この作品は、キュリー研究所、パリ、フランスで設立されました。機械式ストレッチャはダミアンCuvelier（キュリー研究所）によって設計され、GREM（メカニック-grem.com）によって製造されている。 PDMS上のパターニングはアマルAzioune（ボルドーII大学）によって開発されました。

1。 PDMSの不動態化<ol><li>あらかじめ作られたシート（材料の表に示すように、たとえば、GelPak、）からのX 20ミリメートルのPDMSが約35ミリメートルの部分をカット。 </li><li>プラスチックの上部と下部の保護層を除去（必要な場合）、ペトリ皿（処理した培養を細胞ではない）は、プラスチックに、PDMSを配置するためにピンセットを使用しています。 </li><li> /分30振動で回転装置上で5分間、70％エタノールで、PDMSを洗ってください。 </li><li>その上に空気を流すことにより、表面を乾燥させます。 </li><li>約5cmのUVバルブからの距離で5分間、深紫外（λ= 180 nm）をして点灯（ランプリファレンスのための材料のシートを参照してください。パラメータは、異なるランプに異なります）。 </li><li>各PDMSピース用EDC / NHS溶液200μlを準備し（溶液の反応性が時間のうちに崩壊するので、これは使用直前に調製しなければならない）。 pH6.0の0.05MのMES + 0.5 M NaCl緩衝液の1ミリリットルの場合は、スルホ-NHSの11.5 mgおよびEDCを19.2ミリグラムを追加します。 </li><li>転送照らされていないペトリ皿の蓋にペトリ皿からのPDMSシート。これはPDMSの周囲が非常に疎水性であることを確認して、次のステップを容易にするであろう。 </li><li> EDC / NHS溶液を添加し、室温で15分間インキュベートする。 </li><li>水とEDC / NHSを洗浄します。 </li><li> PLL-gの PEG-溶液（10mMのHEPES pHが8.6で0.5 mg / ml）を添加し、室温で一晩に3時間のインキュベート。 </li><li>水でPLL-G-PEGを洗浄します。 （PLL-gの -PEGで官能化された）不動態化されたPDMSは、4℃で数日間保存することができる</li></ol> 2。 PDMSのパターン化<ol><li> PDMSシートを取り、パターニング微小特徴を有する合成石英フォトマスクの上に置き（ 図1を参照）。フォトマスクのクロム側にPLL-G-PEGをもつPDMS側に配置します。 </li><li>約5cmのUVバルブから距離を置いて、フォトマスクを介して7分間点灯します。</li><li>マスク+ PDMS上に水を加え、徐々にマスクオフPDMSを剥離。 </li><li>室温で1時間、pH8.6のHEPESで緩衝液中に50 mg / mlのフィブロネクチン溶液でインキュベートする。それは、他のECMタンパク質を使用することが可能であるが、これらはこのプロトコルで試験されていない。 </li><li> PBSですすいでください。 </li></ol> 3。デバイスの取り付け<ol><li>ストレッチングデバイスに以前に不動態化されたPDMSをマウント（トラブルシューティングのための議論を参照）。 <ol><li>ストレッチャの固定部分にPDMSの1側を接続してください。 </li><li> PDMSシートのあまりをクランプすることなく、ストレッチャの可動部分に反対側を固定します。 </li></ol></li><li>厚手のPDMS刺しにPDMSの長方形（22ミリメートルX 19ミリメートル）をカットします（ 図2を参照）細胞および培地を保持し、プール（またはフレーム）を持つために内部に別の四角形を切った。 </li><li>この下にシリコーングリースを追加し、プールをPDMSおよびMEDを作成するために、PDMSシートの上部に配置しますイウムは、プールを保持。グリースは、延伸時のPDMSシート上のプールの滑空が可能になります。 </li></ol> 4。細胞のパターニング<ol><li>ベルセンと50％の集密度の培養フラスコから細胞を剥離。 </li><li>細胞を計数し、200,000細胞/ mlの濃度で再懸濁します。 </li><li> （詳細は、説明を参照）、プール内の細胞懸濁液の1ミリリットルを追加します。 </li><li>細胞が10〜30分間のパターンに結合する（細胞型に応じて、RPE-1細胞を20分間、10分間およびHeLa細胞がかかります）にしよう。 </li><li>静かに平衡化した培地（培養器内の媒質を平衡化）で浮遊細胞を洗い流す。 </li><li> （RPE-1は、少なくとも2時間が必要になります。HeLa細胞を3時間が必要になります）、細胞がパターンにはいくつかの時間のために普及してみよう。 </li><li> PDMSの改場合の蒸発とメディア漏れを回避するために、プールの上にカバースリップを追加します。 </li><li>倒立顕微鏡上のデバイスを入れて撮影開始。油浸OBの使用を避けるそれが原因で問題が焦点に動作しませんようにjectives。 </li></ol> 5。ストレッチング<ol><li> x軸、y軸、z軸（主にX）で​​のステージ位置を補正しながらマイクロメータネジを回します。ステージ位置は、PDMSの拡大、焦点の損失を打ち消すために補正する必要がある。 （議論では段落「ストレッチ中に」を参照してください）​​。 </li></ol>

<ol>
	<li>Vogel, V., Sheetz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+force+and+geometry+sensing+regulate+cell+functions.">Local force and geometry sensing regulate cell functions.</a> Nat. Rev. Mol. Cell Biol. 7, 265-275 (2006).</li><li>Terenna, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+mechanisms+redirecting+cell+polarity+and+cell+shape+in+fission+yeast.">Physical mechanisms redirecting cell polarity and cell shape in fission yeast.</a> Curr. 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U.S.A. 107 (38), 16518-16523 (2010).</li><li>Irimia, D., Charras, G., Agrawal, N., Mitchison, T., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polar+stimulation+and+constrained+cell+migration+in+microfluidic+channels.">Polar stimulation and constrained cell migration in microfluidic channels.</a> Lab Chip. 12, 1783-1790 (2007).</li><li>Fink, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=External+forces+control+mitotic+spindle+positioning.">External forces control mitotic spindle positioning.</a> Nat. Cell. Biol. 13 (7), 771-778 (2011).</li><li>Azioune, A., Storch, M., Bornens, M., Th&#233;ry, M., Piel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+rapid+process+for+single+cell+micro-patterning.">Simple and rapid process for single cell micro-patterning.</a> Lab Chip. 9 (11), 1640-1642 (2009).</li><li>Azioune, A., Carpi, N., Tseng, Q., Th&#233;ry, M., Piel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+micropatterns:+A+direct+printing+protocol+using+deep+Uvs.">Protein micropatterns: A direct printing protocol using deep Uvs.</a> Methods Cell Biol. 97, 133-146 (2010).</li><li>Azioune, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+method+for+high-throughput+surface+patterning+of+deformable+substrates.">Robust method for high-throughput surface patterning of deformable substrates.</a> Langmuir. 27 (12), 7349-7352 (2011).</li><li>Carpi, N., Piel, M., Azioun, A., Cuvelier, D., Fink, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+on+silicon+elastomer+(PDMS)+with+deep+UVs.">Micropatterning on silicon elastomer (PDMS) with deep UVs.</a> Protoc. 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著者らは、競合する経済的利益を宣言していません。

この技術は、多数回使用されていますし、徹底的にテストされていますが、失敗した実験につながることができ、いくつかの重要なステップがあります。 PDMSについて： この作業では、GelPak、市販の薄いPDMSシートを用いた。別の方法として、PDMSシートは、PDMSミックスから直接鋳造することができる。それは、より再現性があり、カスタムメイドのPDMS...

高等生物の組織を構成する細胞は、機械的緊張にさらされ、外部環境からか、周囲の細胞の1,2のいずれかから来る力を伸ばす。細胞は、に適応し、組織の完全性を維持するためにこれらの力に耐えなければならない。そのような力は、開発中に3,4の組織形態形成のためにも重要である。培養細胞に対する機械的な力を適用すると、組織内で何が起こるか模倣する方法ですが、細胞形状および細胞変形5,6の定量的かつ独立したコントロール機能を備えています。このために、いくつかの技術が使用されてもよい。一つは、AFMまたはその誘導体7,8を使用して、たとえば、細胞（全細胞またはその一部）に押したり、細胞が上に成長している基板を伸ばすことができます。 このホワイトペーパーで説明する方法は、細胞でメッキ面基板をストレッチする方法を示しています。この技術は、元々m個に作用する力の役割を評価するために開発されたitotic、哺乳動物細胞9。有糸分裂細胞は、引込みファイバを介して基板への接続を維持し、膜を延伸は今度は紡錘体の回転を引き起こし、これらの繊維に力を発揮した。接着マイクロパターンを組み合わせて、延伸の関心が力と個々の細胞の形状の独立した制御を達成することである。一軸延伸が適用されている間、それは、完全に等方性の丸い形状に卵形細胞を伸ばすために、例えば、可能である。細胞が微細にplattedされていない場合、ほとんどの細胞は一軸延伸軸と一致長軸を有する、細胞伸長における結果を延伸する。それは長い軸調整や細胞に適用し、ストレッチの効果の影響を分離することが、その後は難しい。 デバイスは、長い時間経過蛍光顕微鏡法を含む任意の生細胞イメージングのために適しており、薬物は、実験中に添加することができる。深いUVの用微細法10ら Aziouneに詳細に記載した。PDMS上11パターニングがAzioune らに記載された。12プロトコルを伸ばし存在カルピらのビデオ版です。13

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GelPak</td>
			<td style="width:71px;">GelPak</td>
			<td style="width:119px;">PF-60-X4</td>
			<td style="width:167px;">Different thickness/stickiness are available. One alternative could be to cast your PDMS yourself.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Silicon grease</td>
			<td style="width:71px;">GE Bayer Silicones</td>
			<td style="width:119px;">Baysilone-Paste</td>
			<td>This one is biocompatible</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Stretching device</td>
			<td style="width:71px;">GREM m&#233;canique</td>
			<td style="width:119px;">Stretcher 2011</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">EDC (N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride)</td>
			<td style="width:71px;">Sigma</td>
			<td align="right" style="width:119px;">3450</td>
			<td>Stable 6 months at -20 &#176;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">NHS (N-Hydroxysulfosuccinimide sodium salt)</td>
			<td style="width:71px;">Sigma</td>
			<td align="right" style="width:119px;">56485</td>
			<td>Protect from humidity</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pll-g-peg (PLL(20)-g[3.5]-PEG(2) 20 mg)</td>
			<td style="width:71px;">SurfaceSolutions (Zurich)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Synthetic Quartz photomask</td>
			<td style="width:71px;">Toppan</td>
			<td style="width:119px;"></td>
			<td>Take standard binary photomask in Quartz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fibronectin from bovine plasma</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">F1141</td>
			<td></td>
		</tr>
	</tbody>
</table>

stretching micropatterned cells on a pdms membrane

機械的力は、細胞に作用するおよび/または組織は、多くのプロセスにおいて重要な役割を果たしている。私たちは、イメージングと互換性ポリジメチルシロキサン（PDMS）膜上で平板培養した細胞を伸ばすためのデバイスを開発した。この手法は、再現性があり、汎用性があります。 PDMS膜は、特定の形状に細胞または組織を閉じ込めるために微細化することができる。最初のステップは、ディープUV技術とPDMS膜上に微細パターンを印刷することである。 PDMS膜は、次いで、機械的なストレッチャーに取り付けられている。室は、ストレッチの際に滑走できるように生体適合性グリース膜の上にバインドされています。細胞は播種し、微細パターン上の数時間広めるために許可されています。サンプルは、マイクロメータねじを用いて複数回延伸·未延伸することができる。それは、その全容（約30％）にストレッチを適用する分もかかります。ここで紹介するテクニックはために必要な電動式の装置が含まれていません迅速および/またはコンピュータ制御された延伸繰り返し延伸サイクルをpplyingが、これは実現することができる。細胞または組織の延伸は、細胞力、機械的なストレスや組織形態形成に対する細胞応答に関連した質問の対象となることができます。このビデオプレゼンテーションでは、一見シンプルなこのタイプの実験を行うときに発生する可能性のある一般的な問題を回避する方法を紹介します。

<html>このビデオプロトコルで提示技術は、有糸分裂、哺乳動物細胞の収縮繊維に力を加えることを可能にした。実際に、細胞分裂の間、有糸分裂の段階では、哺乳動物細胞は、球体の形状をとると、基板に取り付けられた膜で囲まれた細いアクチンケーブルを残すように後退させる。これらのケーブル（後退繊維）、除算に入る前にセル形状のメモリである。 PDMS薄膜上にフォトマスクを通?...

Watch this Scientific Journal Video about Stretching Micropatterned Cells on a PDMS Membrane at JoVE.com

Stretching Micropatterned Cells on a PDMS Membrane

この原稿は、単方向ストレッチを使用して接着細胞または組織に力を適用したり、解放する手法を提示します。

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 ...

stretching-micropatterned-cells-on-a-pdms-membrane

Research

JoVE Journal

Bioengineering

15.2K ビュー.  Institut Curie. この原稿は、単方向ストレッチを使用して接着細胞または組織に力を適用したり、解放する手法を提示します。 

ビデオ: Stretching Micropatterned Cells on a PDMS Membrane

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

癌細胞とヒアルロン酸の相互作用を研究するマイクロパターン表面

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

生物工学

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

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

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

マイクロパターン細胞を用いた薬物の効果の改善可視化と定量分析

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

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

細胞および組織メカノでのアプリケーションのためのプラットフォームをストレッチ小説

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

発光共鳴エネルギーは、哺乳動物細胞で発現膜タンパク質の立体構造変化を研究するために転送

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&amp;#949;-caprolactone) Electrospun Yarns

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

ポリ（εカプロラクトン）エレクトロ糸にヒト間葉系幹細胞の添付ファイルの最適化

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

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

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

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

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

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

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 ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

マイクロパターンマクロスケール3D細胞構造のためのモジュラーヒドロゲルシートの構築

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

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.

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 ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

運動調節血清による靭帯構築物の治療：翻訳組織工学モデル

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

Methods Article

PDMS膜上のマイクロパターン細胞を伸ばす