This work was supported by an NSF/ARRA award (IOS 0920357) and an award from the NIH (5R21MH098463).

1。細胞調製（日-2〜0） <ol><li>分割HEK293細胞1： - 80％コンフルエント（2 - 3日間）および0.05％トリプシン-EDTA溶液を用いて5〜60まで、5％CO 2、37℃で増殖培地中でインキュベートする。各条件については、2×100mm皿内の培養細胞。 </li></ol> 2細胞トランスフェクション（1日目） <ol><li>そのようなリポフェクタミンなどのトランスフェクション試薬を使用してコードするプラスミドFc融合でHEK293細胞をトランスフェクトする。同等のトランスフェクション効率をもたらす代替的な方法を用いてもよい。 </li><li> 24時間インキュベーターにトランスフェクトされた細胞を返します。 </li></ol> 3セルの伝播（2日目） <ol><li> 37℃までのウシ胎児血清（-FBS）なしの予熱増殖培地。 </li><li>料理は10ミリリットルの成長メディア-FBSで2回すすぎ、1時間、37℃のインキュベーターに皿を返す。 </li><li>合計3回の洗浄のために、10ミリリットルの成長メディア-FBSと文化の料理をもう一度洗浄します。 </li><li> Incuお譲りしたメディアを収集する前に、FBSを含まない別の48時間、37℃でトランスフェクトされたHEK293細胞。 </li></ol> 4。ビーズ集約（4日目） <ol><li>培養皿からメディアを収集するには、細胞破片をペレット化し、5分間500×gで50ミリリットルコニカルチューブとスピンに料理の各ペアからメディアを転送します。 </li><li> 30ミリリットルの注射器、0.45μmのシリンジフィルターを用いて遠心フィルターに50ミリリットルコニカルチューブからフィルターメディア。 </li><li>濃縮された培養培地の体積は約500μlの（約15分）まで、4,000×gで、4℃で遠心分離フィルタースピン。すべての培養培地を添加して、濃縮されるまで繰り返します。 </li><li> 、各サンプルについて1.5mlのマイクロチューブに結合バッファーの氷冷1mlにプロテインG磁気ビーズを1.5μl加え、磁石上の場所とバッファを削除します。すぐにプロテインG磁気ビーズに濃縮した培養培地を追加します。 </li><li> 2時間、4℃で管を回転させます。 </li><li> Plの磁石上のエースチューブとは、メディアを取り出します。すばやく結合バッファーの氷冷1で2回ビーズを洗浄した後、結合緩衝300μlのビーズを懸濁します。 </li><li>スプリットは2管、各チューブに150μlの中にビーズを再懸濁し、その後、それぞれ、「カルシウム」と「いいえカルシウム」の条件について200のCaCl 2または200のEDTA、1.5μlを添加する。 </li><li>うつ病への転送、各条件からの100μlをうまくスライドさせ、透過光顕微鏡（ 図2）を使用して、顕微鏡写真を収集します。所望の各時点での各実験のために5視野からの画像を収集します。 </li></ol> 5。データ解析<ol><li> [ファイル ]プルダウンメニューから... ImageJの、または同等の画像解析ソフト、 インポート/イメージシーケンスを使用して、5つの画像データセットのオープン1を使用した。これらは、画像スタックとして開かれます。 </li><li> イメージ / プロパティ...]ダイアログボックスで、私を変更ピクセルにメイジユニットおよび1.0にピクセルの幅と高さをピクセル単位で設定します。 </li><li> 画像プルダウンメニューで調整/しきい値...コマンドを使ってバイナリに画像を変換します。ビーズやビーズ凝集体に貢献する画素が含まれるようにしきい値を設定しますが、その背景と小さい粒子を除外します。スタック内のすべての画像に適用します。 </li><li> セットの測定 ]ダイアログボックスで、 エリアとスタックポジションチェックボックスをオンにします。 </li><li>プルダウンメニューの分析に...粒子を分析し実行します。これは、それらの大きさ（面積）およびそれらが同定された画像を含む同定された粒子のリストを生成する。 </li><li>実験と実験条件について、この手順を繰り返します。 </li></ol>

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The authors declare that they have no competing financial interests.

細胞接着は多細胞生命の本質的な特徴であり、細胞表面タンパク質の広範な配列によって媒介される。このうち、詳細な接着性が比較的小さい割合と理解される。ここでは、便利なエピトープタグに融合された分泌された外部ドメインの同種親和性接着剤能力を調査するための、シンプルで迅速なプロトコルを記載している。このアプローチは、多くの重要な利点を有している。タンパク質?...

細胞間接着は、多細胞生物の発生および完全性に必須であり、細胞表面分子の多様な配列によって媒介される。これらの接着分子の多くは、多くはまだ発見されていないものの、同定され、特徴付けられている。いくつかの方法は、アッセイを、細胞選別、細胞凝集アッセイ1-8と、原子間力顕微鏡、表面力分光法9-12のような生物物理学的方法を含む細胞接着分子（CAM）の特性を調べるために使用される。 偶数細胞株を用いてインビトロ系において簡略化の複雑さは、それが困難な推定上のCAMの接着特性を決定することを可能にする。非接着細胞株にトランスフェクトした場合には細胞凝集を誘導する場合、通常、分子はCAMと考えられている。しかし、これは接着活性の直接的な証拠はないことは明らかである。例えば、細胞表面の配信または安定性を促進するCAMはまた、増加した細胞凝集1,13をもたらすであろう。また、真のCAMは、細胞株は、細胞表面送達または安定化に必要なその他の補助因子を欠いている場合、細胞凝集を媒介することに失敗することがあります。 これらの複雑な要因を回避するために、より直接的なアッセイは、接着性相互作用は、細胞外ドメインの固有の生化学的性質であるべきであるという考えに基づいていることを採用することができる。ビーズは最初に呉-CAM 2を特徴付けるために使用されたが、これらのアッセイは、カドヘリン介在性接着12,14,15を調べるために拡張されている。 C-カドヘリン細胞外ドメイン-Fc融合の融合を使用して、Gumbinerラボは、複数のカドヘリンリピートが相互作用14を同種親和性に寄与することを示した。 ショウジョウバエからプロト1,15-18とDSCAMアイソフォームの数を持っているように、同等のビーズ凝集アッセイ、E-カドヘリンとN-カドヘリン接着を使用すると、、12,15を特徴づけられている<em&gt; 19。ここでは、推定上の同種親和性のCAM（ 図1）の分泌され、エピトープタグの外部ドメインの接着活性を特徴付けるため、比較的簡単かつ迅速なアッセイを記述する。私たちは、カドヘリンスーパ​​ーファミリーのメンバーを特徴づけるために、主にこのアッセイを使用している。

<table border="0" cellpadding="0" cellspacing="0" width="630">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:212px;">Name of Material/ Equipment</td>
			<td style="width:203px;">Catalog Number</td>
			<td style="width:216px;">Company</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">pFc-N1 plasmid</td>
			<td style="width:203px;">To be submitted</td>
			<td style="width:216px;">Addgene</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:212px;">HEK293 cells</td>
			<td style="width:203px;">CRL-1573</td>
			<td style="width:216px;">American Type Culture Collection</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">DMEM</td>
			<td style="width:203px;">10-013-CV</td>
			<td style="width:216px;">Mediatech - Corning</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:203px;">F2442</td>
			<td style="width:216px;">Sigma</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:212px;">100X Pen-Strep (10,000 U/ml)</td>
			<td style="width:203px;">15140-122</td>
			<td style="width:216px;">Life Technologies</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">0.05% Trypsin-EDTA</td>
			<td style="width:203px;">25300054</td>
			<td style="width:216px;">Life Technologies</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">Lipofectamine 2000</td>
			<td style="width:203px;">11668030</td>
			<td style="width:216px;">Life Technologies</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">Dynabeads &#8211; Protein G</td>
			<td style="width:203px;">10003D</td>
			<td style="width:216px;">Life Technologies</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">Bovine Serum Albumin</td>
			<td style="width:203px;">A3294</td>
			<td style="width:216px;">Sigma</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:212px;">Depression slides</td>
			<td style="width:203px;">71878-06</td>
			<td style="width:216px;">Electron Microscopy Sciences</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="2" style="height:43px;width:212px;">Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-10 membrane</td>
			<td rowspan="2" style="width:203px;">UFC901024</td>
			<td rowspan="2" style="width:216px;">Millipore</td>
		</tr>
		<tr height="22">
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;">0.45mm syringe filter</td>
			<td style="width:203px;">83.1826</td>
			<td style="width:216px;">Sarstedt</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;">Dynamag-2 Magnet</td>
			<td>12321D</td>
			<td style="width:216px;">Life Technologies</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:212px;">Fiji (ImageJ) Image Analysis Software</td>
			<td style="width:203px;"></td>
			<td style="width:216px;">http://fiji.sc/Fiji</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;"></td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;"></td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;"></td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;"></td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;">Table of Buffers/Solutions</td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;"></td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:212px;">Growth Media</td>
			<td style="width:203px;">DMEM + 10% FBS + Pen-Strep</td>
			<td style="width:216px;">Filter sterilize and store at 4&#176;C.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:212px;">Growth Media &#8211; FBS</td>
			<td style="width:203px;">DMEM + Pen-Strep</td>
			<td style="width:216px;">Filter sterilize and store at 4&#176;C.</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:212px;">Binding Buffer</td>
			<td style="width:203px;">50 mM Tris, pH 7.4, 100 mM NaCl, 10 mM KCl, 0.2% BSA</td>
			<td style="width:216px;">Vortex until dissolved, keep on ice.</td>
		</tr>
	</tbody>
</table>

bead aggregation assays for the characterization of putative cell adhesion molecules

細胞 - 細胞接着は多細胞生命の基本であり、細胞表面タンパク質の多様な配列によって媒介される。しかし、これらのタンパク質の多くのための接着剤の相互作用はほとんど解明されていない。ここでは、推定される同種親和性細胞接着分子の接着特性を特徴付けるための、シンプルで迅速な方法を提示する。培養されたHEK293細胞は、細胞表面タンパク質の分泌、エピトープタグ外部ドメインをコードするDNAプラスミドでトランスフェクトされる。エピトープタグに特異的な官能化されたビーズを用いて、可溶性の分泌された融合タンパク質を培地から捕捉される。コー​​ティングされたビーズは、その後、ビーズ凝集アッセイにおいて、または同種親和性接着を試験するためのアッセイを選別蛍光ビーズに直接使用することができる。所望の場合、突然変異誘発は、その後の接着に必要な特定のアミノ酸又はドメインを解明するために使用することができる。このアッセイは、発現されたタンパク質の少量のみを必要とする安定な細胞株の産生を必要とせず、アコすることができ4日間でmplished。

<html>実験例は、FC（NcadEC-Fc）のに融合したN-カドヘリンの細胞外ドメインによってカルシウム依存性ビーズの凝集を示した図2に示されている。カルシウムの非存在下では、ビーズは凝集するほとんど又は全く傾向を示す、時間（ 図1A、C）との凝集体サイズの増加はない。カルシウムの存在下では、ビーズは、凝集体サイズが（ 図1B、C）は、時間の経過と...

Watch this Scientific Journal Video about Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules at JoVE.com

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

推定細胞接着分子のキャラクタリゼーションのためのビーズ凝集アッセイ

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

bead-aggregation-assays-for-characterization-putative-cell-adhesion

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Unit 1

Cell-Cell Interactions

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10.4K ビュー.  Ohio State University. Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

ビデオ: Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

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

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

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

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

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

生物工学

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

用接着周波数アッセイその場で</em細胞間のインタフェースにおけるクロス接合分子間相互作用の>速度論解析

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

マトリックス修飾により誘導される細胞接着及び脱接着におけるリアルタイムの変化を測定するために、細胞 - 基質インピーダンスと生細胞イメージングを使用して、

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white blood cells and platelets that are suspended in blood plasma. Due to the abundance of the RBCs (40% to 45% of the blood volume), their behavior dictates the rheological behavior of blood especially in the microcirculation. At very low shear rates, RBCs are seen to assemble and form entities called aggregates, which causes the non-Newtonian behavior of blood. It is important to understand the conditions of the aggregates formation to comprehend the blood rheology in microcirculation. The protocol described here details the experimental procedure to determine quantitatively the RBC aggregates in microcirculation under constant shear rate, based on image processing. For this purpose, RBC-suspensions are tested and analyzed in 120 x 60 &#181;m poly-dimethyl-siloxane (PDMS) microchannels. The RBC-suspensions are entrained using a second fluid in order to obtain a linear velocity profile within the blood layer and thus achieve a wide range of constant shear rates. The shear rate is determined using a micro Particle Image Velocimetry (&#181;PIV) system, while RBC aggregates are visualized using a high speed camera. The videos captured of the RBC aggregates are analyzed using image processing techniques in order to determine the aggregate sizes based on the images intensities.

The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

赤血球凝集の動的な調査のための制御されたマイクロ流体環境

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

改善された細胞接着のためのポリテトラフルオロエチレンにウェットケミストリーおよびペプチドの固定化

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

軟骨再生のための3次元磁気幹細胞凝集およびバイオリアクター成熟

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior in such restrictive conditions. To address this issue, we have developed growth chambers for tip-growing plant cells that contain a series of narrow, micro-fabricated gaps (~1 &#181;m) in a poly-dimethylsiloxane (PDMS) substrate. This transparent material allows the user to monitor tip elongation processes in individual cells during microgap penetration by time-lapse imaging. Using this experimental platform, we observed morphological changes in pollen tubes as they penetrated the microgap. We captured the dynamic changes in the shape of a fluorescently labeled vegetative nucleus and sperm cells in a pollen tube during this process. Furthermore, we demonstrated the capability of root hairs and moss protonemata to penetrate the 1 &#181;m gap. This in vitro platform can be used to study how individual cells respond to physically constrained spaces and may provide insights into tip-growth mechanisms.

We describe a method to investigate the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to elongate through extremely narrow gaps (~1 &#181;m) in a microfluidic device.

非常に小さいスペースで先端成長植物細胞の伸長機能を勉強するマイクロ流体デバイスの開発

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

ナノ製剤中のエンドトキシン検出カブトガニ リムラス ライセート (ラル) 試金を使用して

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

長期無細胞遺伝子発現の伝導のための多層マイクロ流体プラットフォーム

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed from JBNTs and FN through self-assembly in an aqueous solution. JBNTs measure 200-300 &#181;m in length with inner hydrophobic hollow channels and outer hydrophilic surfaces. JBNTs are positively charged and FNs are negatively charged. Therefore, when injected into a neutral aqueous solution, they are bonded together via noncovalent bonding to form the NM bundles. The self-assembly process is completed within a few seconds without any chemical initiators, heat source, or UV light. When the pH of the NM solution is lower than the isoelectric point of FNs (pI 5.5-6.0), the NM bundles will self-release due to the presence of positively charged FN.
NM is known to mimic the extracellular matrix (ECM) morphologically and hence, can be used as an injectable scaffold, which provides an excellent platform to enhance hMSC adhesion. Cell density analysis and fluorescence imaging experiments indicated that the NMs significantly increased the anchorage of hMSCs compared to the negative control.

The goal of this protocol is to show the assembly of a biomimetic nanomatrix (NM) with Janus base nanotubes (JBNTs) and fibronectin (FN). When co-cultured with human mesenchymal stem cells (hMSCs), the NMs exhibit excellent bioactivity in encouraging hMSCs adhesion.

ヤヌスベースナノチューブと幹細胞接着のためのフィブロネクチンによるバイオミメティックナノマトリックスの製造

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed ...