SynVivo技術はNHLBIからの助成金番号2R44HL076034の下で開発されました。

1。プライミングSynVivo-SMNマイクロ流体デバイス<ol><li>デバイスの各ポート（入口/出口）は、2つのパラレルポートから構成されている-表面被覆部分に流れる毎に（接着分子、成長マトリックスなど）および/ ​​または播種アッセイ（ 図1Aを実行するための他のための細胞）。 </li><li>完全滅菌脱イオン（DI）水を含むペトリ皿にSynVivo-SMNマイクロ流体デバイス（ 図1B）を水没し、真空デシケーターにお皿を置く。空気のすべてがデバイスのチャネルから削除されるまでデシケーターを実行させておきます。これは、約15分かかります。 </li><li>水からデバイスを取り外す前に、細かい点ピンセットでデバイスの各ポートに水で下塗りタイゴンチューブ（0.06のOD」と0.02のID」）を配置します。チューブは長さが約1インチでなければなりません。デバイスは、水から除去することができる。 図1Cは、デビックの画像を示すチューブとE。 </li></ol> 2。所望のタンパク質（ 例えば 、アビジン）とマイクロ流体デバイスをコーティング<ol><li>ピペットを用いて、1流入口チューブの根元に水を一滴（約100μl）を配置します。慎重にプライムデバイスに使用されるチューブを取り外します。水滴は、デバイスを空気が入るのを防ぐであろう。 </li><li> 20μg/ mlの濃度でアビジンを搭載し1ミリリットルの注射器を準備します。 24 Gのステンレス鋼針やチューブに注射器を接続します。デバイスの入口ポートにチューブを挿入する。ジョーのクランプで利用されていない入口ポートをクランプ。 </li><li>装置の完全なかん流を可能にするために10分間、1μlの/分の流速でアビジンを注入する。フロー時間の終了時に、顎クランプでチューブをクランプし、4℃で一晩装置を配置する。 </li></ol> 3。接着実験のためのビオチン化粒子を流れる<ol><li>デバイスができるようにする室温に来る。電動ステージと高性能カメラを搭載した倒立蛍光顕微鏡の上には置か。 </li><li>リン酸緩衝生理食塩水（PBS）中5×10 6粒子/ mlの濃度で2μmのビオチン化粒子の溶液を調製する。 1ミリリットルの注射器に粒子をロードします。 PBSのみの第二の1ミリリットルの注射器を準備します。シリンジポンプに各シリンジをロードし、針とチューブに接続します。 </li><li>ピペットを用いて、流入口チューブの根元に水を一滴（約100μl）を配置します。丁寧にコーティングするために、デバイスを使用し、チューブを取り外します。水滴は、デバイスを空気が入るのを防ぐであろう。 </li><li>慎重に入口ポートのそれぞれにステップ3.2からチューブのビオチン化粒子と、PBSのために挿入します。 図2（a）は、セットアップの画像を示す。 </li><li> 2.5μL/分の流量で、ビオチン化粒子を注入開始。顕微鏡上の入口ポートを監視します。最初の兆候で粒子から、タイマーを開始し、3分間の流れを継続する。 </li><li>同時に2.5μlの/分の流速でPBSの流れを凝視しながら3分間の終わりに、ビオチン化された粒子の流れを止める。 PBSを結合していない粒子を洗い流すために3分間デバイス内に流れるようになります。 </li></ol> 4。画像を取得し、イメージングソフトウェア（NIKON要素）を用いて関心領域（AOI）測定の実行<ol><li>装置全体の画像を取得する画像処理ソフトウェアで「スキャン大きなイメージ」機能を使用してください。 </li><li>順次番号分岐装置内のチャンネルの二倍の直径を持つ円形のAOIを作成します。チャンネル径が100μmであるため、この場合は、200μmのAOI径を設定します。 </li><li> MS Excelのシートに各AOIの粒子数をエクスポートするために、イメージングソフトウェアで自動化されたカウント機能を使用してください。 </li><li>同様に、全体の中の粒子の数をエクスポートするために自動化されたカウント機能を使用デバイス。 </li></ol> 5。計算流体力学（CFD）モデルを用いて粒子フラックス解析<ol><li> CFDシミュレーションはSynVivo-SMNのデバイストポロジのために市販のソフト（CFD-ACE +、ESI社）を使用して実行されます。結果は、実験観察を分析するためのデータベースに格納される。壁のせん断速度、速度、粒子束、およびデバイスにおける接着に関するシミュレーション結果情報を格納します。 </li><li>シミュレーション結果は、所与の入口粒子濃度に基づいて、それぞれのAOIに入る粒子の数を決定するために使用される。 </li></ol> 6。生成剪断接着地図<ol><li>式6.1に示すように分岐部を流れる粒子によって分岐内の付着した粒子を分割することにより密着性の％を計算します。 <img alt="式（1）" fo:content-width="3in" src="/files/ftp_upload/51025/51025eq1.jpg" /> 付着粒子の数と粒子が流れることは、pから得られる場合rotocolは、それぞれ4.3と5.2を繰り返します。 </li><li>ステップ（5.1）でデータベースから取得したネットワークおよび式6.1から得％の接着値の各分岐部に剪断速度を用いて剪断接着マップをプロットする。 </li></ol>

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CFDリサーチ社が主催するこの記事の公開手数料。

パラレルプレートフローチャンバーは、細胞 - 細胞および細胞 - 粒子間相互作用に重要な洞察を提供しながら、このような高い試薬の消費および剪断接着マップを生成するために複数の実験ランの必要性など、いくつかの制限を受ける。 SynVivo合成微小血管ネットワーク（SynVivo-SMNs）の使用は、生体内の状態に模倣条件での一回の実験からの剪断接着マップの生成を可能にしま?...

細胞間および粒子 - 細胞相互作用を研究するための現在のアッセイは、典型的には粒子または細胞は、タンパク質マトリックスまたは接着細胞上でインキュベートされた静的なウェルプレートフォーマットを含む。指定されたインキュベーション時間の終了時に、付着粒子または細胞の数を、顕微鏡1を用いて定量する。これらのアッセイは、これらの相互作用の背後にある生物化学的プロセスに重要な洞察を提供するにもかかわらず、主要な制限は、生理的な流体（典型的な微小循環の）流れと粒子の付着への影響がないことである。 この制限を克服するために、 インビトロのフローチャンバーは、近年開発されている。これらのフローチャンバーの共通要素は、 インビボ 2 の血管において観察される壁せん断速度と一致するように、低レイノルズ数で灌流透明な装置である。血管壁は、流れcの一面に生体分子の被膜または細胞の増殖のいずれかによってモデル化されるhamber 3。粒子4-7又は8-16の細胞は、次いで種々の剪断速度下で粒子を付着させるの数を定量化するために流速の所望の範囲内に流される。 しかし、生化学的現象を研究し、検証するための平行板フローチャンバーの使用は、かなり高価であり、時間がかかる。これは、複数の実験が付着粒子/細胞の数に対する流体剪断の地図を生成するために行われる必要があるという事実に主に起因する。また、プレートフローチャンバーは、それらの大きなサイズ（高さ&gt;250μmで、幅&gt; 1mm）のに試薬を大量に必要とする。最後に、これらのデバイスは、正確に生体内に存在する幾何学的特徴（ 例えば 、分岐点）とフロー条件（ 例えば 、フローを発散対収束）をモデル化しないでください。 リソグラフィベースの微細加工17〜19の最近の進歩により、ラボ·オン·チップのフィールドを加速していますデバイス20〜21。これらのデバイスは、マイクロメートル領域における寸法の平行板フローチャンバーの小型化されたバージョンの開発に尽力してきました。ディメンションの減少も試薬、細胞や実験に必要とされる粒子の体積の面で大きなメリットが得られます。しかしながら、現在利用可能なデバイスの重要な制限は、in vivoで観察複合体微小血管系を模倣していない微小血管をモデル化する線形チャネルの使用である。 我々は最近、生体内の条件の合成表現、その結果、使い捨てのプラスチック基板上に微小血管のネットワークを再作成するための新たな方法論を開発しました。これらのデバイスは、SynVivo、合成微小血管ネットワーク（SMN）と呼ばれるPDMSベースのソフトリソグラフィプロセスを使用して開発されている。 SynVivo-SMNデバイスは、細胞/粒子の密着22の剪断接着マップを得るために使用することができ、研究では、薬物送達及びh 23を標的とin vivoデータ24〜25と比較して検証されていAVE。本論文では、それによって、資源と時間の大幅な節約をもたらす1-5μLと小さい容量の中で単一の実験から、せん断接着マップの生成を可能にするプロトコルを提示する。

<table border="0" cellpadding="0" cellspacing="0" width="638">
	<tbody>
		<tr>
			<td>SynVivo-SMN</td>
			<td>CFD Research</td>
			<td>SMN-001</td>
			<td>Exclusive at CFDRC</td>
		</tr>
		<tr>
			<td>CFD-ACE+</td>
			<td>ESI Inc.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Avidin</td>
			<td>Invitrogen</td>
			<td>43-4401</td>
			<td>Any avidin source will work for this assay</td>
		</tr>
		<tr>
			<td>Biotinylated Particles</td>
			<td>Polysciences</td>
			<td>24173-1</td>
			<td>Any source of biotinylated particles will work for the assay</td>
		</tr>
		<tr>
			<td>Tygon Tubing</td>
			<td>VWR</td>
			<td>63018-044</td>
			<td>Size is typical for use with SynVivo-SMN</td>
		</tr>
		<tr>
			<td>NIKON Elements</td>
			<td>NIKON Instruments</td>
			<td>N/A</td>
			<td>Any other imaging software can be used</td>
		</tr>
	</tbody>
</table>

generation of shear adhesion map using synvivo synthetic microvascular networks

セル/粒子接着アッセイは疾患の病態生理に関与して生化学的相互作用を理解する上で重要であり、新たな治療薬の開発のための探求の重要な用途がある。静的条件を用いたアッセイは、 生体内環境との相関性を制限する、剪断に対する密着性の依存性を捕捉することができない。生理学的な流体の流れの下での密着性を定量化する平行板フローチャンバーは、剪断接着マップを生成するための複数の実験を必要とする。加えて、それらはインビボ規模および形態を表し、実験のための試薬 ​​の大量（〜ml）を必要としない。本研究では、マイクロ流体デバイス、SynVivo-SMNベースの微小血管ネットワークを用いて単一の実験からの剪断接着マップの生成を示す。このデバイスは、幾何学的な規模、形態学的要素、フロー機能との細胞間相互作用を含む、 生体内血管系で複合体を再作成in vitroでのフォーマット、それによって基本のため、生物学的に現実的な環境を提供し、細胞挙動、薬物送達、および創薬応用研究。アッセイは、マイクロチップのアビジン被覆表面とのビオチン2μmの被覆粒子との相互作用を研究することによって実証された。微小血管系において観察剪断の全範囲は、生理的条件下で粒子に対するせん断マップ対接着を可能にする単一のアッセイで得られる。

図1Aは、概略的かつSynVivo-SMN装置の明視野像を示す。 図1Bは、スライドガラス上に搭載SynVivo-SMN装置を示す。 図1Cは 、真空デシケーター中で水でプライミング以下のチューブを有する装置を示している。 図2aは、実験的な設定までのイメージを示す。 図2Bは、2ミクロンビオチン化粒子の結合後の典型的なア?...

Watch this Scientific Journal Video about Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks at JoVE.com

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables.

SynVivo合成微小血管網を利用した剪断接着マップの生成

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important ...

generation-shear-adhesion-map-using-synvivo-synthetic-microvascular

Research

JoVE Journal

Bioengineering

8.8K ビュー.  CFD Research Corporation. Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables. 

ビデオ: Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

細胞ネットワークの作成のためのプラズマのリソグラフィ表面パターニング

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

生物工学

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

血液疾患における血管相互作用を研究するためのEndothelializedマイクロフルイディクス

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from regulation of cell migration and proliferation, to secretion and even differentiation. The signals underlying each of these complex processes arise from the molecular interactions of extracellular matrix receptors on the surface of the cell. Integrins are the prototypic receptors and provide a mechanical link between extracellular matrix and the cytoskeleton, as well as initiating some of the adhesion-dependent signaling cascades. However, it is becoming increasingly apparent that additional transmembrane receptors function alongside the integrins to regulate both the integrin itself and signals downstream. The most elegant of these examples is the transmembrane proteoglycan, syndecan-4, which cooperates with &alpha;5&beta;1-integrin during adhesion to fibronectin. In vivo models demonstrate the importance of syndecan-4 signaling, as syndecan-4-knockout mice exhibit healing retardation due to inefficient fibroblast migration1,2. In wild-type animals, migration of fibroblasts toward a wound is triggered by the appearance of fibronectin that leaks from damaged capillaries and is deposited by macrophages in injured tissue. Therefore there is great interest in discovering strategies that enhance fibronectin-dependent signaling and could accelerate repair processes.
The integrin-mediated and syndecan-4-mediated components of fibronectin-dependent signaling can be separated by stimulating cells with recombinant fibronectin fragments. Although integrin engagement is essential for cell adhesion, certain fibronectin-dependent signals are regulated by syndecan-4. Syndecan-4 activates the Rac1 protrusive signal3, causes integrin redistribution1, triggers recruitment of cytoskeletal molecules, such as vinculin, to focal adhesions4, and thereby induces directional migration3. We have looked for alternative strategies for activating such signals and found that low-intensity pulsed ultrasound (LIPUS) can mimic the effects of syndecan-4 engagement5. In this protocol we describe the method by which 30 mW/cm2, 1.5 MHz ultrasound, pulsed at 1 kHz (Fig. 1) can be applied to fibroblasts in culture (Fig. 2) to induce Rac1 activation and focal adhesion formation. Ultrasound stimulation is applied for a maximum of 20 minutes, as this combination of parameters has been found to be most efficacious for acceleration of clinical fracture repair6. The method uses recombinant fibronectin fragments to engage &alpha;5&beta;1-integrin, without engagement of syndecan-4, and requires inhibition of protein synthesis by cycloheximide to block deposition of additional matrix by the fibroblasts., The positive effect of ultrasound on repair mechanisms is well documented7,8, and by understanding the molecular effect of ultrasound in culture we should be able to refine the therapeutic technique to improve clinical outcomes.

This protocol describes the stimulation of cultured fibroblasts with low-intensity pulsed ultrasound, which drives focal adhesion formation and Rac1 activation by mimicking engagement of the transmembrane matrix receptor, syndecan-4. This approach allows investigation of a successful clinical technique at the cellular level, thereby providing opportunities for refinement of the therapy.

低強度の超音波を用いた接着依存性のシグナルの誘導

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from ...

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 ...

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

プログラム可能な材料とのインタフェースリビング細胞を操作するために合成生物学を使用して、

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

剪断応力の複数の平行平板流チャンバーを使用して内皮細胞の遺伝子発現解析

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, including Pseudomonas aeruginosa (P. aeruginosa). Here, we will describe how the system can be used to study the growth kinetics and the morphological properties such as the surface roughness and textural entropy of P. aeruginosa strain PA01 that expresses an enhanced green fluorescent protein (PA01-EGFP). A detailed protocol will describe how to grow and seed PA01-EGFP cultures, how to set up the microscope and autorun, and conduct the image analysis to determine growth rate and morphological properties using a variety of shear forces that are controlled by the microfluidic device. This article will provide a detailed description of a technique to improve the study of PA01-EGFP biofilms which eventually can be applied towards other strains of bacteria, fungi, or algae biofilms using the microfluidic platform.

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

Here, we describe the use of a higher-throughput microfluidic bioreactor coupled with a fluorescent microscope for the analysis of shear stress effects on Pseudomonas aeruginosa biofilms expressing green fluorescent proteins, including instrument set up, the determination of biofilm coverage, growth rate, and morphological properties.

緑膿菌自動の高スループット マイクロ流体システムを使用して上のせん断応力のライブ細胞解析

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, ...

Generation of Transgenic Rats using a Lentiviral Vector Approach

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

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

レンチウイルスベクターアプローチによるトランスジェニックラットの生成

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

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

細胞を含まない細菌抽出物、リポソーム、エマルジョン移動を用いた合成細胞の調製

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous lipid tubules found in eukaryotic cells. However, in vivo LNTs are highly non-equilibrium structures that require chemical energy and molecular motors to be assembled, maintained, and reorganized. Furthermore, the composition of in vivo LNTs is complex, comprising of multiple different lipid species. Typical methods to extrude LNTs are both time- and labor-intensive, and they require optical tweezers, microbeads, and micropipettes to forcibly pull nanotubes from giant lipid vesicles. Presented here is a protocol for the gliding motility assay (GMA), in which large scale LNT networks are rapidly generated from giant unilamellar vesicles (GUVs) using kinesin-powered microtubule motility. Using this method, LNT networks are formed from a wide array of lipid formulations that mimic the complexity of biological LNTs, making them increasingly useful for in vitro studies of lipid biophysics and membrane-associated transport. Additionally, this method is capable of reliably producing LNT networks in a short time (&#60;30 min) using commonly used laboratory equipment. LNT network characteristics such as length, width, and lipid partitioning are also tunable by altering the lipid composition of the GUVs used for fabricating the networks.

This protocol describes a process for fabricating lipid nanotube networks using gliding kinesin motility in conjunction with giant unilamellar lipid vesicles.

グライダーキネシン運動性アッセイを用いた多成分脂質ナノチューブネットワークの構築

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous ...