O. KurtulusとD. Dimliogluは、カリフォルニア大学研究費研究プログラム賞12-LR-237197によってサポートされています。 P. Daggumatiはサイエンス＆エンジニアリング（RISE）賞カリフォルニア州デービス·リサーチ·インベストメンツ大学によってサポートされています。 CAチャップマンは、ナショナルニードフェローシップの教育大学院援助分野の部門によってサポートされています。この作業は、UCラボ料研究プログラム、UCデービスのRISE、工学スタートアップ資金のカリフォルニア大学デ​​ービス校の大学によってサポートされていました。

1。ナノポーラス金の作製<ol><li>ピラニア溶液中のクリーン基板<ol><li>混合物をホットプレート上で65℃に結晶化皿および熱た100mlの硫酸（96％）を25ミリリットルの過酸化水素（30％）を追加します。注意：液体は極めて腐食性であり、慎重に取り扱わなければならない。それが爆発する可能性があるので使用済み溶液を、密封容器に保存されているべきではありません。 </li><li>混合耐酸性ピンセットを用いて、10分間のためにそれらをきれいに3インチ顕微鏡スライドによって場所の1インチ。小さなカバースリップをバッチ洗浄のために磁器の免疫染色のボートを使用します。溶液中に浸漬する前に30秒のために10 Wの空気プラズマの小さなカバースリップを扱う。 </li><li> 3分間脱イオン（DI）流水結晶皿にきれいにサンプルを洗浄します。糸くずの出ないタオルの上に窒素銃ブロー乾燥試料。 </li></ol></li><li> （：ミリスケールのパターンを作成するためにこれを使用方法1）ステンシルマスクを準備<ol><li>パンチ生検パンチと250μmの厚さのシリコーンエラストマーシートおよび/または半硬質プラスチック表面上にメスで地域を切開。窒素銃でイソプロパノール、乾燥70％で処理されたエラストマーシートを清掃してください。 </li><li>リントフリークリーンルームタオルの上にパンチングシートを置き、ステンシルが直面している試料表面とステンシルオーバーピラニア洗浄をカバースリップを合わせます。 </li></ol></li><li>パターンリフトオフレジスト（方法2：サブミリスケールのパターンを作成するために使用します） <ol><li>チャックを回転にピラニア洗浄を顕微鏡スライドを置き、窒素銃で任意の粒子を吹き飛ばす。プラスチックピペットを用いてスライドガラス上に接着促進剤（ヘキサメチルジシラザン）1.5mlを分注する。 5秒、30秒のために1,500 rpmで500rpmで連続的にスライドを回転することによりプロモーターを広める。 7分間115℃のホットプレート上でスライドを焼いて5分間冷ます。 </li><li>スライドガラス上にフォトレジスト4mlのディスペンス（3インチByは1インチ）。接着促進剤の場合と同じプロトコルを使用してスライドを回転させてレジストを広げた。 1.5分間115℃のホットプレート上にフォトレジストを焼くと10分間冷ます。 </li><li> 15秒のために透明マスクを介して：UV光でフォトレジストでコーティングされたスライド（22 MW / cm 2と強度）を公開します。 1.5分間、115℃のホットプレート上にフォトレジストを焼いて45分間待ちます。少なくとも3.5分間現像液に露光されたフォトレジストを溶解する。 DI水で十分に洗い流してください。光学顕微鏡下で開発されたパターンを点検します。 </li></ol></li><li> NP-Auの薄膜を製造するための前駆体の金属を堆積させる<ol><li>独立して、金、銀、クロムを預けることができるスパッタ装置にサンプルをロードします。金属蒸着を開始する前に、25トルアルゴン処理雰囲気下、50 Wで90秒間のサンプルをスパッタクリーン。 </li><li> 10トルアルゴン下300 Wで10分間クロームスパッタ。 400 W国連で90秒間金をスパッタファンデ10トルアルゴン。 100 W.で200 WおよびAuパワーで銀と10分間、金と銀は、金、銀をオフにする前にソース約10秒スパッタ源をスパッタ切り共同スパッタ。 </li></ol></li><li>前駆金属微細パターンを得る<ol><li> 20秒の超音波処理を10サイクルとサイクルの間に2分間の休止のためのフォトレジストストリッパーの〜180ミリリットルでフォトレジストでコーティングされたサンプルを超音波処理。 DI水でサンプルをすすぎ、窒素銃で乾燥させます。顕微鏡下で金属パターンを検査します。 </li><li>堆積した金属を明らかにするために2つのピンセットを使用して非フォトレジスト被覆された試料からの剥離性エラストマーステンシル。 </li></ol></li><li>熱処理経由Dealloy前駆金属および変更ナノ構造<ol><li>硝酸（70％）170 mlのを200mlガラスビーカーを記入し、ホットプレート上で55℃に液温を維持する。注意：硝酸は非常に腐食性であり、適切な保護具を処理する必要があります。 </lI&gt; <li>硝酸に浸漬する前に30秒のために10 Wの空気プラズマの小さなカバースリップを扱う。耐酸性ピンセットを用いてビーカーに置き、3インチ顕微鏡スライド1インチおよび15分のためにそれらをdealloy。バッチ脱合金小さなカバースリップ用磁器免疫染色のボートを使用してください。 </li><li>順次新鮮なDI水で三回充填2ビーカーにそれらを浸漬することによりdealloyedサンプルをリンス。少なくとも1週間DI水のサンプルを保存し、毎日新鮮なDI水で水を交換してください。使用前に糸くずの出ないタオルの上に窒素銃ブロー乾燥試料。 </li><li>急速加熱処理装置におけるクリーンシリコンウェハー上にサンプルをロードする。 200°Cと450°Cとランプ速度との間に温度を調節10℃/秒。窒素雰囲気下で10分間所定の温度にサンプルを公開します。チャンバーは冷ます（&lt;100℃）、サンプルを削除します。また、徐々に熱treatm用ホットプレート上のサンプルを配置ENT。 </li></ol></li></ol> 2。細胞培養<ol><li>細胞培養用NP-Auのサンプルを準備します<ol><li>ポリスチレンディッシュ内の場所NP-Auのサンプルを、30秒間10 Wで空気プラズマで処理し、24ウェル組織培養プレートにサンプルを移す。 </li><li>各ウェルに500μlの完全培地（ダルベッコ改変イーグル培地、10％ウシ胎児血清および1％ペニシリン/ストレプトマイシンを有する）を追加する。 37℃加湿インキュベーターに保管℃、細胞（&lt;1時間）播種まで5％CO 2。 </li></ol></li><li>維持するため、通路や種子細胞<ol><li>培地、細胞が70％コンフルエントである経過とともにT75フラスコ中で目的の細胞（この場合は3T3-NIH線維芽細胞又はマウスアストロサイト）を維持する。 </li><li>細胞を継代については、緩衝生理食塩水（PBS）を10mlのリン酸で二回洗い、フラスコからメディアを取り出し、細胞デタッチ（〜5分）まで、1×トリプシン/ EDTAとインキュベート2 mlを加える。 3ミリリットル新鮮な培地を追加し、3分間1,200 rpmで遠心。上清を吸引除去し、2 mlの培地中でペレットを中断します。 </li><li> 1ミリリットルの最終容量で25,000細胞/ cm 2の密度でカバーガラス上に井戸や種子細胞から過ごしたメディアを取り出します。サンプルの細胞の均一なコーティングを確保するために前進·バックと左右培養プレートを振る。毎日検査しながら分析まで細胞をインキュベート。 </li></ol></li></ol> 3。細胞と材料分析<ol><li>細胞骨格と核を可視化するために細胞を染色<ol><li>井戸から過ごしたメディアを取り出し、PBSで細胞を2回洗浄する。 15分間PBS中4％パラホルムアルデヒドで細胞を固定します。 </li><li> 1％ウシ血清アルブミンを含むPBSで300 nMのアレクサフルーア488ファロイジンの染色溶液を調製。 </li><li> PBSで細胞を2回洗浄し、5分間PBSでトリトンX-100を500μlの0.1％それらを透過性。 </li><li> PBSで細胞を2回洗浄し、井戸をきれいにするために、それらを転送します。しみ50 - サンプルと20分間、暗所で店舗へ200μlの染色液。 </li><li> PBSで5分間PBS中DAPIの3 Nmのカウンター染色で細胞を洗浄します。 </li><li> PBSで細胞を洗浄し、クリアなマニキュアで取り付けメディアやシールでガラスカバースライド上にマウントする前に脱イオン水に浸す。 </li></ol></li><li> NP-Auの表面にNP-Auのサンプルと、細胞培養物の画像を取得する<ol><li>画像NP-Auの二次電子検出器を使用して、10kVの電子エネルギーで50,000 Xの倍率で走査型電子顕微鏡（SEM）による表面。 </li><li>適切なフィルタキューブで10倍の倍率で倒立蛍光顕微鏡を用いてサンプル上の異なる箇所で複合セル画像をキャプチャします。 </li></ol></li><li>プロセスイメージは、毛穴や細胞形態を決定する<ol><li> ImageJのに該当する場合、個々のチャネルに分割オープン画像。 8ビットに画像を変換し、バックグラウンドを減算し、中央値filteriことによって、それらを滑らかにNG。手動または孔（ボイド）と細胞体/核を強調するためにアルゴリズムを閾値ビルトインによってしきい値を調整します。 </li><li>マージされた毛穴や細胞を分離するために流域コマンドを使用します。粒子解析パラメータを設定し、粒子による粒子、平均面積、およびパーセントカバレッジ数を抽出するためにコマンドを実行します。細胞計数およびパーセントセルカバレッジを定量化するためファロイジン染色細胞画像のDAPI染色細胞の画像を使用してください。 </li><li>複数の画像を一括分析を実行するために含まれたマクロファイルを変更する。 </li></ol></li></ol>

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著者は全く相反する経済的利害関係を持っていません。

私たちは、マイクロシステムと生物学的研究におけるこれらのフィルムの使用を拡大するため微細NP-Auの膜には、2つの異なる技術を実証する。スパッタコーティング、金と銀は、スパッタリング、従来の微細加工プロセスと合金組成と厚さを簡単に個々のスパッタ銃のパワーを変化させることにより制御（金と銀の目標のために）とすることができますとの互換性があるように、NP-Auのパタ?...

<p class="jove_content"ナノスケール機能を持​​つ&gt;材料は、燃料電池を含む様々なアプリケーションを高めるのに約束を示している<sup&gt; 1</sup&gt;、センサー<sup&gt; 2,3</sup&gt;、および生物医学装置<sup&gt; 4,5</sup&gt;。比較的新しい材料は、ナノスケールの自己組織化プロセスによって製造されるナノ多孔質金（NP-Au）である。 NP-Auの前駆体は、最も一般的に原子百分率が60％〜80％の銀で構成され、金合金である。銀は強酸によって溶解されるように簡単に説明すると、特徴的な開孔ナノ構造は、クラスタ内の金原子の再配列の結果である（<em&gt;例えば</em&gt;硝酸70％）又は電気化学ポテンシャルの下<sup&gt; 6-8</sup&gt;。大きな有効面積、高い導電率、十分に確立された表面機能化技術、および生体適合性を含むいくつかの望ましい属性から、NP-金給付<sup&gt; 9</sup&gt;。 NP-Auの上の研究の急速な拡大、NP-auの機械的性質に焦点を当て、それらのほとんどがあったにもかかわらず、<sup&gt; 10,11</sup&gt;、触媒活性<sup&gt; 12</sup&gt;、そして生体分子センシング性能<sup&gt; 13-15</sup&gt;。望ましい属性は、いくつかの生物医学的なツールのために非常に有用であるが<sup&gt; 16,17</sup&gt;、この分野における用途が限られている。この理由の1つは、ほとんどの研究は、主にマクロスケール標本を使用している（<em&gt;例えば</em&gt;シート、ホイル、およびインゴット）と小型化システムでは、NP-Auのを組み込むための技術が不十分なままである。実際には、NP-Auの膜を用いる従来の微細加工技術の使用例のほんの一握りがある<sup&gt; 16-20</sup&gt;。小型化技術と新規生物医学ツールの必要性の出現と、それはデバイスに新しい材料を統合できるようにすることが重要になってきている。これは通常、材料が堆積され、従来の微細加工技術を用いてパターニングすることができることを必要とする。また、細胞 - 材料の相互作用の迅速な定量化は、新しい材料の生体適合性を評価するために一般に必要である。本研究の目的は、微細NP-Auの膜への基本的な技術を示し、ナノ構造体及びディジタル画像処理を介して、細胞 - 材料の相互作用の両方を定量化することである。</p

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Gold target</td>
 <td>Lesker</td>
 <td>EJTAUXX403A2</td>
 <td >Precursor to alloy for producing np-Au</td>
 </tr>
 <tr>
 <td>Chrome target</td>
 <td>Lesker</td>
 <td>EJTCRXX353A2</td>
 <td >Adhesive layer</td>
 </tr>
 <tr>
 <td>Silver target</td>
 <td>Lesker</td>
 <td>EJTAGXX403A2</td>
 <td >Precursor to alloy for producing np-Au</td>
 </tr>
 <tr>
 <td>Porcelain boat</td>
 <td>Thomas Scientific </td>
 <td>8542E40</td>
 <td >Used for processing small samples</td>
 </tr>
 <tr>
 <td>Nitric acid</td>
 <td>Sigma-Aldrich</td>
 <td>43873</td>
 <td >Used at 70% for dealloying</td>
 </tr>
 <tr>
 <td>Sulfuric acid</td>
 <td>J.T Baker </td>
 <td>7664-93-9</td>
 <td >Used at 96% for piranha cleaning</td>
 </tr>
 <tr>
 <td>Hydrogen peroxide</td>
 <td>J.T Baker </td>
 <td>7722-84-1</td>
 <td >Used at 30% for piranha cleaning</td>
 </tr>
 <tr>
 <td>Biopsy punches</td>
 <td>Ted Pella</td>
 <td>150xx</td>
 <td >Available in several sizes</td>
 </tr>
 <tr>
 <td>Silicone elastomer sheets</td>
 <td>Rogers Corporation </td>
 <td>HT 6240</td>
 <td >Available in several thicknesses</td>
 </tr>
 <tr>
 <td>Hexamethyldisilazane</td>
 <td>Sigma-Aldrich</td>
 <td>440191-100ML</td>
 <td >Used as adhesion promoter for positive resist</td>
 </tr>
 <tr>
 <td>Microposit MF CD26</td>
 <td>Shipley</td>
 <td>38490</td>
 <td >Positive photoresist developer</td>
 </tr>
 <tr>
 <td>PRS 3000</td>
 <td>J.T Baker</td>
 <td>JT6403-5</td>
 <td >Positive photoresist stripper</td>
 </tr>
 <tr>
 <td>Circular glass coverslips (12 mm)</td>
 <td>Ted Pella</td>
 <td>26023</td>
 <td >Used as substrate for metal patterns and cell culture</td>
 </tr>
 <tr>
 <td>Glass slides (1 x 3 inch)</td>
 <td>Ted Pella</td>
 <td>26007</td>
 <td >Used as substrate for metal patterns</td>
 </tr>
 <tr>
 <td>Kapton polyimide tape</td>
 <td>VWR</td>
 <td>82030-950</td>
 <td >Used for securing elastomer</td>
 </tr>
 <tr>
 <td>Transparency masks</td>
 <td>Output City</td>
 <td></td>
 <td>Used in photolithography <a href="http://www.outputcity.com/" target="_blank">http://www.outputcity.com/</a></td>
 </tr>
 <tr>
 <td>Plasma cleaner</td>
 <td>Harrick Plasma</td>
 <td>PDC-32G</td>
 <td >Used for activating glass surfaces</td>
 </tr>
 <tr>
 <td>Sputtering machine</td>
 <td>Kurt J. Lesker</td>
 <td>LAB18</td>
 <td >Used for depositing metals</td>
 </tr>
 </tbody>
 </table>

microfabrication of nanoporous gold patterns for cell-material interaction studies

数十ナノメートルのフィーチャサイズを有するナノ構造材料は、燃料電池、バイオセンサー、生体医用器具​​用コーティング、および薬物送達ツールを含むいくつかの技術の性能を強化している。ナノスケールの自己組織化プロセスによって生成されたナノ多孔質金（NP-Auが）、大きな実効表面積、高い導電率、および触媒活性を示す比較的新しい材料である。これらのプロパティは、NP-Auのを科学界に魅力材料を作った。 NP-Auの上のほとんどの研究は、材料の基礎科学とその触媒とセンサアプリケーションにマクロスケール標本やフォーカスを採用しています。マクロスケール標本は生物医学デバイスを含む小型化システムにおけるNP-auの可能性を制限する。これらの問題に対処するために、我々は、最初に剛性基板上に微細パターンNP-Auの薄膜を2つの異なる方法が記載されている。最初の方法は、ミリスケールNP-Auのパターンを作成するための薬用手動生産ステンシルマスクを採用していますeは、第二の方法は、パターンのサブミリスケールパターンにリフトオフフォトリソグラフィーを使用しています。 NP-Auの薄膜をスパッタ堆積プロセスによって得られ、それらはマイクロへの容易な統合することにより適した従来の微細加工技術と互換性がある。これらのシステムは高い有効表面積の恩恵、電気伝導性、そして金 - チオール系表面バイオコンジュゲーションその電気的にアドレス可能なバイオセンサーのプラットフォームが含まれています。我々は、いくつかのバイオセンサーのための重要な性能パラメータである哺乳動物細胞でNP-Auからの対話を定量化するために、細胞培養、免疫染色し、画像処理技術を説明する。ここでは、図示の技法は、様々な長さスケールでのプラットフォームやバイオセンサー、エネルギー貯蔵システム、および触媒を含む多数の用途においてNP-Auを統合を支援することを期待している。

図1は 、培養細胞をNP-Auのパターンを作成するナノ構造を定量化し、細胞形態を特徴付ける含めて、主要な処理手順の概要を説明します。 図2aに示したエラストマーステンシル（上面）の下に画像に示すようにNP-Auのパターンを作成するために使用される。 図2bは、バッチ処理の試験片の磁器船の写真である。 図2cは、堆積された金属パター?...

Watch this Scientific Journal Video about Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies at JoVE.com

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

私たちは、ステンシル印刷やフォトリソグラフィーと同様、微細パターン上に培養細胞へのメソッドを経由して微細パターンナノポーラス金薄膜の技術について報告する。また、材料及び走査型電子顕微鏡、蛍光技術を用いて培養細胞の形態を特徴づける画像解析方法が記載されている。

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, ...

microfabrication-nanoporous-gold-patterns-for-cell-material

Research

JoVE Journal

Bioengineering

11.3K ビュー.  University of California, Davis. 私たちは、ステンシル印刷やフォトリソグラフィーと同様、微細パターン上に培養細胞へのメソッドを経由して微細パターンナノポーラス金薄膜の技術について報告する。また、材料及び走査型電子顕微鏡、蛍光技術を用いて培養細胞の形態を特徴づける画像解析方法が記載されている。

ビデオ: Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

非対称受容体のパターンでセルローリング軌道を勉強

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

生物工学

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

合成、組立、および単分子膜のキャラクタリゼーションは、タンパク質単分子層の電気化学のための金ナノ粒子膜を保護

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

生理フローの状況の下で循環細胞のケモカイン指向溢出を研究する新規三次元フローチャンバーデバイス

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.

A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination.

タンパク質分析のための継続的な認識パターンを生成する電子舌

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

サンドイッチ状の微小環境は、Cell /材料の相互作用を活用します

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

ゴールドナノロッドを含む細胞車の作製と光音響分析

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting and drug delivery are some of the applications of these AuNPs, but endothelial extracellular matrices&#39; defensive properties hamper particle uptake. To address this issue, we describe a synthesis method for ultrasmall gold nanoparticles to improve vascular delivery, with customizable functional groups and polymer lengths for further adjustments. The protocol yields 2.5 nm AuNPs that are capped with tetrakis(hydroxymethyl)phosphonium chloride (THPC). The replacement of THPC with hetero-functional polyethylene glycol (PEG) on the surface of the AuNP increases the hydrodynamic radius to 10.5 nm while providing various functional groups on the surface. The last part of the protocol includes an optional addition of a fluorophore to allow the AuNPs to be visualized under fluorescence to track nanoparticle uptake. Dialysis and lyophilization were used to purify and isolate the AuNPs. These fluorescent nanoparticles can be visualized in both in vitro and in vivo experiments due to the biocompatible PEG coating and fluorescent probes. Additionally, the size range of these nanoparticles render them an ideal candidate for probing the glycocalyx without disrupting normal vasculature function, which may lead to improved delivery and therapeutics.

We describe a method of synthesizing biocompatible 10-nm gold nanoparticles, functionalized by coating poly-ethylene glycol onto the surface. These particles can be used in vitro and in vivo for delivering therapeutics to nanoscale cellular and extracellular spaces that are difficult to access with conventional nanoparticle sizes.

粒子の合成機能 10 nm ポリマー コーティング金対象化、血管内皮細胞とドラッグデリバリー

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

多重の人工細胞の微小環境配列の作製

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

近赤外線発光金ナノクラスターの生物応用のための合成

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been ...

Asymmetrical Flow Field-Flow Fractionation for Sizing of Gold Nanoparticles in Suspension

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and size distribution of nanoparticles is of utmost importance for various applications. The size range is also important, as it defines the most &#8220;active&#8221; component of a nanoparticle dose.
Asymmetrical Flow Field-Flow Fractionation (AF4) is a powerful technique for sizing of particles in suspension in the size range of approximately 1&#8211;1000 nm. There are several ways to derive size information from an AF4 experiment. Besides coupling AF4 online with size-sensitive detectors based on the principles of Multi-Angle Light Scattering or Dynamic Light Scattering, there is also the possibility to correlate the size of a sample with its retention time using a well-established theoretical approach (FFF theory) or by comparing it with the retention times of well-defined particle size standards (external size calibration).
We here describe the development and in-house validation of a standard operating procedure (SOP) for sizing of an unknown gold nanoparticle sample by AF4 coupled with UV-vis detection using external size calibration with gold nanoparticle standards in the size range of 20&#8211;100 nm. This procedure provides a detailed description of the developed workflow including sample preparation, AF4 instrument setup and qualification, AF4 method development and fractionation of the unknown gold nanoparticle sample, as well as the correlation of the obtained results with the established external size calibration. The SOP described here was eventually successfully validated in the frame of an interlaboratory comparison study highlighting the excellent robustness and reliability of AF4 for sizing of nanoparticulate samples in suspension.

This protocol describes the use of Asymmetrical Flow Field-Flow Fractionation coupled with UV-vis detection for the determination of the size of an unknown gold nanoparticle sample.

懸濁液中の金ナノ粒子のサイズに対する非対称流量場流分画

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and ...