著者らは、国立科学財団を通じて、材料研究科学工学センターの一部として資金を供給ジョンズホプキンス、少なくとも表面分析実験室の使用を認めます。 LEDはIGERT研修生と国立科学財団大学院のフェローです。この研究は、部分的にNIHの助成金U54CA143868によってサポートされていました。

1。マイクロパターンスタンプ作製のための標準的なフォトリソグラフィー<ol><li>エタノールで新しいシリコンウエハをリンスし、空気の流れで水分を拭き取ります。ウェハを処理し、全体のプロセスの間に表面欠陥を防止するために鉗子を使用してください。 </li><li> SU - 2025ネガ型フォトレジストを塗布し、カバーウエハの表面を回転させるウエハを転送する。フォトレジストとウェーハの少なくとも80％をカバーしています。 3000rpmの時に30秒間続いて600rpmで10秒間スピンコート、。フォトレジストの感光性のために、前方にこの点から、部屋の照明をオフにしてください。 </li><li> 3分間95℃で&quot;ソフトベーク&quot;のためにホットプレートにコーティングされたシリコンウェハをフォトレジスト転送する。ホットプレートから外し、1分を冷却することができます。 </li><li>フォトレジストで覆われたシリコンウェハの上に所望のパターンを持つプレハブマスクを置き、20秒間紫外線（UV）照射（350〜450 nm）にさらす。 </li><li> 110&quot;ハードベーク&quot;のためのホットプレートに転送シリコンウエハ℃で6分間。 </li><li>ライトの電源を入れ、ホットプレートからウェハを取り外す。明確なパターンは、この時点で表示されるはずです。 </li><li>慎重にSU - 8フォトレジストの現像液でシリコンウエハをリンス。現像液の3洗浄した後、エタノールですすいでください。いずれかの白い残留物が存在する場合は、フォトレジスト現像液でリンス繰り返す、または単に水没ウエハを現像液で2分間とすすぎエタノールを進めます。水と空気乾燥で洗浄する。 </li><li>午前10時01分重量比で混合ポリジメチルシロキサン（PDMS）エラストマー溶液と硬化剤。気泡を除去するために5分間900rpmで遠心する。 </li><li>場所は、ペトリ皿の中でウェーハをパターニングしてシリコンウエハ上にPDMSを注ぐ。泡が消えるまで泡がカップル分間desicatorと真空の場所は、現在がある場合。 </li><li>補完的なエラストマーのスタンプを形成するために室温（RT）で一晩硬化する。 PDMSは、完全に30分間60℃のオーブンで行われる12時間、後硬化されていない場合。 </li><li>慎重にシリコンウエハからPDMSを削除します。希望のサイズにスタンプをカットするためにかみそりのエッジを使用してください。 15分間エタノールで超音波洗浄します。ウェーハは、新しいエラストマーのスタンプを作成するために複数回利用することができます。 </li></ol> 2。 HAのマイクロパターニング<ol><li> 5分間のプラズマ清浄なガラススライド。 5分間チャンバーと真空中にすべてのスライドを置きます。その後、酸素の低流量でチャンバーを入力することができます。 5分間のプラズマクリーナーを入れます。鉗子を用いてシャーレにプラズマクリーナーと場所から削除します。 </li><li>プラズマ洗浄の間に、15分間エタノールでPDMSスタンプを超音波で分解する。 </li><li> 15 mLの遠心管の95％v / vエタノールで3 - アミノプロピルトリメトキシシラン（APTMS）の新鮮な3パーセントv / v溶液を準備します。シラノールを形成するために室温で5分間反応することができます。 </li><li>パターンアップスピンコーターチャックとカバーの上に置いてPDMSスタンプは、APTMS液で表面をパターン化。 30秒間3500rpmでSpincoat。 PDMSスタンプの唯一の側面は、切手の表面に触れないでください。 </li><li>パターン化された側の一方の端に下向きにセットのスタンプを、それが1分間、ガラス表面に接触して完全になるように静かにPDMSを押すと、GLIDE：プラズマへ転送APTMSソリューションは、ガラス面を掃除した。 </li><li> PDMSは優しくスタンプと削除、およびパターン化されたスライドガラスは、30分間室温でインキュベートすることができます。エタノールと空気乾燥でパターン化表面を洗浄します。過剰APTMSを削除するには、15分間PDMSスタンプを超音波処理してください。 </li><li> 115℃で1時間オーブンでまたはホットプレート上に熱表面℃のエタノールでリンスし、空気で乾燥させます。 </li><li>トルエンの[メトキシ（ポリエチレンオキシ）プロピル]トリメトキシシラン - 新鮮なポリエチレングリコール（PEG） - シラン20パーセントV / V 2の溶液を調製します。これは、パターンを囲む非接着領域として機能します。 </li><li> 75℃で45分間、PEG -シラン溶液中でガラスシャーレのインキュベートに転送パターン化されたスライドガラスをホットプレート上に° C。トルエン、水、および空気と乾燥ですすいでください。 </li><li>生物学的安全キャビネット内の紫外線殺菌照射を使用して1時間滅菌する。無菌性を維持するために前方にこの時点から、生物学的安全キャビネットに進みます。 </li><li>水性滅菌HAソリューションを準備。 1 - エチル-3 - （3 - ジメチルアミノプロピル）カルボジイミド（EDC）の10mMの N -ヒドロキシスクシンイミドの5mMの 50μg/ mLのフルオレセイン標識HA </li><li>無菌状態でパターニングされたガラス基板にHAソリューションを適用し、光から保護。パターニングされた基板は、16〜24時間静にする必要があります。 </li><li> HAソリューションをオフ吸引、で洗浄し、リン酸緩衝生理食塩水を（PBS）、及び1時間PBSで（BSA）の1％ウシ血清アルブミンとの表面を覆う。表面は細胞培養の準備ができています。 </li></ol> 3。 HAマイクロパターン表面上における細胞培養<ol><li>製造業者の指示に従って、MDA - MB - 231ヒト乳癌細胞とLS174T大腸癌細胞を展開。 </li><li> 0.4 × 10の間に密度のがん細胞のシード単細胞懸濁液<sup18.75センチメートル2ガラスの表面積当たりとHA固定化表面へ&gt; 6癌細胞。 5％CO 2で維持雰囲気で加湿インキュベーター（37℃）でインキュベートする。細胞接着は、24時間以内に発生する必要がありますし、倒立光学顕微鏡を用いて観察することができます。 </li><li>細胞培養表面は、標準的なインキュベータの条件で5日間まで維持することができます。一日おきにメディアを変更します。細胞の形態と成長は、倒立光学顕微鏡を用いて観察することができます。追加の細胞アッセイは、HAの表面への応答を分析するために適用することができます。 </li></ol> 4。代表的な結果： 共有結合APTMSパターニングされたガラススライドにHAを固定するために使用されるカルボジイミド化学を図1Aに示されています。 EDCは、アミン反応性中間体を形成するために、HAのカルボキシル基と反応して長さゼロの架橋剤です。この中間体が加水分解を受けやすいとして、NHSは、カルボジイミド反応効率を高めるために追加されます。 HAソリューションはAPTMS微細パターン、HAの中間とATPMSの第一級アミンとの間の安定したアミド結合の形態と相互作用として。 HAパターン表面の例は、HAのパターン化された固定化を示す蛍光顕微鏡とImageJの蛍光強度の解析を用いて可視化（ 図1B - C）が表示されます。 HAマイクロパターン表面はexpgenous HAと癌細胞の相互作用の研究を可能にします。 MDA - MB - 231ヒト乳癌とLS174Tヒト大腸癌細胞株の両方が優先的にHAマイクロパターン領域（ 図2）に従っている。走査型電子顕微鏡を用いた高分解能イメージングは、HA固定化表面（ 図3）による培養細胞の相互作用を分析するために使用することができます。 <img src="/files/ftp_upload/2413/2413fig1.jpg" alt="図1" /> 図1 HAマイクロパターン表面のHA機能面の開発を記述する（）回路図、フルオレセインの（B）蛍光画像（緑）で標識されたHAマイクロパターン化表面。離散的なHAのパターンを示すImageJのソフトウェアを使用して（C）3Dピクセルの分布図。スケールバー=100μmの。エルゼビアから許可を得て9日から変更。 <img src="/files/ftp_upload/2413/2413fig2.jpg" alt="図2" /> HAマイクロパターン表面上に図2。癌細胞接着。コロン（）と乳房（B）癌が優先的に文化の24時間とHAのパッチに付着両方。エルゼビアから許可を得て9日から変更。 <img src="/files/ftp_upload/2413/2413fig3.jpg" alt="図3" /> HAと図3。癌細胞の相互作用。 （A）大腸癌細胞の低倍率（左）と高倍率（右、左の上の正方形の）に示すように、HAの表面上に（）培養の電子顕微鏡像を走査し、乳癌細胞の（B）に示すように、HA表面上で培養低倍率（左）と高倍率（右、左の上の正方形の）。

<ol>
	<li>Toole, B. P., Wight, T. N., Tammi, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan-cell+interactions+in+cancer+and+vascular+disease.">Hyaluronan-cell interactions in cancer and vascular disease.</a> Journal of Biological Chemistry. 277, 4593-4596 (2002).</li><li>Hamilton, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hyaluronan+Receptors+CD44+and+Rhamm+(CD168)+Form+Complexes+with+ERK1,2+That+Sustain+High+Basal+Motility+in+Breast+Cancer+Cells.">The Hyaluronan Receptors CD44 and Rhamm (CD168) Form Complexes with ERK1,2 That Sustain High Basal Motility in Breast Cancer Cells.</a> Journal of Biological Chemistry. 282, 16667-16680 (2007).</li><li>G&#246;tte, M., Yip, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparanase,+Hyaluronan,+and+CD44+in+Cancers:+A+Breast+Carcinoma+Perspective.">Heparanase, Hyaluronan, and CD44 in Cancers: A Breast Carcinoma Perspective.</a> Cancer Research. 66, 10233-10237 (2006).</li><li>Lord, M. S., Pasqui, D., Barbucci, R., Milthorpe, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+adsorption+on+derivatives+of+hyaluronic+acid+and+subsequent+cellular+response.">Protein adsorption on derivatives of hyaluronic acid and subsequent cellular response.</a> Journal of Biomedical Materials Research. Part A 91, 635-646 (2009).</li><li>Barbucci, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+hyaluronic+acid+by+sulphate+groups+insertion+to+obtain+a+heparin-like+molecule.">Modification of hyaluronic acid by sulphate groups insertion to obtain a heparin-like molecule.</a> Gazz. Chim. Ital. 125, 169-180 (1995).</li><li>Morra, M., Cassinelli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-fouling+properties+of+polysaccharide-coated+surfaces.">Non-fouling properties of polysaccharide-coated surfaces.</a> Journal of Biomaterials Science, Polymer Edition. 10, 1107-1124 (1999).</li><li>Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Layer-by-layer+deposition+of+hyaluronic+acid+and+poly-L-lysine+for+patterned+cell+co-cultures.">Layer-by-layer deposition of hyaluronic acid and poly-L-lysine for patterned cell co-cultures.</a> Biomaterials. 25, 3583-3592 (2004).</li><li>Ibrahim, S., Joddar, B., Craps, M., Ramamurthi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+surface-tethered+model+to+assess+size-specific+effects+of+hyaluronan+(HA)+on+endothelial+cells.">A surface-tethered model to assess size-specific effects of hyaluronan (HA) on endothelial cells.</a> Biomaterials. 28, 825-835 (2007).</li><li>Dickinson, L. E., Ho, C. C., Wang, G. M., Stebe, K. J., Gerecht, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+surfaces+for+high-resolution+analysis+of+cancer+cell+interactions+on+exogenous+hyaluronic+acid.">Functional surfaces for high-resolution analysis of cancer cell interactions on exogenous hyaluronic acid.</a> Biomaterials. 31, 5472-5478 (2010).</li><li>Gao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan+oligosaccharides+promote+excisional+wound+healing+through+enhanced+angiogenesis.">Hyaluronan oligosaccharides promote excisional wound healing through enhanced angiogenesis.</a> Matrix Biology. 29, 107-116 (2010).</li><li>Slevin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan-mediated+angiogenesis+in+vascular+disease:+Uncovering+RHAMM+and+CD44+receptor+signaling+pathways.">Hyaluronan-mediated angiogenesis in vascular disease: Uncovering RHAMM and CD44 receptor signaling pathways.</a> Matrix Biology. 26, 58-68 (2007).</li></ol>

許可を得て複製したビデオの概略図;著作権ワイリー- VCH出版社＆Co KGaA社。ディッキンソンLE、マラヤS、生体材料を使用して、胚性幹細胞の分化のニッチを再構築Gerecht S.。 Macromol。バイオサイエンス。 2010; 10月21日。 [印刷の前にトップ|]。

提示HAの微細方法は、外因性HAと細胞の相互作用の研究が可能になります。 HAは、癌の進行1に重要な役割を果たすことが知られているしかし、二次元のHAパターニング表面上での癌細胞の相互作用を調査する限られた研究が行われている。外因性HAの微細パターンで制御可能研究は癌細胞の接着、成長、および移行の高解像度可視化を可能にし、癌のさらなる基礎を解明することがで?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>SU-2025 photoresist</td>
<td>&nbsp;</td>
<td>MicroChem Corp.</td>
<td>Y111069</td>
<td>&nbsp;</td>
<tr>
<td>SU-8 developer</td>
<td>&nbsp;</td>
<td>MicroChem Corp.</td>
<td>Y020100</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>3-aminopropyltrimethoxysilane (APTMS)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>281778</td>
<td>&nbsp;</td>
<tr>
<td>2- [methoxy(polyethyleneoxy) propyl] trimethoxysilane (Peg-silane)</td>
<td>&nbsp;</td>
<td>Gelest Inc</td>
<td>SIM6492.7</td>
<td>&nbsp;</td>
<tr>
<td>1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>22980</td>
<td>&nbsp;</td>
<tr>
<td>N-hydroxysuccinimide (NHS)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>24500</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescein labeled hyaluronic acid (FL-HA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrick</td>
<td>F1177</td>
<td>Reconstitute with 10ml of DI water </td>
</tr>
<tr>
<td>MDA-MB-231 breast carcinoma cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>HTB-26</td>
<td>&nbsp;</td>
<tr>
<td>LS174t colon carcinoma cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>Cl-188</td>
<td>&nbsp;</td>		</tbody>
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micropatterned surfaces to study hyaluronic acid interactions with cancer cells

癌の浸潤や進行は、腫瘍の微小環境内での増殖因子/サイトカインや細胞外マトリックス（ECM）成分による複雑な規制下にある運動性細胞の表現型を伴います。ヒアルロン酸（HA）は、強化の侵入、増殖、および血管新生1で腫瘍の進行を容易にするために確認されている間質ECMコンポーネントです。その細胞表面の受容体CD44とHAの相互作用は、それによって転移拡散2-3増加、腫瘍細胞の増殖、生存、および移行を促進するシグナル伝達事象を誘導する。 HAは、D -グルクロン酸とDN -アセチルグルコサミンの繰り返し単位から構成される陰イオン性、nonsulfatedグリコサミノグリカンである。 、二糖繰り返し単位のカルボキシル及びヒドロキシル基の存在のためにネイティブHAの大部分は親水性とphotoreative固定化4-5硫酸基を導入する化学修飾の影響を受けやすいです。表面にHAの固定化を含むこれまでの研究では6-7表面上に細胞接着を制御するために、HAのbioresistant挙動とその硫酸化誘導体を利用する。これらの研究では細胞接着には、優先的に非HA模様の領域で発生します。 外因性のHAと細胞間相互作用を分析するために、我々は、制御の研究とHAと癌細胞の相互作用の高解像度可視化を可能にするパターン化された官能化された表面を開発しました。我々は、ガラスの表面にHAの離散パターン領域を定義するためにマイクロコンタクトプリンティング（UCP）を利用。 HAを固定化するカルボジイミドリンク化学を適用する&quot;テザリング&quot;のアプローチは8を使用しました。ガラスの表面は、マイクロコンタクトをアミノシランで印刷し、パターン化された配列にHA固定化を有効にするEDCとNHSの最適比率のHA溶液と反応させた。 MCPを搭載したカルボジイミド化学を組み込むと、in vitroでのアプリケーションに適したサーフェスを作成、定義された領域にHAの固定化を可能にした。両方の結腸癌細胞および乳癌細胞は、暗黙的にHAマイクロパターン表面と相互作用した。癌細胞の接着は、48時間で増殖して24時間以内に起こった。 HAマイクロパターン表面を使用して、我々は癌細胞の接着は、HA受容体CD44を介して起こることを示した。さらに、HA柄の表面は電子顕微鏡（SEM）と癌細胞の接着剤の突起の許容される高解像度の画像をスキャンし、外因性のHA上で癌細胞の運動性を分析するためにHAパターンで拡散との互換性があった。

Watch this Scientific Journal Video about Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells at JoVE.com

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

外因性ヒアルロン酸（HA）と癌細胞の相互作用の高分解能解析を可能にする斬新なアプローチが説明されています。パターニングされた表面は、カルボジイミド化学とマイクロコンタクトプリンティングを組み合わせることによって作製されています。

癌細胞とヒアルロン酸の相互作用を研究するマイクロパターン表面

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

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15.0K ビュー.  Johns Hopkins University. 外因性ヒアルロン酸（HA）と癌細胞の相互作用の高分解能解析を可能にする斬新なアプローチが説明されています。パターニングされた表面は、カルボジイミド化学とマイクロコンタクトプリンティングを組み合わせることによって作製されています。

ビデオ: Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

マイクロパターン細胞を用いた薬物の効果の改善可視化と定量分析

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

生物工学

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

のための周期的圧力のバイオリアクターの設計生体外で</em大動脈心臓弁の>研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

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 ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

癌細胞浸潤への応用による腫瘍間質微小環境の組織工学

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

遺伝的に改変された細胞移植のためのマイクロパターン培養プレート上スフェロイド細胞に対する遺伝子導入

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

組織工学のための熱応答性ナノ構造表面の調製

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D相互作用の研究のための3Dコラーゲンゲルおよびマイクロチャネルの作製<em&gt;インビボ</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities.

骨形成タンパク質 2 固体表面にクリックケミストリーによるサイト向け固定

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

ヒアルロン酸を用いた神経膠芽腫患者由来細胞の 3次元培養ヒドロゲル

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

ヒト人工多能性幹細胞由来心筋細胞における単細胞光学作用電位測定

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...