SynVivo teknoloji enstitünün hibe # 2R44HL076034 altında geliştirilmiştir.

1.. Astar SynVivo-SMN mikroakışkan Cihazı <ol><li> Cihazın her bir bağlantı noktası (giriş / çıkış), iki paralel bağlantı noktaları oluşur - yüzey kaplama parçaları içinde akan için, bir (yapışma molekülleri, büyüme matrisler, vb) ve / veya tohumlama ile analizi (Şekil 1A çalıştırmak için diğer için hücrelerin .) </li><li> Tamamen steril deiyonize (DI) su içeren bir Petri kabındaki SynVivo-SMN mikroakışkan cihazı (Şekil 1B) daldırın ve bir vakum desikatöre koyunuz. Hava tüm cihazın kanalları kaldırılır kadar desikatör çalışmasına izin verir. Bu yaklaşık 15 dakika sürer. </li><li> Su cihazını çıkarmadan önce, ince nokta forseps ile cihazın her noktasına su ile astarlanmalıdır Tygon tüpünü (0.06 OD &quot;ve 0.02 kimliği&quot;) koyun. Boru uzunluğu, yaklaşık 1 inç olmalıdır. Cihaz artık sudan uzaklaştırılabilir. Şekil 1C deva ve görüntüyü gösterirboru ile e. </li></ol> 2.. Istenen protein ile mikroakışkan Cihaz kaplama (örneğin, avidin) <ol><li> Bir pipet kullanarak, bir giriş deliği boru tabanı etrafında, su (yaklaşık 100 ul) bir damla yerleştirin. Dikkatle asal cihaza kullanılan boru çıkarın. Suyun damla cihazı hava girmesini engeller. </li><li> 20 ug / ml &#39;lik bir konsantrasyonda avidin ile yüklü bir 1 ml şırınga hazırlayın. Bir 24 G, paslanmaz çelik boru ve iğne için şırınga bağlayın. Cihazın giriş bağlantı noktalarından birine boru yerleştirin. Bir çene kelepçe ile kullanılmaktadır olmayan giriş noktası sıkıştırın. </li><li> Cihazın tam perfüzyon izin vermek için 10 dakika için 1 ml / dk &#39;lık bir akış hızında enjekte edilir avidin. Akış süresi sonunda, çene kelepçe ile boru kelepçe ve gece boyunca 4 ° C&#39;de cihazı yerleştirin. </li></ol> 3.. Yapışma Deneyleri Biyotinilat Partiküllerini Akan <ol><li> Cihazı izin verOda sıcaklığına kadar gelir. , Bir motorlu kaide ile yüksek performanslı bir kamera ile donatılmış bir ters flüoresan mikroskop üzerine yerleştiriniz. </li><li> Fosfat tamponlu salin (PBS) içinde 5 x 10 6 partikül / ml &#39;lik bir konsantrasyonda biyotinile edilmiş 2 um parçacıklar içinde bir çözeltisi hazırlandı. Bir 1 ml şırınga içine parçacıkların yerleştirin. Sadece PBS ikinci bir 1 ml şırınga hazırlayın. Bir şırınga pompa üzerine yerleştirin ve her bir şırınga ve iğne boru bağlanabilir. </li><li> Bir pipet kullanarak, giriş deliği boru tabanı etrafında, su (yaklaşık 100 ul) bir damla yerleştirin. Dikkatlice kat cihaz için kullanılan boru çıkarın. Suyun damla cihazı hava girmesini engeller. </li><li> Dikkatle giriş portları her birine adım 3.2 &#39;den biyotinilatlı parçacıklar ve PBS için boru adlı yerleştirin. Şekil 2A set-up görüntü gösterir. </li><li> 2.5 ul / dk &#39;lık bir akış hızında enjekte biyotinile parçacıkları başlatın. Mikroskop giriş noktasını izleyin. Ilk işaretiparçacıkların, zamanlayıcı başlar ve 3 dakika boyunca akışı devam eder. </li><li> Eş zamanlı olarak 2,5 ul / dk &#39;lık bir akış hızında PBS akışını bakan ise 3 dakika sonunda, biyotinlenmiş parçacıkların akışını durdurmak. PBS bağlanmamış parçacıkları yıkamak için 3 dakika boyunca cihazdaki akmasına izin verin. </li></ol> 4. Görüntüler Kazanılması ve Görüntüleme Yazılımı (NIKON Elemanları) kullanarak Faiz (AOI) Ölçümlerinin Alanı Yapımı <ol><li> Tüm cihazın görüntüsünü elde etmek için görüntüleme yazılımı &quot;tarama büyük resim&quot; işlevini kullanın. </li><li> Cihazdaki Sırasıyla sayı çatallanma ve kanalların iki katı çapa sahip olan dairesel AOI oluşturun. Kanal çapı 100 um olduğu için, bu durumda, 200 um çapı AOI ayarlayın. </li><li> MS Excel sayfası her AOI parçacıkların sayısını ihracat görüntüleme yazılımı otomatik sayım işlevini kullanın. </li><li> Benzer şekilde, in the entire parçacıkların sayısını vermek için otomatik sayım özelliği kullanmakcihaz. </li></ol> 5.. Computational Fluid Dynamics (CFD) modelleri kullanarak Partikül Akı Analizi <ol><li> CFD simülasyonları SynVivo-SMN cihaz topoloji için mevcut ticari yazılım (CFD-ACE + ESI, Inc) kullanılarak çalıştırılır. Sonuçlar deneysel gözlemleri analiz etmek için bir veritabanında saklanır. Duvar kayma oranları, hız, parçacık akı, ve cihazda yapışması üzerinde simülasyon sonuçları saklar. </li><li> Simülasyon sonuçları, belirli bir giriş partikül konsantrasyonuna bağlı olarak, her AOI giren parçacıkların sayısını belirlemek için kullanılır. </li></ol> 6.. Yaratma Kesme Yapışma Haritası <ol><li> Denklem 6.1 &#39;de gösterildiği gibi, çatallanma akan parçacıkların çatallanma olarak yapışmış parçacıklar bölünmesiyle yapışmasının% hesaplayın. <img alt="Denklem 1" fo:content-width="3in" src="/files/ftp_upload/51025/51025eq1.jpg" /> yapışmış parçacıklar ve akan parçacık sayısı p elde edilir buradaotokol sırasıyla, 4.3 ve 5.2 adımları. </li><li> Basamak (5.1) &#39;de veri tabanından elde ağları ve denklem 6.1&#39; den elde edilen% yapışma değerlerinin her bifürkasyonunda kesilme hızı kullanılarak ilk kesme yapışma çizilir. </li></ol>

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CFD Research Corporation tarafından desteklenen bu yazı için yayın ücreti.

Paralel plaka akış odası, hücre-hücre ve hücre-parçacık etkileşimleri hakkında önemli bilgiler sağlarken, bu tür yüksek reaktif maddelerin tüketimi ve bir kesme yapışma haritası oluşturmak için birden fazla deney çalışmaları için ihtiyaç olarak çeşitli kısıtlamalara maruz kalmıştır. SynVivo Sentetik Mikrovasküler Ağlar (SynVivo-SMNs) kullanılması vivo koşullarında taklit eden koşullarda tek bir deneyden gelen bir kesme yapışma haritanın üretilmesini sağlar. Ayrıca, ...

Hücre-hücre ve hücre-partikül etkileşimlerini incelemek için deneyler, tipik haliyle Mevcut parçacıklar ya da hücrelerin protein matrisleri ya da yapışık hücreler üzerinde inkübe edildiği statik oyuklu plaka formatı içerir. Belirtilen inkübasyon süresi sonunda, yapışkan parçacıkların ya da hücre sayısı 1 mikroskopi kullanılarak ölçülür. Bu deneyler, bu etkileşimlerin arkasında biyokimyasal süreçlerin önemli fikir olsa da, önemli bir sınırlama (mikrodolaşımında tipik), fizyolojik sıvı akışının olmaması ve parçacık yapışması üzerindeki etkisidir. Bu sınırlamayı aşmak için, in vitro akış gözlerinden son yıllarda geliştirilmiştir. Bu akış gözlerinden bir ortak öğesi 2, in vivo olarak kan damarlarında görülen duvar kesme hızları aynı düşük Reynolds sayılarında perfüze şeffaf bir aparattır. Damar duvarı akış c bir yüzeyi üzerinde biyomoleküllerin kaplama veya hücrelerin büyümesi biri ile modellenirhamber 3. Parçacıklar 4-7 veya 8-16 hücreler daha sonra çeşitli kayma oranlarının altında parçacıkları kalarak sayısını ölçmek için akış oranları istenilen aralığında aktı vardır. Ancak, biyokimyasal olayları incelemek ve doğrulamak için paralel plaka akış odaları kullanımı oldukça pahalı ve zaman alıcıdır. Bu deneyler, çoklu partiküller yapışmış / hücrelerin sayısı genel akışkan kesme bir haritasını oluşturmak için yapılması gerekir olmasından kaynaklanmaktadır. Buna ek olarak, plaka akış odalar nedeniyle büyük boyutu (yükseklik&gt; 250 mikron genişlik ve&gt; 1 mm) için reaktifler büyük miktarda gerektirir. Son olarak, bu cihazlar doğru in vivo mevcut geometrik özellikleri (örneğin, çatallanma) ve akış koşulları (örn. akışlarını uzaklaşan vs yakınsak) modeli yok. Litografi temelli Mikrofabrikasyona 17-19 son gelişmeler laboratuvarı-on-a-chip alanını hızlandırmıştırcihazlar 20-21. Bu cihazlar mikrometre rejiminde boyutları ile paralel plaka akış odasının bir minyatür versiyonu gelişmekte vesile olmuştur. Boyutunda azalma da reaktifler, hücreler ya da deneyler için gerekli olan parçacık büyüklükleri açısından önemli avantajlar elde edilir. Bununla birlikte, şu anda mevcut cihazlar önemli bir sınırlama, in vivo olarak gözlenen kompleks mikrovaskuleterini taklit etmez mikrodamarlar, Modele doğrusal kanallarının kullanılmasıdır. Son zamanlarda in vivo koşulları sentetik temsil edilen bir tek kullanımlık plastik substratlar üzerine mikrovasküler ağlar yeniden oluşturmak için yeni bir metodoloji geliştirdik. Bu cihazlar SynVivo-Sentetik Microvascular Ağları (SMN) yumuşak litografi süreci tabanlı PDMS kullanılarak geliştirilmiştir adlandırılır. SynVivo-SMN cihazlar hücre / parçacık yapışmasının 22 kesme yapışma haritasını elde etmek için kullanılabilir, çalışma ilacı teslimat 23 ve h hedeflenenin vivo veri 24-25 karşı valide edilmiştir ave. Bu yazıda, 1-5 ul böylece kaynak ve zaman önemli tasarruflar elde kadar küçük hacimlerde tek bir deneyden elde kesme yapışma haritanın nesil sağlayan bir protokol mevcut.

<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

Hücre / tanecik yapışma deneyleri, hastalığın patofizyolojisinde rol biyokimyasal etkileşimleri anlaşılması için kritik olan ve yeni tedavilerin geliştirilmesi için arayışı içinde önemli bir uygulamaya sahiptir. Statik koşullar kullanılarak deneyler in vivo ortamı ile korelasyon sınırlama, kesme yapışması bağımlılığını yakalamak için başarısız. Fizyolojik sıvı akımı altında yapışma ölçmek paralel plaka akış oda, bir kesme yapışma haritasının üretilmesi için çok sayıda deney gerekir. Buna ek olarak, in vivo ölçek ve morfolojisi temsil etmez ve deneyler için reaktifler büyük miktarda (~ mi) gerektirir. Bu çalışmada, mikroakışkan cihaz, SynVivo-SMN dayalı bir mikrovasküler ağını kullanarak tek bir deneyden elde kesme yapışma haritanın üretilmesini göstermektedir. Bu cihaz, geometrik ölçek, morfolojik elemanlar, akış özellikleri ve bir hücresel etkileşimler de dahil olmak üzere in vivo damar karmaşık yenidenin vitro biçimi, böylece temel bir biyolojik gerçekçi ortam sağlayarak ve hücresel davranışı, ilaç dağıtım, ve ilaç keşfi uygulamalı araştırma. Deney mikroçip avidin kaplı yüzeyler ile 2 mikron biyotin kaplı partiküllerin etkileşimlerini incelemek ile gösterilmiştir. Mikrovasküler gözlenen makaslama tüm aralığı, fizyolojik koşullar altında parçacıkları için kesme harita karşı tek bir deney sağlayan bir yapışma elde edilir.

Şekil 1A-SynVivo SMN cihazının bir şematik ve parlak saha görüntüsünü gösterir. Şekil 1B, bir cam slayt üzerine monte SynVivo-SMN cihazı. Şekil 1C bir vakumlu desikatör içinde su ile astar boru takımı ile birlikte cihazı göstermektedir göstermektedir. Şekil 2A, 2 um biyotinile edilmiş parçacıkların bağlayıcı Aşağıda, tipik bir avidin kaplı SynVivo-SMN cihazını göstermektedir, deney set up....

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 Sentetik Mikrovasküler Ağları kullanma Kesme Yapışma Haritası Üretimi

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 Görüntüleme.  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. 

Video: 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.

Plazma Litografi Yüzey Desenlendirme Hücre Ağları Oluşturma

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

Biyomühendislik

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.

Hematolojik Hastalıklarında Mikrovasküler Etkileşimleri Öğrenmenin Endothelialized Mikroakiskan

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.

Düşük yoğunluklu Ultrason Kullanımı Yapışma bağımlı Sinyalleri indüksiyonu

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.

Matrix Modifikasyon Bağlı Hücresel Yapışma ve De-yapışma Real-time Değişiklikleri Tedbir Hücre-substrat Empedans ve Canlı Hücre Görüntüleme kullanma

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.

Yaşayan Hücreleri Mühendise Sentetik Biyoloji kullanarak That Programlanabilir Malzemelerle Arayüzü

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.

Stres birden çok paralel-plaka akışı Chambers kullanarak yamultmak için maruz endotel hücrelerinin gen ifade Analizi

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.

Canlı hücre analizi, yamultma stres bir otomatik yüksek üretilen iş mikrosıvısal sistemi kullanarak Pseudomonas aeruginosa

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.

Lentiviral Vektör Yaklaşımı Kullanarak Transgenik Sıçanların Üretimi

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.

Hücre Serbest Bakteriyel Özler, Lipozomlar ve Emülsiyon Transferi Kullanarak Protein Üreten Sentetik Hücreler Hazırlama

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.

Kayan Kinesin Motilite Testi Kullanılarak Çok Bileşenli Lipid Nanotüp Ağlarının Üretilmesi

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