この研究のための資金調達（FHKに）脊髄研究（NJCSCR）にニュージャージー州委員会によって提供された、（BLFに）CSCR14IRG005を付与し、NIHは（CHCに）R15NS087501を付与し、（ETA）はFMカービー財団。

注：使用前に、関連するすべての物質安全データシート（MSDS）を参照してください。このプロトコルで使用される化学物質の一部は有毒で発ガン性があります。毒性または酸/基材を使用した場合、すべての適切な安全対策（ヒュームフード、グローブボックス）および個人用保護具（安全眼鏡、手袋、白衣、完全長ズボン、閉じたつま先の靴）を使用してください。 フォトリソグラフィを用いて、ソフトリソグラフィのためのマスター金型の1製作<ol><li>コンピュータ支援設計（CAD）図面ツールを使用してマイクロチャネルのレイアウトを描きます。 </li><li>レーザーマスク描画装置を用いて、マスクブランク板にレイアウトを印刷します。 </li><li>いかなる溶媒をウェハ上に残っていないことを確実にするために窒素エアガンイソプロパノール及びドライ続いアセトンで4インチシリコンウエハをすすぎます。 </li><li> 5分間、200℃のホットプレート上で焼成することにより、ウエハを脱水。 </li><li>マイクロチャネルの所望の高さのために設計されたネガ型フォトレジストを選択します。 Fまたは、例えば、50μmの高さのマイクロチャネルを得るために、ネガティブフォトレジストSU-8 50を使用します。 </li><li>ウェハを室温に戻します、その後、スピンコーターのチャック上にウエハを合わせます。 </li><li>ウェーハの中心に、ネガ型フォトレジストの4mLの（ウェーハの1ミリリットル/インチ直径）を分配します。 </li><li>スピナーを用いた2段階の回転周期にコートにウェハをスピン。まず、10秒間100回転/秒の加速度で500回転の広がりサイクルを適用します。その後、ウエハ上のフォトレジストの50μmのコーティングを得るために、30秒間300回転/秒2の加速度で2000回転の回転サイクルを適用します。 </li><li>ソフトメーカーの指示に従って、ホットプレート上の2つのステップでウェハを焼きます。フォトレジストの50μmのコーティングについては、6分間の65℃でのウエハプリベーク最初にして、すぐに20分間95℃でポストベークするウエハホットプレートの温度を上昇します。 </li><li>ウェハを室温まで冷却した後、ロードしますリソグラフィステージ上にウエハ。 </li><li>マスクアライナを用いてウェハ上にマスクを合わせ、ために必要な220ミリジュール/ cm 2の総UV線量を適用するために146秒間、1.5ミリワット/ cm 2の強度で（製造業者の指示に従って）UV光にウェハを公開フォトレジストの厚さ50μmの層。 </li><li>ホットプレート上の2つのステップでウェハに露光後ベークを適用します。フォトレジストの50μmのコーティングのために、1分間65℃、ウェハプリベーク、直ちに最初のポストベークすること、95℃で5分間、ホットプレートの温度をランプアップ。 </li><li> SU-8現像液にウェハを浸漬し、特徴は、ウェハ上明確になるまで、穏やかに振とうすることにより、ウェーハを開発します。厚さ50μmのフォトレジスト用〜6分間ウェハを開発します。 </li><li>その後、窒素エアガンでウェハを乾燥、イソプロパノール及びエタノールで余剰の現像を洗い流してください。 </li><li>ハード15分間、150℃のホットプレート上にシリコンウェハを焼きます。 </li><li>置きシラン化剤25μLのトリデカフルオロ-1,1,2,2-テトラヒドロオクチル-1-トリクロロシランでデシケーター中でウェハ。 注：シラン化剤は、腐食性のある、このようにして、適切な個人用保護具（安全眼鏡、手袋、白衣、完全長ズボン、閉じたつま先の靴）と、酸/塩基ドラフト内で使用されるべきです。 </li><li> 5分間の真空を適用し、真空を解除せずに30分間シランの蒸気にウェハを公開します。 </li><li>適切なサイズのペトリ皿にウェハを転送します。 </li></ol>粘着テープを使用したソフトリソグラフィ用のマスター金型の2製作<ol><li> CADの描画ツールを使用して、マイクロチャネルのレイアウトを描き、白い紙に図面を印刷します。 </li><li>イソプロパノールで設計を収容した後、空気銃でスライドを乾燥するのに十分な大きスライドガラスを清掃してください。 </li><li>清浄なガラススライド上に粘着テープを取り付けます。任意の気泡をトラップに注意しないこと。 </li><li> GLAの両側をテープssはテープ側を上にして、デザインを紙の上にスライドさせます。 </li><li>参考として、紙のデザインを使用して、スライドガラス上にテープをカットした後、スライドガラスの不要な領域からテープを剥離するメスを使用してください。 </li><li>イソプロパノールでガラススライドを洗浄した後、エアガンで乾燥。 30分間65℃のオーブン中にスライドガラスを焼きます。 </li><li>穏やかに気泡を除去し、スライドを室温に冷却させるために、ゴムローラーを用いてテープの上に転がります。 </li></ol> PDMSデバイスの3ソフトリソグラフィ製作<ol><li> 10の比でPDMSエラストマーと硬化剤を混合する：1（W：W）、混合物を激しく撹拌し、その後に気泡を混合物に表示されなくなるまで真空チャンバ内に配置することによって混合物を脱気。 </li><li>すべての気泡が消えるまで、PDMSと脱ガスの〜2ミリメートルの厚さの層を得るために、ペトリ皿の中でシラン化されたシリコン型や粘着テープの金型に混合物を注ぎます。 </li><li>オーブンでPDMSを治します2時間65℃。 </li><li>離れの特徴から少なくとも5ミリメートルPDMS層をカットした後、ピンセットを用いて、金型から硬化PDMSを剥離するメスを使用してください。 </li><li>好ましくは、 図2a及び図4bに見られるように、マイクロチャネルのネットワークの端部で、任意の場所にマイクロチャネルに、1mmの生検パンチを備えた単一の入口穴を開ける。 </li><li>デバイス上に吸着ダスト粒子を除去するために、粘着テープでキャストPDMSを清掃してください。滅菌脱イオン（DI）水、続いて70％エタノール溶液ですすぐことにより、PDMSマイクロチャネルデバイス（PDMSキャスト）を滅菌し、30分間UVにさらします。 </li><li>使用するまで滅菌ペトリ皿中の滅菌PDMSデバイスを保管してください。 </li></ol> 4.基板のパターニング<ol><li>滅菌ガラスカバースリップまたはペトリ皿の上にピンセットを用いて鋳造滅菌PDMSを置き、ピンセットの先端を使用して穏やかな圧力を適用します。 注：PDMSキャストはGLと共形が、可逆的シールを作りますお尻のカバースリップまたは任意の接着剤を使用せずにマイクロチャネルを形成するペトリ皿。 </li><li>完全入口孔を覆って、入口に基質溶液の - （40μL20）の液滴を配置します。マイクロチャネルの入口に四ホウ酸ナトリウム緩衝液中のPDLの20μLの液滴を配置することにより、パターンポリ-D-リジン（PDL）。 </li><li> 10分間（生物学研究所内の真空を収容するのに相当）、および気泡の形でチャネルから出てくる空気を観察〜254 mmHgAの真空中でペトリ皿を置きます。 </li><li>真空を解除し、マイクロチャネルに流入基質溶液を観察します。 </li><li>基板密着性のための適切な条件で皿をインキュベートします。 （これらは基板に応じて変化）インキュベーション条件を表1及び表 2を参照のこと。 PDLのパターニングに、37℃で1時間インキュベートします。 </li><li>慎重に滅菌ピンセットを使用してPDMSスタンプをはがしし、DI水で3回カバーガラス上にパターンを洗います。 </li&gt; <li>皿またはガラス製カバースリップをカバーし、37℃で一晩インキュベートするペトリ皿に、1％ウシ血清アルブミン（BSA）溶液を加えます。 注：これはコーティングされていない地域での非特異的付着を軽減します。 </li><li> 1％BSA溶液を吸引除去し、パターニングされた基板を使用する準備が整いました。 </li></ol>細胞の5.間接パターニング<ol><li>無血清培地中の細胞懸濁液を調製します。細胞型および細胞密度を表1に示します。 </li><li>パターン化されたペトリ皿に細胞懸濁液を添加し、完全に細胞懸濁液中のパターン領域を水没。 </li><li>細胞がパターン化された基板に付着することを可能にするために15分間インキュベーター中で37℃でペトリ皿をインキュベートします。 </li><li>ペトリ皿からの過剰の細胞懸濁液を吸引し、穏やかに付着していない細胞を除去するために10秒間振盪しながらリン酸緩衝生理食塩水（PBS）でパターン形成細胞を3回洗浄します。 </li><li> Aを追加パターン化された細胞培養物にppropriate培地及びCO 2インキュベーター中37℃でパターン化細胞をインキュベートします。 </li></ol>細胞の6.ダイレクトパターニング注：この方法は、しかし、ステップ5とは異なり、この技術では、細胞を基板コーティングの有無にかかわらず、組織培養表面上にパターン化された手順5で説明した間接的な細胞パターン形成に代わるものです。 <ol><li> DI水、続いて70％エタノールの溶液で洗浄することにより、マイクロ流体デバイスを滅菌します。 </li><li> PDMSへの細胞接着を防止するために、1％BSAの溶液中で一晩デバイスを浸します。 </li><li>室温でデバイスを乾燥させ、組織培養処理ペトリ皿の底に取り付けます。 </li><li>無血清培地中の細胞懸濁液を調製します。細胞型および細胞密度を表2に示します。 </li><li>満たすのに十分の細胞懸濁液 - （8μL4）、液滴を配置マイクロチャネルは、入口に完全に入口穴をカバーしています。 </li><li> 10分間真空中でペトリ皿を入れ、泡の形でチャネルから出てくる空気を観察します。真空を解除し、マイクロ流路に流入する細胞懸濁液を観察します。 </li><li> 表2に記載された（パターン化細胞のタイプに依存）インキュベーション条件に従って、細胞接着を促進するためのインキュベーターでペトリ皿をインキュベートします。 PDMSデバイスを沈めるシャーレにPBSを追加します。 </li><li>ピールPDMS慎重にピンセットでシャーレを切り、PBSでパターンを洗います。ペトリ皿に細胞培養培地を加え、インキュベーター内で細胞培養を置きます。 </li></ol>

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D., Pinnau, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+sorption,+diffusion,+and+permeation+in+poly(dimethylsiloxane).">Gas sorption, diffusion, and permeation in poly(dimethylsiloxane).</a> J Polym Sci Part B Polym Phys. 38 (3), 415-434 (2000).</li><li>Shrirao, A. B., Hussain, A., Cho, C. H., Perez-Castillejos, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesive-tape+soft+lithography+for+patterning+mammalian+cells:+application+to+wound-healing+assays.">Adhesive-tape soft lithography for patterning mammalian cells: application to wound-healing assays.</a> Biotechniques. 53 (5), 315-318 (2012).</li><li>Shrirao, A. B., Perez-Castillejos, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chips+&amp;+tips:+simple+fabrication+of+microfluidic+devices+by+replicating+scotch-tape+masters.">Chips &amp; tips: simple fabrication of microfluidic devices by replicating scotch-tape masters.</a> Lab Chip. , (2010).</li><li>Anil, B. S., Frank, H. K., Derek, Y., Cheul, H. C., Ellen, T. -. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vacuum-assisted+fluid+flow+in+microchannels+to+pattern+substrates+and+cells.">Vacuum-assisted fluid flow in microchannels to pattern substrates and cells.</a> Biofabrication. 6 (3), 035016 (2014).</li><li>Yuen, P. K., Goral, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-cost+rapid+prototyping+of+flexible+microfluidic+devices+using+a+desktop+digital+craft+cutter.">Low-cost rapid prototyping of flexible microfluidic devices using a desktop digital craft cutter.</a> Lab on a Chip. 10 (3), 384-387 (2010).</li><li>Wang, L., 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=Self-loading+and+cell+culture+in+one+layer+microfluidic+devices.">Self-loading and cell culture in one layer microfluidic devices.</a> Biomed Microdevices. 11 (3), 679-684 (2009).</li><li>Feng, 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=Survival+of+mammalian+cells+under+high+vacuum+condition+for+ion+bombardment.">Survival of mammalian cells under high vacuum condition for ion bombardment.</a> Cryobiology. 49 (3), 241-249 (2004).</li><li>Haubert, K., Drier, T., Beebe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDMS+bonding+by+means+of+a+portable,+low-cost+corona+system.">PDMS bonding by means of a portable, low-cost corona system.</a> Lab on a Chip. 6 (12), 1548-1549 (2006).</li><li>Fan, D. -. H., Yuan, S. -. W., Shen, Y. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+modification+with+BSA+blocking+based+on+in+situ+synthesized+gold+nanoparticles+in+poly+(dimethylsiloxane)+microchip.">Surface modification with BSA blocking based on in situ synthesized gold nanoparticles in poly (dimethylsiloxane) microchip.</a> Colloids Surf, B. 75 (2), 608-611 (2010).</li><li>Hideshima, S., Sato, R., Inoue, S., Kuroiwa, S., Osaka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+tumor+marker+in+blood+serum+using+antibody-modified+field+effect+transistor+with+optimized+BSA+blocking.">Detection of tumor marker in blood serum using antibody-modified field effect transistor with optimized BSA blocking.</a> Sens Actuator B-Chem. 161 (1), 146-150 (2012).</li><li>Zheng, 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=High-throughput+immunoassay+through+in-channel+microfluidic+patterning.">High-throughput immunoassay through in-channel microfluidic patterning.</a> Lab on a Chip. 12 (14), 2487-2490 (2012).</li><li>MacLeish, P., Barnstable, C., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+monoclonal+antibody+as+a+substrate+for+mature+neurons+in+vitro.">Use of a monoclonal antibody as a substrate for mature neurons in vitro.</a> Procs Nat Acad of Sci. 80 (22), 7014-7018 (1983).</li><li>Suchodolskis, 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=Elastic+properties+of+chemically+modified+baker's+yeast+cells+studied+by+AFM.">Elastic properties of chemically modified baker's yeast cells studied by AFM.</a> Surf Interface Anal. 43 (13), 1636-1640 (2011).</li></ol>

The authors declare no competing financial interests.

従来のフォトリソグラフィは、ソフトリソグラフィ用の金型を作成するための十分に確立された技術、設備、材料、従来のフォトリソグラフィを使用するために必要なスキルですが、ほとんどの研究室に容易に入手できません。これらのリソースへのアクセスのない研究室のために、我々は、マイクロ流体デバイスのための比較的単純な機能を備えた金型を作成する方法として、粘着テープ?...

組織工学とバイオセンシングでは、ミクロンスケールのタンパク質や細胞の空間的な組織を制御する能力は、最後の四十年1、2、3の上にますます重要になってきています。タンパク質および細胞の正確な空間的な組織は、研究者は、細胞増殖を誘導すること、およびバイオセンサー4、5、6、7、8の製造のために生体分子を固定化するために、細胞の同様のまたは異なる種類を含む細胞と基板の間の相互作用を調べることを可能にしました9。 パターニングタンパク質の現在の方法は、光パターン及びマイクロコンタクト印刷が挙げられます。フォトパターンはultrに曝露されると架橋されている感光材料を利用しますバイオレット（UV）光。 （UV光の透過を防止するために、暗い領域と透明な領域からなる）、フォトマスクに向けられたUV光は、生体材料またはセル10,11の後続の結合のために使用することができる特定の領域に架橋させます。この方式は非常に正確であり、培養表面のトポグラフィーを正確に制御することができるが、それは、UV放射12によってパターニングすることができるUV感受性生体分子に限定されます。マイクロコンタクトプリント法は、特定のタンパク質13、14をパターニングする別の一般的な方法です。この方法では、ポリジメチルシロキサン（PDMS）スタンプは、選択された生体分子の基質の溶液に浸漬される前に、表面改質剤の様々な治療されます。次に、それを静かにこのように培養表面上に生体分子を「スタンプ」カバーガラスまたは他の表面に押し付けられます。ホーweverは、スタンプはPDMS 15をスタンプの表面への生体分子の濡れ性と同様に転写することができる材料の種類に限定されます。 細胞の直接パターニングがより困難になり、そのような特定の細胞接着分子16、17と切り替え可能基板、ステンシルベースの方法、またはパターニングのような複雑な方法に依存していることができます。これらの方法は、原因互換性細胞接着基板の欠如、敏感な生体細胞および制約で動作するプロセスの不適合、パターニング再生の矛盾、及び手順の複雑さにパターン細胞の能力が制限されています。例えば、切替可能な基質と、カスタム基板は、&lt;、プロセス17において使用されるUV光及び熱への暴露時に劣化することなく、特定の細胞型への付着を切り替え、すべての細胞型のために設計される必要があります<html> SUPクラス= &quot;外部参照&quot;&gt; 18、19、20。ステンシルベースのパターニング方法は、パターンのセルにその能力に多目的です。しかしながら、使用16、21のために適切な厚さでPDMSステンシルを製造することは困難です。 1）マイクロ流体チャネルの製造及び2に容易に）多くの異なる細胞と基質のための適性：PDMSマイクロ流体チャネルへの細胞の直接注射は、次のようないくつかの利点を持っています。しかし、注入プロセス中に気泡の捕捉の流行問題は、気泡を減少させるプラズマ洗浄、または他の方法を使用せずにPDMSの疎水性のために、それが困難一貫ガラスまたはプラスチック表面21上にパターン化された細胞を作製することができます。 この作品は、毛細管マイクロモールド22、23と膨張します小娘= &quot;外部参照&quot;&gt; 24、25、26およびマイクロチャネルへのタンパク質や細胞懸濁液を注入する方法を報告します。ここで使用される方法は、基板のパターン形成および特定の細胞型の両方の直接的および間接的なパターニングを示しています。この技術は、PDMSの高い疎水性を克服し、PDMS 27のガス透過性を利用して基板または細胞のいずれかの注入時の気泡の存在を排除します。本稿では、いくつかの異なる基質と細胞型と技術の使用を示しています。記事はまた、従来のフォトリソグラフィと同様に、リソースの限定された設定28、29で有用なシンプルかつ低コストの粘着テープ法を用いたソフトリソグラフィ用の金型の製造を強調しています。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CorelDRAW X4 CAD Drawing Tools</td>
			<td>Corel Corporation, Canada</td>
			<td>X4 Version 14.0.0.701</td>
			<td>CAD tool used to draw the layout of the microfluidic device</td>
		</tr>
		<tr>
			<td>Laser Printer HP</td>
			<td>Hewlett Packard, CA</td>
			<td>1739629</td>
			<td>Used to print the layout of microfluidic device for adhesive tape technique</td>
		</tr>
		<tr>
			<td>Bel-Art Dessicator</td>
			<td>Fisher Scientific, MA</td>
			<td>08-594-16B</td>
			<td>Used to degass the PDMS mixture</td>
		</tr>
		<tr>
			<td>Adhesive Scotch Tape</td>
			<td>3M Product, MN</td>
			<td>Tape 600</td>
			<td>Used to fabricate adhesive tape Master</td>
		</tr>
		<tr>
			<td>PDMS Sylgard 184</td>
			<td>Dow Corning, MI</td>
			<td>1064291</td>
			<td>Casting polymer</td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>Fisher Scientific, MA</td>
			<td>08-772-23</td>
			<td>Used to keep the mold to cast with PDMS</td>
		</tr>
		<tr>
			<td>Stainless steel Scalpel (#3) with blade (# 11)</td>
			<td>Feather Safety Razor Co. Ltd. Japan</td>
			<td>2976#11</td>
			<td>Used to cut the PDMS</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Ted Pella, CA</td>
			<td>5627-07</td>
			<td>Used to handle the PDMS cast during peeling</td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher Scientific, MA</td>
			<td>12-546-2</td>
			<td>Used as surface to pattern the Substrate</td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher Scientific, MA</td>
			<td>12-544-4</td>
			<td>Used as surface to pattern the Substrate</td>
		</tr>
		<tr>
			<td>Rubber Roller</td>
			<td>Dick Blick Art Materials, IL</td>
			<td>40104-1004</td>
			<td>Used to attach adhesive tape on glass without trapping air bubbles</td>
		</tr>
		<tr>
			<td>Laser Mask Writer</td>
			<td>Heidelberg Instruments, Germany</td>
			<td>DWL66fs</td>
			<td>Used to fabricate quartz mask used in photolithography fabrication process</td>
		</tr>
		<tr>
			<td>EVG Mask Aligner (Photolithography UV exposure tool)</td>
			<td>EV Group, Germany</td>
			<td>EVG 620T(B)</td>
			<td>Used to expose the photoresist to UV light</td>
		</tr>
		<tr>
			<td>Spin Coater Headway</td>
			<td>Headway Research Inc, TX</td>
			<td>PWM32-PS-CB15PL</td>
			<td>Used to spin coat the photoresist on silicon wafer</td>
		</tr>
		<tr>
			<td>Photoresists SU-8 50</td>
			<td>MicroChem, MA</td>
			<td>Y131269</td>
			<td>Negative photoresist used for mold fabrication</td>
		</tr>
		<tr>
			<td>SU-8 Devloper</td>
			<td>MicroChem, MA</td>
			<td>Y020100</td>
			<td>Photoresist developer</td>
		</tr>
		<tr>
			<td>Tridecafluoro-1,1,2,2-Tetrahydrooctyl-1-Trichlorosilane</td>
			<td>UCT Specialties, PA</td>
			<td>T2492-KG</td>
			<td>Coat mold to avoid PDMS adhesion</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma-Aldrich, MO</td>
			<td>190764</td>
			<td>Cleaning Solvent</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich, MO</td>
			<td>24102</td>
			<td>Sterilization Solvent</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine hydrobromide (PDL)</td>
			<td>Sigma-Aldrich, MO</td>
			<td>P0899-10MG</td>
			<td>PDL solution is made at 0.1 mg/mL in Sodium Tetraborate Buffer</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich, MO</td>
			<td>L2020</td>
			<td>Laminin aliquoted into 10 &#181;L aliquots and diluted to 20 &#181;g/&#181;L in PBS prior to use</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Fisher Scientific, MA</td>
			<td>BP1605100</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>C2C12 Myoblast cell lline</td>
			<td>ATCC, VA</td>
			<td>CRL-1722</td>
			<td>Used to demonstrate C2C12 patterning</td>
		</tr>
		<tr>
			<td>PC12 Cell Line</td>
			<td>ATCC, VA</td>
			<td>CRL-1721</td>
			<td>Used to demonstrate PC12 patterning</td>
		</tr>
		<tr>
			<td>Collagen type 1, rat tail</td>
			<td>BD Biosciences</td>
			<td>40236</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>GIBCO, MA</td>
			<td>11965-084</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Horse Serum, heat inactivated</td>
			<td>Fisher Scientific, MA</td>
			<td>26050-070</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Phalloidin-tetramethylrhodamine B isothiocyanate (TRITC)</td>
			<td>Sigma-Aldrich, MO</td>
			<td>P1951</td>
			<td>To label cells</td>
		</tr>
		<tr>
			<td>Calcein-AM live dead cell Assay kit</td>
			<td>Invitrogen, MA</td>
			<td>L-3224</td>
			<td>Cell viability Assay</td>
		</tr>
		<tr>
			<td>Biopsy Hole Punch</td>
			<td>Ted Pella, CA</td>
			<td>15110-10</td>
			<td>Punched hole in PDMS</td>
		</tr>
	</tbody>
</table>

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.

この方法は、タンパク質およびマスターモールドが行われた後、ほぼすべての生物学研究室では10μmと利用可能な機器と小さい寸法のデッドエンドマイクロ流体チャネルを用いた細胞の間接的なパターニングのパターニングを可能にします。この技術は、伝統的なソフトフォトリソグラフィ、または粘着テープの製造（ 図1）28、29<...

Watch this Scientific Journal Video about A Versatile Method of Patterning Proteins and Cells at JoVE.com

A Versatile Method of Patterning Proteins and Cells

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 ...

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Research

JoVE Journal

Bioengineering

9.3K ビュー.  Rutgers University. This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

ビデオ: A Versatile Method of Patterning Proteins and Cells

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

細胞ネットワークの作成のためのプラズマのリソグラフィ表面パターニング

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

生物工学

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

ソフトリソグラフィ機能化およびパターニング酸化物フリーシリコンとゲルマニウム

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

全内部反射蛍光顕微鏡を用いた微生物のCortex組織とダイナミクスの可視化、

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

マイクロスケール細胞刺激のための実験のための多目的自動化プラットフォーム

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

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

単一細胞の多彩な化学分析マイクロ流体チップ

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

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 ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the efficiency with which macromolecules penetrate the cytosolic space of cells. Recently, we have circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We have generated a fluorescently labeled dimer of the prototypical cell-penetrating peptide (CPP) TAT, dfTAT, which penetrates live cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT is achieved in multiple cell lines, including primary cells. Moreover, delivery does not noticeably impact cell viability, proliferation or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in dfTAT synthesis and cellular delivery. The manuscript describes how to control the amount of protein delivered to the cytosolic space of cells by varying the amount of protein administered extracellularly. Finally, the current limitations of this new technology and steps involved in validating delivery are discussed. The described protocols should be extremely useful for cell-based assays as well as for the ex vivo manipulation and reprogramming of cells.

We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky.

エンドソーム溶解試薬dfTATとのインキュベーションによって生細胞へのタンパク質、ペプチドまたは細胞不浸透性小分子の送達

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

マイクロ流体プローブとのライブの細胞層の急速なサブトラクティブパターニング

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

細胞培養アプリケーションのためのヒドロゲルの光媒介形成とパターニング

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

生理活性タンパク質や生物学的応用の光化学ハイドロゲルのペプチドをパターニング

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...