li>慎重にピンセットを用いて、シリコンモールドからPDMSモールドをはがし。 注：PDMS型は最大1週間、周囲条件で保存することができます。硬化後、PDMS金型30の一部の未架橋のPDMS樹脂分子と残留硬化剤があります。低分子量の分子が徐々に外に拡散し、時間の経過とともに表面に蓄積します。これは、PDMS表面31の地形学的および機械的特性に影響を与えます。 DIF融合は、1週間以内に重要ではありません。 </li></ol></li></ol>大型、シングルモールドにPDMS金型の2ステッチ<ol><li>ステップ1.4を繰り返して、複数のPDMS金型を準備します。 注：PDMS混合物の同じ量、同じ厚さのPDMSモールドを得るたびに秤量します。 </li><li>光学顕微鏡下で、このようなnanogratingsなどの異方性のPDMSナノパターンの向きを決定し、マーカーペンでPDMSモールドの裏面にマークします。 注：それはそのようなナノピラー等方性ナノトポグラフィの向きをマークする必要はありません。 </li><li>ドラフト内でエタノールとSi基板をきれいにし、圧縮空気で乾燥させました。 </li><li>ブレードで各PDMSモールドのパターン化されていない部分を切り落とします。 注：ステッチ金型の周囲に配置されますPDMS金型については、他の人と接触している唯一のパターン化されていない領域がトリミングされるべきです。 </li><li>上のナノフェイスダウンでトリミングPDMSモールドを配置その後、ミラーSi基板の側との近くに他の金型を揃えるが、周囲の金型（複数可）には触れていません。 </li><li> PDMS接着剤層を調製します<ol><li>厚さ0.5mmの層を形成するために清浄なガラススライドインチ（2.5cm×7.5センチ）の1gをPDMSプレポリマー（1.05：10 PDMS樹脂と硬化剤の比率）を脱気キャスト。 </li><li> 3-5分間ホットプレート上で100℃でPDMS層焼きます。層をタッチして、層が部分的にではなく、完全に硬化されることを保証するために針を使用してください。 注：部分的に硬化したPDMSは、未硬化PDMSプレポリマーのように流れることができないが、それは硬化PDMSと比較して粘着性です。 </li></ol></li><li>整列PDMSモールドの裏面にPDMS層を配置し、すぐにこのアセンブリを反転し、ホットプレートに移します。 </li><li> PDMS接着剤層とPDMSモールドの裏面との間の良好な接触を確実にし、1時間100℃でPDMS接着剤層を硬化させるために、アセンブリの上に金属ブロックを用いて、圧縮力（5キロパスカル）を適用します。&lt;BR /&gt;注：慎重にアセンブリの傾きを避けるために、金属ブロックの位置を調整します。 </li><li>金属ブロックを削除し、Si基板からのシングル、ステッチPDMSモールドを剥がします。 </li></ol> PS基板上のマスターモールドの3世代注：スライドガラス上に固定化されたステッチPDMSモールドは、PS版や作業ナノ基材を製造することが可能なPS薄膜上にマスターモールドを生成するために使用することができます。 <ol><li> PS版上のマスターモールドを生成します<ol><li> PS版を準備<ol><li>二日間80℃の真空オーブン中でPSペレットを乾燥させます。 </li><li> 230℃でプレス機を予熱。 </li><li>下から上へ順に、アルミニウム板、ポリテトラフルオロエチレン（PTFE）シートとアルミニウムスペーサーを組み立てます。 </li><li> 3センチメートル（L）×3センチメートル（W）×0.3センチメートル（H）の正方形の開口部を有するアルミニウムスペーサーで負荷3.5グラムのPSペレット。 注：スペーサーがほぼ0です。1 PDMSモールドよりも厚くcmであり、従って、最終的なナノPSは、基板は、約0.1センチメートルの厚さです。 </li><li>アルミスペーサー上の別のPTFEシートとし、別のアルミニウム板を置きます。 </li><li>プレス機械にアセンブリを配置します。 </li><li> 30分間230℃でPSペレットを予熱。 </li><li> 5分間のアセンブリ上の圧縮圧力（0.1MPaに）適用します。 </li><li>圧力を解放した後、アセンブリ上に圧力0.5MPaの圧縮圧力を再適用します。 </li><li> 1.5 MPaでの望ましい圧力まで圧力0.5MPaの増加に伴って繰り返しステップ3.1.1.9に到達します。 </li><li>プレス機のヒーターの電源をオフにして、1.5 MPaでの一定の圧力で70℃以下に冷却します。 </li><li>アセンブリを取り出し、PS版を再入力から水分を防ぐために、80℃の真空オーブン中でPS版を保存します。 </li></ol></li><li> PS版へのナノインプリントステッチPDMSモールド<ol><li>アルミスペーサーでPS版を置き3インチのSiウェハ上に設定。 注：スペーサの内側の寸法は、PS版と同じであるので、PS版は、右スペーサに収まります。 </li><li> 30分間250℃でホットプレート上でPS版を加熱します。 </li><li>溶融したPS版上のナノパターンのフェースダウンで縫製PDMSモールドを置きます。 注：PDMSモールドの片側は最初のPS版の表面と接触して置かれ、他方は、界面での気泡の形成を回避するために、PSの表面に接触して徐々に低下します。 注意：ホットプレートの表面が高温になっています。ナノインプリントプロセス中thermoglovesを着用してください。 </li><li>ステッチPDMSモールドのスライドガラス上にアルミ板を置きます。 </li><li>アルミ板上に金属ブロックを使用して圧縮圧力（12.5キロパスカル）を適用し、3分間待ちます。 注：アルミニウム板が傾いていないことを確認してください。 </li><li>持ち上げて、アルミ板から金属ブロックを交換し、私25キロパスカルに圧縮圧力をncrease。 </li><li>圧力とを繰り返し、ステップ3.1.2.6は50キロパスカルまで上昇しました。 注：この手順は、PDMSモールドとPSプレートとの間に閉じ込められた空気を除去することです。 </li><li> 15分間、50 kPaでの一貫した圧力下240と250℃の間のホットプレートの温度を維持します。 </li><li>ホットプレートの電源をオフにして、全体のセットアップをクールダウン。 注：ファンは、冷却プロセスを促進するために使用することができます。 </li><li> PS版からステッチPDMSモールドを剥がし慎重に温度が50℃以下になった後の金属ブロックを削除し、。 注：PS基板が逆ナノパターンを有し、作業PDMS基板を製造するためにマスターモールドとして使用することができます。 </li></ol></li></ol></li><li> PS薄膜上にマスターモールドを生成します<ol><li> PS薄膜を作製<ol><li>ドラフト内で10ミリリットルのトルエン中の1グラムPSを溶解させます。 注意：トルエンは皮膚を引き起こす可能性がありますirritationおよび重篤な眼の損傷、および長期または反復暴露による臓器の障害を引き起こす可能性があります。適切なPPEを着用してください。 </li><li> 1分間2500 rpmでのウエハ2で上にスピンコート1ミリリットルのPS溶液を、〜1μmの厚さのPS薄膜を形成しました。 </li><li> 3日間、ドラフトのSiウェハ上のPSフィルムを設定することにより、膜からトルエンを蒸発させます。 </li><li>一晩80℃の真空オーブン中でPS薄膜をアニールします。 </li></ol></li><li>ナノインプリントPS薄膜上にPDMSモールド<ol><li>ホットプレート上で設定されているPS薄膜、上のナノトポグラフィフェースダウンで縫製PDMSモールドを置きます。 </li><li> PDMSモールドのガラス側の金属ブロックを使用して、PDMSモールドに12キロパスカルの圧縮圧力を適用します。 </li><li> 180℃にホットプレートの温度を上昇させ、15分間それを維持します。 注意：溶融PSフィルムは、潤滑剤として機能することができます。滑り落ちるから金属ブロックを防ぐために注意を払います。 </li><li>ホットプレートの電源を切り全体のセットアップをクールダウン。 注：ファンは、冷却プロセスを促進するために使用することができます。 </li><li>温度が50℃以下に低下した後、金属ブロックを外し、慎重にPSフィルムからステッチPDMSモールドを剥がします。 注：ナノPSフィルムは、作業PDMS基板を製造するためのマスター型として機能します。 </li></ol></li></ol></li></ol>細胞の挙動の4 Nanotopographical変調注：ヒト上皮細胞は、細胞拡散のnanotopographical変調を実証するために、代表nanotopographies上で培養されます。 <ol><li>用途に応じて、ステップ3.1または3.2のいずれかから生成されたマスターモールドからPDMS作業基板をキャストします。 </li><li>中空鋼アーチパンチを使用して、特定のマルチウェルプレート（ 例えば 、24ウェルプレート）の構成に合わせてディスクにナノPDMS基板をカット。 </li><li> OウェルにPDMSディスクを配置するためにピンセットを使用して、FAマルチウェルプレート。 </li><li> 、30分間毎に70％エタノール、次いで、UV露光を使用してPDMS基板を滅菌します。 </li><li> 1X滅菌リン酸緩衝食塩水（PBS）で3回PDMS基板を洗浄します。 </li><li>コート室温で30分間、細胞外マトリックスタンパク質（ すなわち 、20 / mlのフィブロネクチン）を有するPDMS基板。 </li><li> 5分間PDMS基板を滅菌PBSで3回、それぞれを洗い流します。 </li><li> 10％ウシ胎児血清を含むダルベッコ改変イーグル培地中で、ヒト肺癌細胞A549を中断し、血球計数器を用いて細胞を数えます。 </li><li>一日、5％CO 2を含む加湿雰囲気中、37℃でPDMS基板と培養それらを2,000細胞/ cm 2の播種密度で細胞をプレート。 </li><li> PBSで細胞を3回洗浄します。 </li><li> 4時間、PBS中の4％パラホルムアルデヒドおよび2％グルタルアルデヒドの混合物中の細胞を固定し、CO 2の重要なPOを用いて細胞を脱水電子顕微鏡観察29を走査するための int型乾燥機。 注意：パラホルムアルデヒドおよびグルタルアルデヒドは、重篤な皮膚の薬傷・眼の損傷を引き起こす可能性があります。化学フード内で動作し、適切なPPEを着用してください。 </li></ol>

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Rev. 105 (4), 1171-1196 (2005).</li><li>Yin, Y., Lu, Y., Gates, B., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Template-Assisted+Self-Assembly:+A+Practical+Route+to+Complex+Aggregates+of+Monodispersed+Colloids+with+Well-Defined+Sizes,+Shapes,+and+Structures.">Template-Assisted Self-Assembly: A Practical Route to Complex Aggregates of Monodispersed Colloids with Well-Defined Sizes, Shapes, and Structures.</a> J. Am. Chem. Soc. 123 (36), 8718-8729 (2001).</li><li>Tada, Y., 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=Directed+Self-Assembly+of+Diblock+Copolymer+Thin+Films+on+Chemically-Patterned+Substrates+for+Defect-Free+Nano-Patterning.">Directed Self-Assembly of Diblock Copolymer Thin Films on Chemically-Patterned Substrates for Defect-Free Nano-Patterning.</a> Macromolecules. 41 (23), 9267-9276 (2008).</li><li>Cheng, J. Y., Rettner, C. T., Sanders, D. P., Kim, H. C., Hinsberg, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dense+self-assembly+on+sparse+chemical+patterns:+rectifying+and+multiplying+lithographic+patterns+using+block+copolymers.">Dense self-assembly on sparse chemical patterns: rectifying and multiplying lithographic patterns using block copolymers.</a> Adv. Mater. 20 (16), 3155-3158 (2008).</li><li>Colburn, 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=Step+and+flash+imprint+lithography:+a+new+approach+to+high-resolution+patterning.">Step and flash imprint lithography: a new approach to high-resolution patterning.</a> Proc. SPIE. 3676 ((Pt. 1, Emerging Lithographic Technologies III)), 379-389 (1999).</li><li>Ahn, S. H., Guo, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-speed+roll-to-roll+nanoimprint+lithography+on+flexible+plastic+substrates.">High-speed roll-to-roll nanoimprint lithography on flexible plastic substrates.</a> Adv. Mater. 20 (11), 2044-2049 (2008).</li><li>Vieu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+beam+lithography:+resolution+limits+and+applications.">Electron beam lithography: resolution limits and applications.</a> Appl. Surf. Sci. 164, 111-117 (2000).</li><li>Nagase, T., Gamo, K., Kubota, T., Mashiko, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+fabrication+of+nano-gap+electrodes+by+focused+ion+beam+etching.">Direct fabrication of nano-gap electrodes by focused ion beam etching.</a> Thin Solid Films. 499 (1-2), 279-284 (2006).</li><li>Juodkazis, S., 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=Two-photon+lithography+of+nanorods+in+SU-8+photoresist.">Two-photon lithography of nanorods in SU-8 photoresist.</a> Nanotechnology. 16 (6), 846 (2005).</li><li>Yang, Y., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+surfacing+for+regenerative+medicine.">Nanoscale surfacing for regenerative medicine.</a> Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2 (5), 478-495 (2010).</li><li>Yang, Y., Kulangara, K., Sia, J., Wang, L., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+a+Microfluidic+Cell+Culture+Platform+Embedded+with+Nanoscale+Features.">Engineering of a Microfluidic Cell Culture Platform Embedded with Nanoscale Features.</a> Lab Chip. 11 (9), 1638-1646 (2011).</li><li>Wang, 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=Nanotopographical+modulation+of+cell+function+through+nuclear+deformation.">Nanotopographical modulation of cell function through nuclear deformation.</a> Acs Appl. Mater. Inter. 8 (8), 5082-5092 (2016).</li><li>Lee, J. N., Park, C., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solvent+Compatibility+of+Poly(dimethylsiloxane)-Based+Microfluidic+Devices.">Solvent Compatibility of Poly(dimethylsiloxane)-Based Microfluidic Devices.</a> Anal. Chem. 75 (23), 6544-6554 (2003).</li><li>Yang, Y., Kulangara, K., Lam, R. T. S., Dharmawan, R., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Topographical+and+Mechanical+Property+Alterations+Induced+by+Oxygen+Plasma+Modification+on+Stem+Cell+Behavior.">Effects of Topographical and Mechanical Property Alterations Induced by Oxygen Plasma Modification on Stem Cell Behavior.</a> ACS Nano. 6 (10), 8591-8598 (2012).</li><li>Hua, F., 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=Polymer+Imprint+Lithography+with+Molecular-Scale+Resolution.">Polymer Imprint Lithography with Molecular-Scale Resolution.</a> Nano Lett. 4 (12), 2467-2471 (2004).</li><li>Delamarche, E., Schmid, H., Michel, B., Biebuyck, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+molded+polydimethylsiloxane+microstructures.">Stability of molded polydimethylsiloxane microstructures.</a> Adv. Mater. 9 (9), 741-746 (1997).</li></ol>

The authors have nothing to disclose.

我々は、ナノパターン基板の大面積を生成するために、簡単な手頃な価格の、まだ汎用性の高い方法を提示します。忠実に非常に定義されたナノパターンを展開するには、細心の注意は、いくつかの重要なステップに支払われるべきです。第1は、複数のPDMS金型をトリミングすることです。 PDMSモールドのパターン化されていない領域を除去する必要があります。また、金型の側壁には、金型...

A number of recent findings reveal that substrate nanotopography has pronounced influence on cell behavior, from cell adhesion, spreading and migration, to proliferation and differentiation1-6. For instance, a smaller cell size and lower proliferation rate have been observed in cells cultured on deep nanogratings, even leading to apoptosis although the cell alignment, elongation and migration were enhanced, compared with the flat controls2,7-10. Moreover, nanotopography has been shown to facilitate the differentiation of stem cells into certain lineages such as neuron2,11,12, muscle13, and bone3,4. In addition, because of increasing concerns on the toxicity of engineered nanomaterials14,15, there is a need to incorporate nanotopography into physiologically relevant in vitro models for accurate risk assessment of nanomaterials. To fulfil the biochemical and molecular biology analyses, enough cells are needed to be grown on a large area of nanopatterned substrate. However, conventional nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area.
Self-assembly including colloid lithography16 and polymer demixing17 can readily generate large-area nanostructures at low costs. Because self-assembly relies on interactions between the assembling elements such as colloidal particles and macromolecules, and possible interactions between these elements and substrate, it cannot be a stand-alone method of producing nanostructures with precise spatial positioning and arbitrary shapes18. The accompanied high density of defects is also a drawback. Precise spatial control of nanopatterns can be achieved by employing templated self-assembly, which uses top-down lithographic approaches to provide the topographical and/or chemical template to guide the bottom-up assembly of the assembling elements19-21. Alternative nanofabrication techniques such as step-and-flash lithography22 and a roll-to-roll nanoimprinting lithography23 have been developed but have limited use because of their sophisticated process or the requirement of specialized equipment. Nevertheless, a template or a master mold with defined nanoscale patterns is needed for templated or alternative nanofabrication techniques. 
Such templates and master molds are conventionally generated by using focused electron, ion, or photon beam lithography. For instance, electron beam lithography (EBL)24 and focused ion beam lithography25 can generate defined patterns with a sub-5 nm resolution. Two-photon lithography has demonstrated a feature size as small as 30 nm26. Although the focused beam lithography techniques enable generation of well-defined nanoscale structures, the capital investment and the time-consuming, costly process restrict their widespread use in academic research27. Therefore, it is highly desirable to develop enabling yet affordable techniques to produce a large area of nanopatterned surfaces with high fidelity.
We have reported a simple, cost-effective stitch technique for generating a large area of nanopatterned surface from a small well-defined mold28. This protocol provides step-by-step procedure from replication of poly(dimethylsiloxane) (PDMS) molds using an EBL-written pattern, to assembly of multiple PDMS molds into a single large mold, to generation of a master mold on polymeric such as polystyrene (PS) substrates, to production of working substrates. With the expanded nanopatterned substrates, we demonstrated nanotopographical modulation of cell spreading.

<table><tbody><tr><td>JEOL field emission SEM</td><td>JEOL</td><td>JSM-7600F</td><td>EBL</td></tr><tr><td>E-beam evaporator</td><td>Kurt J. Lesker</td><td>Model: LAB 18 e-beam evaporator</td><td>nickel deposition</td></tr><tr><td>Trion Minilock III ICP/RIE</td><td>Trion technology</td><td>Model: Minilock-phantom III</td><td></td></tr><tr><td>Press machine</td><td>PHI Hydraulic Press</td><td>Molde: SQ-230H</td><td></td></tr><tr><td>Spin coater</td><td>Laurell Technologies</td><td>Modle: WS-400A-6NPP-LITE</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; critical dryer</td><td>Tousimis</td><td>Modle: Autosamdri-815</td><td></td></tr><tr><td>Silicon wafer</td><td>University Wafer</td><td>1080</td><td></td></tr><tr><td>Aluminum plates</td><td>McMaster-carr</td><td>9057K123</td><td></td></tr><tr><td>Teflon sheets</td><td>McMaster-carr</td><td>8711K92</td><td></td></tr><tr><td>100 mm Petri dish</td><td>FALCON</td><td>353003</td><td></td></tr><tr><td>60 mm Petri dish</td><td>FALCON</td><td>353004</td><td></td></tr><tr><td>Glass coverslip</td><td>Fisher Scientific</td><td>12-542-B</td><td></td></tr><tr><td>Glass slide</td><td>Fisher Scientific</td><td>12-550-34</td><td></td></tr><tr><td>Disposable weighing boats</td><td>Fisher Scientific</td><td>13-735-743</td><td></td></tr><tr><td>Glass desiccator</td><td>Fisher Scientific</td><td>02-913-360</td><td></td></tr><tr><td>Plastic desiccator</td><td>Bel-Art Products</td><td>F42025-000</td><td></td></tr><tr><td>Hotplate</td><td>Fisher Scientific</td><td>1110049SH</td><td></td></tr><tr><td>Tweezer</td><td>Ted Pella, inc.</td><td>5726</td><td></td></tr><tr><td>Blade</td><td>Fisher Scientific</td><td>S17302</td><td></td></tr><tr><td>Metal blocks</td><td>McMaster-carr</td><td></td><td></td></tr><tr><td>Punch</td><td>Brettuns Village Leather Craft Supplies</td><td>Arch punch</td><td></td></tr><tr><td>Poly(methyl methacrylate)</td><td>MicroChem</td><td>495 PMMA A4</td><td></td></tr><tr><td>PDMS</td><td>Dow Corning</td><td>Sylgard 184 kit</td><td></td></tr><tr><td>Polystyrene</td><td>Dow Chemical</td><td>Styron 685D</td><td></td></tr><tr><td>1H,1H,2H,2H-perfluorooctylmethyldichlorosilane</td><td>Oakwood Chemical</td><td>7142</td><td></td></tr><tr><td>Developer</td><td>MicroChem</td><td>MIBK/IPA at 1: 3 ratio</td><td></td></tr><tr><td>Remover</td><td>MicroChem</td><td>Remover PG</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>BP2818500</td><td></td></tr><tr><td>Toluene</td><td>Fisher Scientific</td><td>T324-500</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma Aldrich</td><td>D8537</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified eagle medium</td><td>Sigma Aldrich</td><td>D5796</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Atlanta Biologicals</td><td>S11550</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Electron Microsopy Science</td><td>15712-S</td><td></td></tr><tr><td>Glutaraldehyde&amp;nbsp;</td><td>Fisher Chemical</td><td>G151-1</td><td></td></tr><tr><td>Fibronectin</td><td>Corning</td><td>356008</td><td></td></tr><tr><td>A549 cells</td><td>ATCC</td><td>ATCC CCL-185</td><td></td></tr></tbody></table>

expanding nanopatterned substrates using stitch technique for nanotopographical modulation of cell behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

<html>ステッチ技術は、高い忠実度でナノ基板の大面積を生成することができます。 図1aは、それぞれ、Si基板上のPS版とPS薄膜に縫い付けPDMS型から転送ナノ大きな領域を表示1B。元EBL-書かれた金型（ 図1c）と、最終的なPDMS作業基板（ 図1D）との比較は、EBL-書かれたナノパターンを忠実に作業基板に転写することがで?...

Watch this Scientific Journal Video about Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior at JoVE.com

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

細胞の挙動のNanotopographical変調のステッチ法を用いたナノパターン基板を拡大

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

expanding-nanopatterned-substrates-using-stitch-technique-for

Research

JoVE Journal

Bioengineering

6.5K ビュー.  West Virginia University. A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

ビデオ: Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into all three germ layers. This makes them an attractive cell source for regenerative medicine and experimental modeling of normal and diseased organogenesis. However, the differentiation of hPSCs in vitro is heterogeneous and spatially disordered. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Micropatterning hPSCs needs to take into account the stringent requirements for hPSC survival and maintenance. Here, we describe stencil micropatterning as a method that is highly compatible with hPSCs. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where hPSCs can access and attach to the underlying extracellular matrix-coated substrate. Due to this mode of operation, there is greater flexibility to use substrates that can adequately support hPSCs as compared to other cell micropatterning methods. We also highlight critical steps for the successful generation of hPSC micropatterns. As an example, we demonstrate that stencil micropatterning of hPSCs can be used to modulate spatial polarization of cell-cell and cell-matrix adhesions, which in turn determines mesoendoderm differentiation patterns. This simple and robust method to micropattern hPSCs widens the prospects of establishing experimental models to investigate tissue organization and patterning during early embryonic development.

Human pluripotent stem cells (hPSCs) have the intrinsic ability to differentiate and self-organize into distinct tissue patterns; although this requires the presentation of spatial environmental gradients. We present stencil micropatterning as a simple and robust method to generate biochemical and mechanical gradients for controlling hPSC differentiation patterns.

分化運命の空間組織を探るためのヒト多能性幹細胞のステンシルマイクロパターニング

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into ...

発生生物学

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

心臓組織工学のためのキャピラリーフォースリソグラフィ

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

生物工学

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

物質 - 細胞相互作用研究ナノポーラス金パターンの微細加工

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

はSiO：フォトリソグラフィによって画定されたパリレン-C上の細胞のパターニング<sub&gt; 2</sub&gt;基板

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

PDMS膜上のマイクロパターン細胞を伸ばす

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.

This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes.

レーザーカットステンシルを用いたシンプルリソグラフィフリーシングルセルマイクロパターニング

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single ...

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 ...

Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular reactions. However, it is difficult to determine the relation between the size of a nanostructure and the responses of cells owing to the lack of proper screening tools. Here, we show the development of reproducible and cost-effective gradient nanopattern plates for the manipulation of cellular responses. Using anodic aluminum oxide (AAO) as a master mold, gradient nanopattern plates with nanopillars of increasing diameter ranges [120-200&#8239;nm (GP 120/200), 200-280&#8239;nm (GP 200/280), and 280-360&#8239;nm (GP 280/360)] were fabricated by a thermal imprinting technique. These gradient nanopattern plates were designed to mimic the various sizes of nanotopography in the ECM and were used to screen the responses of human endothelial colony-forming cells (hECFCs). In this protocol, we describe the step-by-step procedure of fabricating gradient nanopattern plates for cell engineering, techniques of cultivating hECFCs from human peripheral blood, and culturing hECFCs on nanopattern plates.

Here, we present a protocol for the fabrication of gradient nanopattern plates via thermal nanoimprinting and the method of screening responses of human endothelial progenitor cells to the nanostructures. By using the described technology, it is possible to produce a scaffold that can manipulate cell behavior by physical stimuli.

熱ナノイン プリント技術とヒトの内皮細胞コロニー形成細胞の応答のスクリーニングによるグラデーションによるナノパターンの作製

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

赤外線レーザー支援マイクロパターンによる細胞形状の制御

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

マイクロパターントラクション顕微鏡のパターン生成

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

細胞の挙動のNanotopographical変調のステッチ法を用いたナノパターン基板を拡大

要約

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この動画の章

分化運命の空間組織を探るためのヒト多能性幹細胞のステンシルマイクロパターニング

心臓組織工学のためのキャピラリーフォースリソグラフィ

物質 - 細胞相互作用研究ナノポーラス金パターンの微細加工

はSiO：フォトリソグラフィによって画定されたパリレン-C上の細胞のパターニング

PDMS膜上のマイクロパターン細胞を伸ばす

レーザーカットステンシルを用いたシンプルリソグラフィフリーシングルセルマイクロパターニング

パターニングタンパク質および細胞の多彩な方法

熱ナノインプリント技術とヒトの内皮細胞コロニー形成細胞の応答のスクリーニングによるグラデーションによるナノパターンの作製

赤外線レーザー支援マイクロパターンによる細胞形状の制御

マイクロパターントラクション顕微鏡のパターン生成

細胞の挙動のNanotopographical変調のステッチ法を用いたナノパターン基板を拡大

要約

さらに動画を探す

この動画の章

分化運命の空間組織を探るためのヒト多能性幹細胞のステンシルマイクロパターニング

心臓組織工学のためのキャピラリーフォースリソグラフィ

物質 - 細胞相互作用研究ナノポーラス金パターンの微細加工

はSiO：フォトリソグラフィによって画定されたパリレン-C上の細胞のパターニング

PDMS膜上のマイクロパターン細胞を伸ばす

レーザーカットステンシルを用いたシンプルリソグラフィフリーシングルセルマイクロパターニング

パターニングタンパク質および細胞の多彩な方法

熱ナノイン プリント技術とヒトの内皮細胞コロニー形成細胞の応答のスクリーニングによるグラデーションによるナノパターンの作製

赤外線レーザー支援マイクロパターンによる細胞形状の制御

マイクロパターントラクション顕微鏡のパターン生成

熱ナノインプリント技術とヒトの内皮細胞コロニー形成細胞の応答のスクリーニングによるグラデーションによるナノパターンの作製