This work was supported by grants from the National Institutes of Health, including a microphysiological systems consortium grant from the National Center for Advancing Translational Sciences (UH2TR000503), a Ruth L. Kirschstein National Research Service Award Postdoctoral Fellowship (F32DK098905 for WJM) and pathway to independence award (DK095984 for AB) from the National Institute of Diabetes and Digestive and Kidney Diseases.

ネイティブ可溶性コラーゲン溶液の調製<ol><li>準備または私は1-3ミリグラム/などPiez らによって報告され、標準の単離プロトコルを使用してML 15でラットの尾からコラーゲン酸性化し、可溶性、タイプの200 mgの購入</li><li>修正されたコラーゲン溶液の所望の最終容積に基づいて、出発物質の量をスケーリングします。約可溶性天然コラーゲンを200mgから、3 mg / mlの時に25〜30のメチル化のml及びスクシニルコラーゲン溶液の25〜30ミリリットル、それぞれを行います。 </li></ol> 2.コラーゲンのメチル化<ol><li>氷冷たい滅菌水でミリリットルの0.5mg /の濃度にネイティブ、酸性化（pHは2-3）コラーゲン溶液100mgを希釈し、ゲル化を防止するために氷の上にソリューションを続けます。 </li><li> 1 N NaOHを数滴の9-10のコラーゲン溶液のpHを調整し、室温で30分間攪拌します。白濁するために溶液を引き起こし、コラーゲン沈殿物を観察します。 </lI&gt; <li> 25分間3000×gで沈殿したコラーゲン溶液をスピンダウン。明確な、ゲル状の沈殿物がチューブの底に表示されるはずです。吸引し、適切に上澄み液を廃棄。 </li><li> 0.1 N HClで200mlのメタノールで沈殿コラーゲンを再懸濁し、メチル化反応を4日間室温で撹拌しながら発生することを可能にします。コラーゲンは溶解しないが、解決策は曇りになります非常に小さく見える片にばらばらにする必要があります。 </li><li>メチル化した後、遠心分離器25分間3000×gでソリューションは、メチル化コラーゲンを沈殿させます。吸引除去は、酸性化メタノール上清を処分。 </li><li>繰り返しのピペッティングで約3 mg / mlでの濃度を与える、無菌PBS 25ml中のメチル化コラーゲンを溶解し、60μmの細胞ストレーナーを通して溶液を濾過します。 1 N NaOHを20μlの増分を用いて7.3から7.4への溶液のpHを調整します。 </li><li> concentratioを評価商業ラットコラーゲンELISAキットまたはヒドロキシプロリンアッセイキットを使用して溶液をN。 3ミリグラム/ミリリットル滅菌PBSでの解決策を希釈します。 </li><li>慎重にボトルの底にクロロホルム3mlのを積層、スクリューキャップ付きガラス瓶に移すことにより、メチル化コラーゲン溶液を滅菌、ボトルを4℃でO / Nを設定し、無菌的に除去して保存することを可能にしますメチル化コラーゲンであるトップ層、。 </li><li> 1ヶ月以内に使用するために4℃で溶液を保管してください。 </li></ol> 3.コラーゲンスクシニル<ol><li>氷冷たい滅菌水でミリリットルの0.5mg /の濃度にネイティブ、酸性化（pHは2-3）コラーゲン溶液の他の100ミリグラムを希釈し、ゲル化を防止するために氷の上にソリューションを続けます。 </li><li> 1 N NaOHを数滴の9-10のコラーゲン溶液のpHを調整し、室温で30分間攪拌します。コラーゲンは白濁するソリューションを引き起こして、沈殿させる必要があります。 </li><li> SUCの40 mgの溶かします（コラーゲン溶液の体積番目の 1/20）をアセトン10ml中cinic無水物（コラーゲン1mg当たり0.4ミリグラム）。ゆっくりと（約0.5ミリリットルずつ）を連続的にpHをモニターしながら、攪拌しながら、コラーゲン溶液に、この混合物を追加します。 pHが9.0に近づくにつれて1 NのNaOHの1または2滴を添加することにより9.0を超えるpHを維持します。 </li><li>アセトン中の無水コハク酸のすべてを追加した後、120分間室温で撹拌し続けます。スクシニル化コラーゲンが溶解するよう明確になるために解決策を確認します。定期的にそれが9.0を超えたまま確保するためのpHを確認してください。 </li><li> 1 N HClを20μlの刻みを4.0に溶液のpHを調整します。スクシニル化コラーゲン沈殿物として、再び濁った溶液を観察します。 </li><li>スクシニル化した後、スクシニル化コラーゲンをペレット化するために25分間3000×gで遠心分離ソリューション。未反応の無水コハク酸を用いて酸性化上清を吸引除去し、廃棄してください。 </li><li>スクシニル化コラーゲンを溶かします無菌PBSの25ミリリットル、60ミクロンセルストレーナーを通して溶液をピペッティングを繰り返すと、ミリリットル/約3ミリグラムの濃度を与え、フィルターインチ1 N NaOHを20μlの増分を用いて7.3から7.4への溶液のpHを調整します。 </li><li>商業コラーゲンラットELISAキットまたはヒドロキシプロリンアッセイキットを用いて溶液の濃度を評価します。 3ミリグラム/ミリリットル滅菌PBSでの解決策を希釈します。 </li><li>慎重にボトルの底にクロロホルム3mlのを積層、スクリューキャップ付きガラス瓶に移すことによって、スクシニル化コラーゲン溶液を滅菌、ボトルを4℃でO / Nを設定し、無菌的に除去して保存することを可能にしますスクシニル化コラーゲンであるトップ層、。 </li><li> 1ヶ月以内に使用するために4℃で溶液を保管してください。 </li></ol>コラーゲン修正4.検証<ol><li> 、ネイティブのメチル化、およびスクシニル化コラーゲン溶液のそれぞれから1ミリリットルのサンプルを準備し、濃に希釈します水素イオン滴定のための滅菌純水でミリリットルを0.1mg /のentration。また、少なくとも4時間ごとに、10kDaのカットオフmembraneusing 3溶液の変化を介して水に対して透析によってバッファ、少なくとも1000倍に希釈。 </li><li> NaOHおよびHClを少量の7.3に溶液のpHを調整します。天然のメチル化、およびスクシニル化コラーゲン溶液のための滴定曲線を作成するために、タンフォード16で説明したように任意の基準として、pHが7.3を使用して、試料のそれぞれに水素イオン滴定を行います。 </li><li>バインドされたH +分子当たりのイオン数に対する酸の添加量あたりのpHの変化をプロットします。高pH「アミノ」の範囲は、中性点に向かってスクシニル化コラーゲンのシフト（アミン基の損失）を示すべきであり、低pH、「カルボキシル」の範囲は、メチル化コラーゲン中左シフト（カルボキシル基の損失を示す必要があります）およびスクシニル化コラーゲンの右シフト（カルボキシル基のゲイン秒）、天然コラーゲンと比較しました。 </li><li>標準的なプロトコール17,18以下、2,4,6-トリニトロベンゼンスルホン酸（TNBA）比色法を用いて、スクシニル化によって置き換え天然コラーゲンでのアミノ基の％を決定することによって、サクシニル化反応の有効性を評価します。 </li></ol> 5.マイクロ流体デバイスの作製と細胞播種<ol><li>標準的な方法9を使用して 、マイクロ流体デバイスを製造。 100μmで、背の高い0.4〜1.5ミリメートル幅の広い、および細胞増殖のための長いチャンネル1〜10ミリメートルで、マイクロ流体細胞培養室を作成するために、フォトリソグラフィを使用して定義されたシリコン上にSU-8マスターからPDMSのレプリカ成形を使​​用してください。 </li><li>デバイスとスライドガラスの表面を酸化するためにプラズマクリーナーを使用し、さらに結合を一緒に押します。少なくとも30分間、UV光に露光することによって、デバイスを滅菌した後、滅菌PBS中の50μg/ mlのフィブロネクチンでチャンバを充填し、45分間37℃でインキュベートします。 </li&gt; <li>表現型または分化状態の安定化のためのコラーゲンゲルを必要とするような初代ラットやヒト肝細胞などの細胞、を備えたデバイスをシード。私たちは、デバイスあたり14×10 6細胞/ mlで播種新たに単離した初代ラット肝細胞7,19または市販の凍結保存された初代ヒト肝細胞の20μLを使用しています。 </li><li>細胞は4-6時間アタッチした後、付着していない細胞を洗い流すと、増殖培地でメッキメディアを交換できるようにします。拡散フルセルを確保し、細胞のコンフルエントな単層を作成するために、O / Nインキュベートします。 </li></ol> 6.レイヤー・バイ・レイヤーコラーゲン沈着<ol><li>層流の組織培養フードでは、デバイスごとに各溶液の10アプリケーションのためのメチル化およびスクシニル化コラーゲン溶液の十分なボリュームだけでなく、メディアのいくつかのMLSを準備します。氷の上で解決策をしてください。私たちは、デバイスごとに1層当たりのコラーゲンの20μlの（10〜15回総デバイス・ボリューム）を使用します。 </li><li>うメチル化された20μlのデバイスをフラッシュメチル（ポリカチオン）溶液に、代替と綿繰りし、各アプリケーションとの間で1分を待って、コラーゲン溶液をスクシニル。デバイスに約20分を取る必要があります解決策につき10回、の合計をフラッシュします。細胞は、培地なしている時間の量を最小にするためにすぐに働きます。 </li><li>ゆっくりとその大きさに応じて、入口/出口に蓄積コラーゲンを守ってください。流体の流れが増加に対する耐性は、メディアに一度か二度のデバイスをフラッシュし、場合には、階層化を続けます。 </li><li>全ての層を適用した後、新鮮な培地で二回デバイスをすすぎ、インキュベーターに戻します。細胞の種類に応じて、そのような肝細胞で強化された偏光のような細胞外マトリックスにより誘導される形態学的変化は、数時間以内に表示されるはずです。 </li><li>コラーゲンマトリックスの存在を確認するために、標準的な方法9を用いて 、透過型電子顕微鏡のための代表的な装置を準備培養細胞の上に組み立て。 </li></ol>細胞の表現型および機能の安定化7 <ol><li>画像の細胞型のための標準的な方法を用いて細胞の形態、生存率、および極性。肝細胞は、コラーゲン沈着の効果を実証するために、頂端の偏光に対する位相顕微鏡、生存のためにLIVE / DEAD染色し、CMFDA色素を使用して、14日間に渡って画像を収集。 </li><li>細胞形態学のために、リンス20μlのPBS、及び画像位相差顕微鏡を使用してデバイスを注入するためにPBS20μlをピペットで注入口を介してデバイスを洗い流します。 </li><li> LIVE / DEAD染色のために、すすぎ、製造元の指示に従って調製し、DAPIでLIVE / DEAD染色溶液20μlを注入する20μlのPBSでピペットで注入口を介してデバイスをフラッシュし、37℃で30分間インキュベートし、すすぎデバイスを再び20μlのPBS、及び画像蛍光顕微鏡を用いた装置です。 </li><li>胆汁カナリについてCuLi等の染色、DAPIで2μMCMFDA染色溶液20μlを注入、すすぐために20μlのPBSをピペットで注入口を介してデバイスをフラッシュするには、製造元の指示に従って調製し、37℃で30分間インキュベートし、再びデバイスをすすぎます20μlのPBS、及び画像蛍光顕微鏡を使用してデバイス。 </li><li>使用済み培地を収集し、細胞機能を決定するための培養期間にわたって、適切な時点での細胞代謝産物を測定します。肝細胞の場合は、使用済み培地中のアルブミンおよび尿素の量を測定します。 </li><li>このような酵素活性レベル、抗体染色、またはRNA分析を評価するように細胞自体上のエンドポイント機能解析を実行します。肝細胞は、誘導し、シトクロムP450酵素活性または第II相抱合酵素グルタチオンSトランスフェラーゼを測定します。 </li></ol>

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The authors have nothing to disclose.

極薄純粋なコラーゲンアセンブリが​​変更されたコラーゲンのレイヤーバイレイヤー堆積を用いて帯電した細胞または材料表面上に堆積することができます。この研究の結果は、メチル化及び天然コラーゲンのスクシニル化は、細胞（ 図2）上の極薄コラーゲンマトリックスアセンブリを堆積させるために層ごとの手法で使用することができる（ 図1）または他の?...

Although microfluidics allows for the exquisite control of the cellular microenvironment, culturing cells, especially primary cells, within microfluidic devices can be challenging. Many traditional cell culture techniques have been developed to sustain and stabilize cell function when cultured in tissue culture plates, but translating those techniques to microfluidic devices is often difficult. 
One such technique is the culture of cells on or sandwiched between collagen gels as a model of the physiological 3D cell environment.1 Type I collagen is one of the most frequently used proteins for biomaterials applications because of its ubiquity in extracellular matrix, natural abundance, robust cell attachment sites, and biocompatibility.2 Many cells benefit from 3D culture with collagen, including cancer cells3,45, microvascular endothelial cells6, and hepatocytes7, among others. While the use of collagen gels is easy in open formats, such as tissue culture plates, the limited channel dimensions and enclosed nature of microfluidic devices makes the use of liquids that gel impractical without blocking the entire channel.
To overcome this problem, we combined the layer-by-layer deposition technique8 with chemical modifications of native collagen solutions to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These layers can stabilize cell morphology and function similar to collagen gels and can be deposited on cells in microfluidic devices without blocking the channels with polymerized matrix. The goal of this method is to modify native collagen to create polycationic and polyanionic collagen solutions and to stabilize cells in microfluidic culture by depositing thin collagen matrix assemblies onto the cells. This technique has been used to stabilize the morphology and function of primary hepatocytes in microfluidic devices.9
Although layer-by-layer deposition has previously been reported with natural and synthetic polyelectrolytes10 to cover hepatocytes in plate culture11,12 and as a seeding layer for hepatocytes in microfluidic devices13,14, this method describes the deposition of a pure collagen layer on top of hepatocytes, mimicking the 3D collagen culture techniques. In this protocol, we use hepatocytes as example cells that can be maintained using 3D collagen layers. The many other types of cells that benefit from 3D culture in collagen may similarly benefit from culture after layer-by-layer deposition of an ultrathin collagen matrix assembly.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>collagen type I, rat tail</td>
			<td>Life Technologies</td>
			<td>A1048301</td>
			<td>option for concentrated rat tail collagen</td>
		</tr>
		<tr>
			<td>collagen type I, rat tail</td>
			<td>Sigma-Aldrich</td>
			<td>C3867-1VL</td>
			<td>option for concentrated rat tail collagen</td>
		</tr>
		<tr>
			<td>collagen type I, rat tail</td>
			<td>EMD Millipore</td>
			<td>08-115</td>
			<td>option for concentrated rat tail collagen</td>
		</tr>
		<tr>
			<td>collagen type I, rat tail</td>
			<td>R%D Systems</td>
			<td>3440-100-01</td>
			<td>option for concentrated rat tail collagen</td>
		</tr>
		<tr>
			<td>succinic anhydride</td>
			<td>Sigma-Aldrich</td>
			<td>239690-50G</td>
			<td>succinylation reagent</td>
		</tr>
		<tr>
			<td>anhydrous methanol</td>
			<td>Sigma-Aldrich</td>
			<td>322415-100ML</td>
			<td>methylation reagent</td>
		</tr>
		<tr>
			<td>sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S5881-500G</td>
			<td>pH precipitation reagent</td>
		</tr>
		<tr>
			<td>hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>320331-500ML</td>
			<td>pH precipitation reagent</td>
		</tr>
		<tr>
			<td>rat collagen type I ELISA</td>
			<td>Chondrex</td>
			<td>6013</td>
			<td>option for detecting collagen content</td>
		</tr>
		<tr>
			<td>hydroxyproline assay kit</td>
			<td>Sigma-Aldrich</td>
			<td>MAK008-1KT</td>
			<td>option for detecting collagen content</td>
		</tr>
		<tr>
			<td>hydroxyproline assay kit</td>
			<td>Quickzyme Biosciences</td>
			<td>QZBtotcol1</td>
			<td>option for detecting collagen content</td>
		</tr>
	</tbody>
</table>

layer-by-layer collagen deposition in microfluidic devices for microtissue stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

天然コラーゲンは、層ごとの堆積に使用するためのポリカチオンおよびポリアニオンコラーゲン溶液を作成するために、メチル化及びスクシニルを使用して変更することができます。サクシニル化は、スクシニル基を有する天然コラーゲンのεアミノ基を変更して、メチル化は、メチル基（ 図1A）と天然コラーゲンのカルボキシル基を修飾します。コラーゲンタンパク質のアミノ...

Watch this Scientific Journal Video about Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization at JoVE.com

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Microtissue安定化のためのマイクロ流体デバイスの層ごとのコラーゲン沈着

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

layer-layer-collagen-deposition-microfluidic-devices-for-microtissue

Research

JoVE Journal

Bioengineering

9.3K ビュー.  Massachusetts General Hospital, Harvard Medical School. The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

ビデオ: Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

誘電泳動と層流を用いたマルチチャネルマイクロ流体デバイスの分離ビーズと細胞

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

生物工学

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

のためのシンプルなマイクロ流体デバイス<em生体内で&gt;</emの&gt;イメージング<em&gt; Cエレガンス</em&gt;<em&gt;ショウジョウバエ</em&gt;とゼブラフィッシュ

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

エンジニアリング3D細胞化血管組織再生のためのコラーゲンゲル

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

マイクロパターン血管筋薄膜の拡張文化ためのマイクロ流体ゲニピン堆積技術

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

3Dペーパーマイクロ流体デバイスを構築するために接着剤パターニングを使用して、

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

シングルまたは共存化学/電気/せん断応力刺激下で細胞応答を研究するためのマイクロ流体デバイスを設計

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

バルブ付き多層マイクロ流体デバイスのための多段階可変高さのフォトリソグラフィ

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

イムノアッセイのための3次元紙ベースのマイクロ流体デバイスの作製

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under combinations of chemical and oxygen gradients. Both chemical and oxygen gradients can greatly affect cell migration in vivo; however, due to technical limitations, very little research has been performed to investigate their effects in vitro. The device developed in this research takes advantage of a series of serpentine-shaped channels to generate the desired chemical gradients and exploits a spatially confined chemical reaction method for oxygen gradient generation. The directions of the chemical and oxygen gradients are perpendicular to each other to enable straightforward migration result interpretation. In order to efficiently generate the oxygen gradients with minimal chemical consumption, the embedded PC thin film is utilized as a gas diffusion barrier. The developed microfluidic device can be actuated by syringe pumps and placed into a conventional cell incubator during cell migration experiments to allow for setup simplification and optimized cell culture conditions. In cell experiments, we used the device to study migrations of adenocarcinomic human alveolar basal epithelial cells, A549, under combinations of chemokine (stromal cell-derived factor, SDF-1&#945;) and oxygen gradients. The experimental results show that the device can stably generate perpendicular chemokine and oxygen gradients and is compatible with cells. The migration study results indicate that oxygen gradients may play an essential role in guiding cell migration, and cellular behavior under combinations of gradients cannot be predicted from those under single gradients. The device provides a powerful and practical tool for researchers to study interactions between chemical and oxygen gradients in cell culture, which can promote better cell migration studies in more in vivo-like microenvironments.

The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.

垂直化学的酸素勾配の下で細胞移動研究のためのポリジメチルシロキサン - ポリカーボネートマイクロ流体デバイス

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

血小板輸血や止血のためのマイクロ流体フローチャンバーモデルは、リアルタイムでの血小板沈着およびフィブリン形成を測定します

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...