Technologie SynVivo a été développé sous subvention # 2R44HL076034 du NHLBI.

1. D&#39;amorçage du dispositif microfluidique SynVivo-SMN <ol><li> Chaque port (entrée / sortie) de l&#39;appareil est constitué de deux ports parallèles - une pour l&#39;écoulement dans des fragments de revêtement de surface (molécules d&#39;adhésion, des matrices de croissance, etc) et / ou des cellules destinées à l&#39;ensemencement et l&#39;autre pour l&#39;exécution de l&#39;essai (figure 1A ). </li><li> Immerger complètement le dispositif microfluidique SynVivo-SMN (figure 1B) dans une boîte de Pétri contenant déminéralisée stérile (DI) et placez le plat dans un dessiccateur à vide. Laisser le dessiccateur à fonctionner jusqu&#39;à ce que tout l&#39;air est éliminé à partir des canaux de l&#39;appareil. Cela devrait prendre environ 15 min. </li><li> Avant de retirer l&#39;appareil de l&#39;eau, placer le tuyau Tygon (OD de 0,06 &quot;et ID de 0,02&quot;) amorcée avec de l&#39;eau dans chaque port de l&#39;appareil avec pointes fines pinces. Le tube doit être d&#39;environ 1 pouce de longueur. L&#39;appareil peut maintenant être retiré de l&#39;eau. Figure 1C montre l&#39;image de la Device avec le tube. </li></ol> 2. Revêtement du dispositif microfluidique avec de la protéine souhaitée (par exemple, avidine) <ol><li> En utilisant une pipette, déposer une goutte de l&#39;eau (environ 100 pi) autour de la base du tube d&#39;un orifice d&#39;entrée. Retirez délicatement le tube utilisé pour amorcer le dispositif. La goutte d&#39;eau empêche l&#39;air d&#39;entrer dans le dispositif. </li><li> Préparer une seringue de 1 ml chargé avec de l&#39;avidine à une concentration de 20 pg / ml. Connectez la seringue à une aiguille et un tube en acier inoxydable 24 G. Insérer le tube pour l&#39;un des ports du dispositif d&#39;entrée. Serrer l&#39;orifice d&#39;entrée n&#39;étant pas utilisée avec un dispositif de serrage de la mâchoire. </li><li> Injecter de l&#39;avidine à un débit de 1 pl / min pendant 10 min pour permettre la perfusion complète du dispositif. A la fin de la durée d&#39;écoulement, serrer le tube avec la pince à mâchoires et placer le dispositif à 4 ° C pendant une nuit. </li></ol> 3. Circuler particules biotinylé pour les expériences d&#39;adhérence <ol><li> Permettre au dispositif derevenir à la température ambiante. Placer l&#39;appareil sur un microscope inversé à fluorescence équipé d&#39;une platine motorisée et une caméra à haute performance. </li><li> Préparer une solution de 2 um particules biotinylés à une concentration de 5 x 10 6 particules / ml dans le tampon phosphate salin (PBS). Charger les particules dans une seringue de 1 ml. Préparer une deuxième seringue de 1 ml de PBS seulement. Chargez chaque seringue sur une pompe à seringue et se connecter à l&#39;aiguille et la tubulure. </li><li> En utilisant une pipette, déposer une goutte de l&#39;eau (environ 100 pi) autour de la base du tube de l&#39;orifice d&#39;entrée. Retirez délicatement le tube utilisé pour recouvrir le dispositif. La goutte d&#39;eau empêche l&#39;air d&#39;entrer dans le dispositif. </li><li> Insérez délicatement le tube de particules pour biotinylés et PBS de l&#39;étape 3.2 dans chacun des ports d&#39;entrée. Figure 2A montre l&#39;image de la mise en place. </li><li> Commencer l&#39;injection de particules biotinylés à un débit de 2,5 ul / min. Surveillance de l&#39;orifice d&#39;entrée sur le microscope. Au premier signede particules, commencer la minuterie et continue de couler pendant 3 min. </li><li> A la fin de la 3 min, arrêter le flux de particules tout en fixant simultanément biotinylés l&#39;écoulement du PBS à un débit de 2,5 ul / min. Laisser PBS de s&#39;écouler dans l&#39;appareil pendant 3 minutes pour se laver de particules libres. </li></ol> 4. Acquisition d&#39;images et effectuer une zone d&#39;intérêt (AOI) Mesures Utilisation du logiciel d&#39;imagerie (NIKON Elements) <ol><li> Utilisez la fonction &quot;scan image de grande taille&quot; dans le logiciel d&#39;imagerie pour acquérir l&#39;image de l&#39;ensemble du dispositif. </li><li> nombre séquentielle de bifurcations dans le dispositif et créer un AOI circulaire avec deux fois le diamètre des canaux. Dans ce cas, régler le diamètre AOI à 200 um depuis diamètre du canal est de 100 um. </li><li> Utilisez la fonction de comptage automatisé dans le logiciel d&#39;imagerie pour exporter le nombre de particules dans chaque AOI à une feuille Excel. </li><li> De même, utiliser la fonction de comptage automatisé pour exporter le nombre de particules dans l&#39;ensembledispositif. </li></ol> 5. Analyse de particules Flux Utilisation Computational Fluid Dynamics (CFD) Modèles <ol><li> Simulations CFD sont gérés en utilisant un logiciel disponible dans le commerce (CFD-ACE +, ESI Inc.) pour le dispositif topologie SynVivo-SMN. Les résultats sont stockés dans une base de données pour l&#39;analyse des observations expérimentales. Le résultats de la simulation de stocker des informations sur les taux de cisaillement de paroi, la vitesse, le flux de particules, et l&#39;adhérence dans le dispositif. </li><li> Les résultats de simulation sont utilisées pour déterminer le nombre de particules entrant dans chaque zone d&#39;intérêt sur la base de la concentration d&#39;entrée de particules donnée. </li></ol> 6. Génération cisaillement adhérence Carte <ol><li> Calculer le% d&#39;adhérence en divisant les particules adhérentes dans la bifurcation par les particules s&#39;écoulant dans la bifurcation, comme indiqué dans l&#39;équation 6.1. <img alt="Equation 1" fo:content-width="3in" src="/files/ftp_upload/51025/51025eq1.jpg" /> où le nombre de particules adhérées et les particules fluides sont obtenus à partir de la protocol les étapes 4.3 et 5.2, respectivement. </li><li> Tracer la carte d&#39;adhésion de cisaillement en utilisant le taux de cisaillement à chaque bifurcation des réseaux obtenus à partir de la base de données à l&#39;étape (5.1) et les valeurs d&#39;adhérence% obtenus à partir de l&#39;équation 6.1. </li></ol>

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Frais de publication de cet article parrainé par CFD Research Corporation.

Parallèles chambres d&#39;écoulement de la plaque, tout en fournissant des indications importantes sur les interactions cellule-cellule et cellule-particules, souffrent de plusieurs limitations telles que la consommation élevée de réactifs et de la nécessité de multiples essais expérimentaux pour générer une carte d&#39;adhérence au cisaillement. L&#39;utilisation des réseaux microvasculaires SynVivo-synthétiques (SynVivo-SMNS) permet la génération d&#39;une carte d&#39;adhésion au cisaillement d&#39;une...

Les tests actuellement pour étudier à cellule-cellule et les interactions particule-cellulaires impliquent généralement statique format de plaque de puits dans lesquels les particules ou les cellules sont incubées sur des matrices de protéines ou de cellules adhérentes. A la fin du temps d&#39;incubation spécifié, les nombres de particules ou de cellules adhérentes sont quantifiés en utilisant une microscopie. Même si ces tests donnent un aperçu significatif dans les processus biochimiques sous-tendent ces interactions, une limitation clé est l&#39;absence de flux de liquide physiologique (typique de la microcirculation) et son impact sur l&#39;adhérence des particules. Pour surmonter cette limitation, in vitro dans les chambres d&#39;écoulement ont été développés ces dernières années. Un élément commun de ces chambres d&#39;écoulement est un dispositif transparent perfusé à faibles nombres de Reynolds pour correspondre à des taux de cisaillement de paroi observées dans les vaisseaux sanguins in vivo 2. La paroi de la cuve est modélisée par une ou l&#39;autre revêtement de biomolécules ou de la croissance de cellules sur une surface de l&#39;écoulement chambre 3. Particules 4-7 ou 8-16 cellules sont ensuite coulé dans au gamme désirée de débits de quantifier le nombre de particules adhérentes sous différents taux de cisaillement. Cependant, l&#39;utilisation de chambres d&#39;écoulement parallèles de la plaque d&#39;étudier et de valider les phénomènes biochimiques est assez cher et prend du temps. Ceci est principalement dû au fait que de multiples expériences doivent être réalisées pour générer un plan de cisaillement fluidique par rapport au nombre de particules / cellules adhérant. De plus, les chambres d&#39;écoulement à plaques nécessitent de grands volumes de réactifs en raison de leur grande taille (hauteur&gt; 250 pm et une largeur&gt; 1 mm). Enfin, ces dispositifs ne modélisent pas exactement les caractéristiques géométriques (par exemple, des bifurcations) et des conditions de flux (par exemple, la convergence vs flux divergent) qui sont présents in vivo. Les progrès récents dans la lithographie basée microfabrication 17-19 ont accéléré le domaine de lab-on-a-chiples dispositifs 20 à 21. Ces dispositifs ont contribué à l&#39;élaboration d&#39;une version miniaturisée de la chambre parallèle des flux de plaque avec des dimensions dans le régime de micromètre. La réduction de la dimension donne également des avantages importants en termes de volumes de réactifs, de cellules ou de particules requise pour les expériences. Cependant, une limitation essentielle des dispositifs actuellement disponibles est l&#39;utilisation des canaux linéaires pour modéliser les microvaisseaux, qui ne reproduisent pas la microvascularisation complexe observé in vivo. Nous avons récemment développé une nouvelle méthode pour recréer des réseaux microvasculaires sur des substrats en plastique jetables résultant en une représentation synthétique des conditions in vivo. Ces dispositifs appelés réseaux microvasculaires SynVivo-synthétiques (SMN) sont développés en utilisant PDMS processus lithographie douce à base. Dispositifs SynVivo-SMN peuvent être utilisés pour obtenir cisaillement carte d&#39;adhésion de l&#39;adhérence cellulaire / particules 22, étude l&#39;administration de médicaments et 23 h cibléave été validé par des données in vivo 24-25. Dans cet article, nous présentons un protocole qui permet la génération de la carte d&#39;adhésion de cisaillement d&#39;une expérience unique dans des volumes aussi petits que 1-5 pi ce qui conduit à d&#39;importantes économies de temps et de ressources.

<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

Cellules / particules tests d&#39;adhésion sont essentielles à la compréhension des interactions biochimiques impliqués dans la physiopathologie de la maladie et des applications importantes dans la recherche pour le développement de nouveaux produits thérapeutiques. Tests utilisant des conditions statiques ne parviennent pas à capturer la dépendance de l&#39;adhérence sur cisaillement, ce qui limite leur corrélation avec l&#39;environnement in vivo. Parallèles chambres d&#39;écoulement à plaques qui permettent de quantifier l&#39;adhérence sous l&#39;écoulement du fluide physiologique besoin de plusieurs expériences pour la génération d&#39;une carte d&#39;adhérence au cisaillement. En outre, ils ne représentent pas l&#39;ampleur et de la morphologie in vivo et nécessitent de grands volumes (~ ml) de réactifs pour les expériences. Dans cette étude, nous avons démontré la génération de la carte d&#39;adhérence au cisaillement à partir d&#39;une seule expérience en utilisant un réseau microvasculaire basé dispositif microfluidique, SynVivo-SMN. Ce dispositif recrée le complexe dans le système vasculaire in vivo, y compris l&#39;échelle géométrique, éléments morphologiques, les caractéristiques d&#39;écoulement et les interactions cellulaires dans unFormat in vitro, fournissant ainsi un environnement biologiquement réaliste pour la recherche fondamentale et appliquée dans le comportement cellulaire, l&#39;administration de médicaments, et la découverte de médicaments. Le dosage a été démontrée par l&#39;étude de l&#39;interaction des particules 2 um revêtue de la biotine avec des surfaces revêtues d&#39;avidine de la micro-puce. L&#39;ensemble de la gamme de cisaillement observée dans le système microvasculaire est obtenu en permettant à un seul essai d&#39;adhérence par rapport à la carte de cisaillement pour les particules dans des conditions physiologiques.

La figure 1A montre une image de champ lumineux et schématique de dispositif SynVivo-SMN. Figure 1B montre le dispositif SynVivo-SMN monté sur une lame de verre. Figure 1C montre le dispositif avec des tubes après sensibilisation avec de l&#39;eau dans un dessicateur sous vide. La figure 2A montre une image de l&#39;expérimental-mis en place. Figure 2B montre un dispositif typique de l&#39;avidine revêt...

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.

Génération de cisaillement d&#39;adhésion Carte Utilisation SynVivo réseaux microvasculaires synthétique

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 vues.  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. 

Vidéo: 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.

Modélisation Plasma surface de lithographie pour la création des réseaux cellulaires

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

Recherche

Bio-ingénierie

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.

Microfluidique endothélialisée pour étudier les interactions microvasculaires dans Hémopathies

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.

L&#39;induction de signaux d&#39;adhésion dépendant de l&#39;aide ultrasons à faible intensité

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.

Utilisant une impédance Cell-substrat et imagerie de cellules vivantes pour mesurer les changements en temps réel dans adhésion cellulaire et De-adhérence induites par 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 ...

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.

Utilisation de la biologie synthétique à l&#39;ingénieur cellules vivantes Interface avec les matériaux programmables

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.

Analyse de l’Expression génique des cellules endothéliales exposées au Stress à l’aide de plusieurs chambres à plaques parallèles flux de cisaillement

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.

Cellule analyse de contrainte de cisaillement en direct sur Pseudomonas aeruginosa en utilisant un système automatisé de microfluidique de débit supérieur

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.

Génération de rats transgéniques utilisant une approche vectorielle lentivirale

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.

Préparation des cellules synthétiques produisant des protéines à l’aide d’extraits bactériens exempts de cellules, de liposomes et de transfert d’émulsion

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.

Fabrication de réseaux de nanotubes lipidiques multi-composants à l’aide du test de motilité de la kinésine glissante

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