この作品はを通じてロジャースファミリー財団、画期的な生物医学研究のためのUCSF /サンドラー財団プログラム、BASF社からの助成金、およびNSFからギャップ賞を埋める、定量的バイオサイエンス（QB3）のためのカリフォルニア工科大学から研究賞によってサポートされていました学部早期キャリア開発（CAREER）プログラム（DBI-1253293）。

1. SU8マスター製作<ol><li> AutoCADのソフトウェアを使用して2つの層の製造のためのマイクロ流体構造を設計し、10μmの分解能で回路基板フィルム上に、ベンダーによって印刷されたデザインを持っています。デバイスの設計の詳細は、添付の参考文献11に記載されているチャネルの形状は、 図1に示されている。層は、各加工層12の機能を連結するために役立つ位置合わせマークを含むべきです。 </li><li>スピンコーターに予め清浄3インチ径のシリコンウエハを置き、チャックに固定する真空をオンにします。 50μmの層厚を提供し、2000rpmで、30秒間、500rpmで20秒間ウェハと回転の中心にSU8-3035を1ml適用します。 </li><li>ウェハを取り外し、30分間135℃のホットプレート上で焼きます。ウエハは次のステップに移動する前に室温に冷却してください。 </li><li> 第 1 層マスク（ 図2Aに塗布されたウェハを公開</stronG&gt;）90秒間LED nmのコリメート190ミリワット、365の下。露光後、次のステップに進む前に、室温まで冷却、1分間135℃のホットプレート上でウェーハを配置します。 </li><li>スピンコーターにウェハを置き、チャックに固定する真空をオンにします。 135ミクロンの厚さの追加を提供する層で、その結果、1,375 rpmで、30秒、500 rpmで20秒間ウェハとスピンの中心にSU8-2050の1ミリリットルを適用します。 </li><li>次のステップに移動する前に、室温まで冷却後、ウエハを取り出して、30分間135℃のホットプレート上で焼きます。 </li><li> 1.3にパターニングジオメトリ上に第 2 層マスク（ 図2B）を合わせ、3分間のLED NMコリメート190ミリワット、365に塗布されたウェハを公開します。暴露した後、1分間135℃のホットプレート上の場所は、次のステップに進む前に、室温まで冷却。 </li><li> 30分間のプロピレングリコールモノメチルエーテルアセテートの撹拌浴中に浸漬することによりマスク​​を開発します。ウェハを洗浄1分間135℃のホットプレート上でイソプロパノール及びベークインチPDMS成形用の100ミリメートルペトリ皿に開発されたマスターを配置します。 </li></ol> 2. PDMSデバイスの製造<ol><li>プラスチックカップに硬化剤を5gとシリコーンベースの50グラムを組み合わせることにより1 PDMS：10を準備します。攪拌棒を装着した回転工具で内容を混ぜます。 30分間、デシケータ内部に、または全ての気泡が除去されるまで、混合物を脱気。 </li><li>マスターの上に3mmの厚さを与え、さらにガス抜きのために戻ってデシケーターに配置するために、PDMSを注ぎます。一旦全ての気泡が除去され、2時間60℃でデバイスを焼きます。 </li><li>パターン化された面を上にして清浄な表面上のメスと場所を使用して金型からデバイスをカットします。マスター2（ 図3a）からマスター1を分離するためにかみそりの刃で半分にPDMSモールドをカットします。マスター1によって刻印さ50μmの流体ハンドリングジオメトリを含む片に、0.7との流体入口および出口をパンチ5ミリメートルの生検パンチ。 </li><li>プラズマは、300Wのプラズマクリーナーで60秒間、1ミリバールのO 2プラズマでデバイスを扱います。一時的にPDMS-PDMS結合を遅らせると、潤滑剤として機能するDI水の滴でPDMSの非パンチ片の表面を濡らし。マスター2面にステレオ顕微鏡、場所のマスター1を介して表示し、機械的なロックが達成されるまで、比較的表面をスライドさせながら時凹んフレームと図3Aメイトで突出フレーム。 </li><li> 60℃のオーブン内で装置を配置し、水で完全な結合を蒸発させ、60℃で2日間組み立てられた装置（ 図3B）を焼きます。 </li></ol>試薬の調製<ol><li>内相のための蒸留水で1mlシリンジを埋めます。 </li><li> 1重量％とHFE 7500フッ素系オイルで1 mlシリンジを埋めます。中間相のための生体適合性界面活性剤％の界面活性剤13。 </li><li> 10重量％で10 mlシリンジを埋めます。 ％polyethylen1重量％を含有する水溶液で電子グリコール（PEG）。 ％のTween 20および1重量連続相の％ドデシル硫酸ナトリウム。 </li></ol> 4.システムの準備<ol><li> &lt;100μ秒のシャッター速度が可能、デジタルカメラと接続された倒立顕微鏡のステージ上にマイクロ流体チップを置きます。 </li><li>シリンジポンプのすべての注射器を取り付け、27 Gの針を取り付けます。針の上に、PE-2チューブの約30センチの長さを接続し、適切に緩い端を挿入し、デバイスに穴をパンチ。 </li><li>デバイスの出口ポートにPE-2の10cmの長さを挿入し、廃棄物収集容器内のもう一方の端を置きます。 </li><li>プライム管セグメント内の流体が装置の入口ポートに到達するまでの速度率の高さ（2000μL/分）でシリンジポンプを実行して、デバイス。 </li></ol> 5.エマルジョンの生成<ol><li> 50ミクロン×50ミクロンのオリフィスを含んでおり、地域に顕微鏡をフォーカス下流の出口チャネル。 </li><li>連続相250μL/内部相のための時間、100μL/中間相のための時間、および700μL/ hrの流量で二重エマルジョン発生器に流体を提供し、平衡化のために10分を待つためにシリンジポンプを設定します。 </li><li>それぞれ、250μL/ hrであり、100μL/時間で、内側と中間相の流量を維持します。 1,050μL/ hrで外側相の流量を設定します。流れ条件のセットの下で安定させるためのダブルエマルションの生成のための3-5分待ってください。 </li><li>手動画像分析によるオフライン処理のために30 Hzでビデオ画像の5秒を取得します。 </li><li> 表1に示す流量の5.3および5.4を繰り返す。内側及び中間相の流量が一定に保持され、搬送波位相流量は、シリンジポンプの設定を調整することによって変化します。 </li></ol>

<ol>
	<li>Van Der Graaf, S., Schro&#235;n, C. G. P. H., Boom, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+double+emulsions+by+membrane+emulsification+-+A+review.">Preparation of double emulsions by membrane emulsification - A review.</a> J. Membrane Sci. 251 (1-2), 7-15 (2005).</li><li>Laugel, C., Baillet, A. P., Youenang Piemi, M., Marty, J., Ferrier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oil-water-oil+multiple+emulsions+for+prolonged+delivery+of+hydrocortisone+after+topical+application:+comparison+with+simple+emulsions.">Oil-water-oil multiple emulsions for prolonged delivery of hydrocortisone after topical application: comparison with simple emulsions.</a> Int. J. Pharm. 160 (1), 109-117 (1998).</li><li>Kim, S. H., Kim, J. W., Cho, J. C., Weitz, D. 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The authors have nothing to disclose.

ここで説明する二重エマルジ ​​ョン生成ジオメトリはガラスキャピラリー装置8の物理学を模倣するように設計されています。これらでは、位置合わせ円筒形ガラスキャピラリーは、均一な二重エマルジョン液滴に剪断された3相の同軸噴流を生成するために使用されます。当社の3D PDMSデバイスの機能は、全体の高さは320ミクロンであり、キャリア位相チャンネルと50μmの背の高い?...

<html>ダブルエマルションは、それらの工業的、医薬における潜在的な用途、及び生物学的用途1中間、非混和性流体層によりキャリア相から分離した小滴からなり、特に重要です。いくつかの例では、ダブルエマルションのコア内の高価値な化合物をカプセル化する能力は、材料を保護し、制御された様式で放出されることを可能にします。例えば、薬物は、外部のキャリア流体2には適していない溶解性条件下でカプセル化することができます。また、中間油層は、薬物、化粧品、および栄養3のカプセル化及び送達のためのカプセルのテンプレートとして使用することができます。これらは、サブナノリットル実験の膨大な数を行うことができるため、生物学において、ダブルエマルションは、その後、検出され、（FACS）装置4,5を蛍光活性化細胞選別を用いて選別、ハイスループットスクリーニングにも有用です。 ENT &quot;&gt;所望の性能特性を有するダブルエマルションの設計は二重エマルジョンサイズ、組成、及び均一性の精密な制御を必要とする。そのような膜乳化などのバルク、乳化プロセスは、業界で使用されているが、得られたエマルジョンは非常に多分散であり、展示機能特性1の多種多様。液滴マイクロフルイディクスのフィールドは自然に慎重に制御組成物6を単分散エマルションの生成を適しています。マイクロ流体ダブルエマルションの生成は、次の2つの主要な戦略、シーケンシャルドロップ作り、ガラス毛細管流動焦点を実現しています。ダブルエマルションことができます処理を行う二段階のドロップを使用して平坦なPDMSデバイスで生成される。第一に、水性の油エマルジョンは、疎水性チャンネル壁と装置の油中水型のドロップ作る領域を用いて作成される。次に、エマルジョンであることができます流しや水の中に油に適した親水性の壁とドロップ作る領域に再注入ドロップすること4。しかし、PMDSの親水性表面処理は、付加的な製造工程を必要とし、多くの場合、一時的な7です。ダブルエマルションを形成するほとんどの制御可能かつ再現方法は、同軸流が集束することによる、技術は、三相を含む同心ジェットは単分散液滴8を生成するために 、小さなオリフィスを通して剪断されるガラスキャピラリーマイクロフルイディクスを使用して開拓しました。この技術は、各相の流量の関数であるダブルエマルションの正確なサイズおよび組成で、チャネルの寸法よりもはるかに小さい液滴の生成を可能にします。滴とチャネルサイズおよび保護外側シースフローとの間の大きな違いは、不必要な表面処理をレンダリングする、チャネル壁に接触する液滴を防ぎます。しかし、そのようなガラスデバイスは、慎重な組立とシールと一緒に、テーパーキャピラリ先端のカスタム製造を必要とします。前の研究者は、3Dソフトリソグラフィを使用していましたグラフィーは、物理学を中心に流れを使用してダブルエマルションを生成するが、これらのデバイスは、直径&gt; 150μmの9,10、通常、FACSでソートしたオブジェクトよりも大きい大きさの約順序でエマルジ ​​ョンを生成しました。魅力的な代替手段は、PDMSソフトリソグラフィの製造の容易さとピントガラスキャピラリー同軸流れの堅牢な機能と小滴の生成を含むことになります。 本稿では、50ミクロンエマルジ ​​ョン≤生成するために焦点を当て同軸フローを使用し、完全に3Dソフトリソグラフィー11を用いて構成されている二重エマルジ ​​ョンジェネレータについて説明します。我々のデバイスは、引っ張らガラスキャピラリーノズルにおけるエマルションの形成過程を近似するために小さなせん断流路（ 図1）を含むデバイスを製造するためにクラムシェル方式を使用します。さらに重要なことは、これらのデバイスは、特定の表面処理を必要とせず、すべてのポリマー構造が簡単で再現可能な製造を提供するSC重複した多数のデバイスにalable。ここでは、二重エマルジョン発電機の設計、製造、テストの概要を説明します。ダブルエマルションの生成は14ミクロンの液滴直径まで、堅牢かつ再現性であることが示されています。製造の容易さと機能性の結合は、このデバイスの新しい二重エマルジョンアプリケーションの開発のための魅力的なオプションになります。

<table border="0" cellpadding="0" cellspacing="0" width="691">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Photomasks</td>
			<td style="width:135px;">CadArt Servcies</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">3&#34; silicon wafers, P type, virgin test grade</td>
			<td style="width:135px;">University Wafers</td>
			<td style="width:119px;">447</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">SU-8 3035</td>
			<td style="width:135px;">Microchem</td>
			<td style="width:119px;">Y311074</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">SU-8 2050</td>
			<td style="width:135px;">Microchem</td>
			<td style="width:119px;">Y111072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Sylgard 184 silicone elastomer kit</td>
			<td style="width:135px;">Krayden</td>
			<td style="width:119px;">4019862</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">1 ml syringes</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">309628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">10 ml syringes</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">309604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">27 gaugue needles</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">305109</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">PE 2 polyethylene tubing</td>
			<td style="width:135px;">Scientific Commodities, Inc.</td>
			<td style="width:119px;">B31695-PE/2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Novec 7500</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">98-0212-2928-5</td>
			<td>Commonly knowns as HFE 7500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Biocompatable surfactant</td>
			<td style="width:135px;">Ran Biotechnologies</td>
			<td style="width:119px;">008-FluoroSurfactant</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">35,000 MW PEG</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">1546660</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Tween 20</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">P1369</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Sodium dodecyl sulfate&#160;</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">L3771</td>
			<td></td>
		</tr>
	</tbody>
</table>

double emulsion generation using a polydimethylsiloxane (pdms) co-axial flow focus device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

ダブルエマルションの生成器は、3D PDMSの製造（ 図1A）を使用して作成したデバイスの焦点を合わせる同軸流から成ります。ジオメトリは、三相の同軸噴流の形成は、水/油/水ダブルエマルション（ 図1B、 図1C）の形成を可能にする、正方形、×50ミクロンのオリフィス50ミクロンに剪断することが可能になります。内水相及び中間?...

Watch this Scientific Journal Video about Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device at JoVE.com

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

ポリジメチルシロキサン（PDMS）同軸フローフォーカス・デバイスを使用したダブルエマルションの生成

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

double-emulsion-generation-using-polydimethylsiloxane-pdms-co-axial

Research

JoVE Journal

Bioengineering

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

15.9K ビュー.  University of California, San Francisco. Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 µm.

ビデオ: Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

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 ...

生物工学

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

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 ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

マイクロ流体ジェッティングを使用して、脂質二重層小胞の生成

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

ポリジメチルシロキサン薄膜を用いた自動化された脂質二重膜の形成

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

マイクロ コント ローラーは従来のタバコの煙、電子たばこエアロゾル液体抽出物の世代のデバイスを運営

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

極薄ポリジメチルシロキサン マイクロ流体チップを用いた保水ライブ線虫個人の固定化

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Generation of Transgenic Rats using a Lentiviral Vector Approach

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is an invaluable tool for gene function analysis in living organisms. The standard transgenesis method is based on microinjecting foreign DNA fragments into a pronucleus of a fertilized oocyte. This technique is widely used in mice but remains relatively inefficient and technically demanding in other animal species. The transgene can also be introduced into one-cell-stage embryos via lentiviral infection, providing an effective alternative to standard pronuclear injections, especially in species or strains with a more challenging embryo structure. In this approach, a suspension that contains lentiviral vectors is injected into the perivitelline space of a fertilized rat embryo, which is technically less demanding and has a higher success rate. Lentiviral vectors were shown to efficiently incorporate the transgene into the genome to determine the generation of stable transgenic lines. Despite some limitations (e.g., Biosafety Level 2 requirements, DNA fragment size limits), lentiviral transgenesis is a rapid and efficient transgenesis method. Additionally, using female rats that are mated with a fertile male strain with a different dominant fur color is presented as an alternative to generate pseudopregnant foster mothers.

This article aims to provide the methodology for lentiviral transgenesis in rat embryos using multiple injections of a virus suspension into the zygote perivitelline space. Female rats that are mated with a fertile male strain with a different dominant fur color is used to generate pseudopregnant foster mothers.

レンチウイルスベクターアプローチによるトランスジェニックラットの生成

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

ハイスループットマイクロ流体装置を用いた動的環境条件の生成

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Contrast-Enhanced Subharmonic Aided Pressure Estimation (SHAPE) Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound imaging are gas-filled, encapsulated microbubbles (diameter &#60; 10 &#956;m) that traverse the entire vasculature and enhance signals by up to 30 dB. These microbubbles also produce nonlinear oscillations at frequencies ranging from the subharmonic (half of the transmit frequency) to higher harmonics. The subharmonic amplitude has an inverse linear relationship with the ambient hydrostatic pressure. Here an ultrasound system capable of performing real-time, subharmonic aided pressure estimation (SHAPE) is presented. During ultrasound contrast agent infusion, an algorithm for optimizing acoustic outputs is activated. Following this calibration, subharmonic microbubble signals (i.e., SHAPE) have the highest sensitivity to pressure changes and can be used to noninvasively quantify pressure. The utility of the SHAPE procedure for identifying portal hypertension in the liver is the emphasis here, but the technique has applicability across many clinical scenarios.

Contrast-Enhanced Subharmonic Aided Pressure Estimation SHAPE Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

A protocol for noninvasively estimating ambient pressures utilizing subharmonic ultrasound imaging of infused contrast microbubbles (following appropriate calibration) is described with examples from human patients with chronic liver disease.

門脈圧亢進症の同定に着目した超音波画像を用いた造影補助圧力推定(SHAPE)

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound ...