Tecnología SynVivo fue desarrollada bajo la concesión # 2R44HL076034 del NHLBI.

1. Cebado del dispositivo de microfluidos SynVivo-SMN <ol><li> Cada puerto (entrada / salida) del dispositivo se compone de dos puertos paralelos - uno para hacer fluir en restos de revestimiento de superficies (moléculas de adhesión, matrices de crecimiento, etc) y / o células para la siembra y el otro para realizar el ensayo (Figura 1A ). </li><li> Sumerja completamente el dispositivo de microfluidos SynVivo-SMN (Figura 1B) en una placa de Petri que contiene desionizada estéril (DI) y colocar el plato en un desecador de vacío. Permitir que el desecador a funcionar hasta que todo el aire se elimina de los canales del dispositivo. Esto debe tomar unos 15 min. </li><li> Antes de extraer el dispositivo del agua, coloque la tubería de Tygon (OD de 0,06 &quot;y el ID de 0,02&quot;) cebado con agua en cada puerto del dispositivo con una pinza de punta fina. El tubo debe ser de aproximadamente 1 pulgada de longitud. El dispositivo puede ahora ser retirado del agua. Figura 1C muestra la imagen de la DevicE con la tubería. </li></ol> 2. Revestimiento del dispositivo de microfluidos con proteína deseada (por ejemplo, avidina) <ol><li> Con una pipeta, coloque una gota de agua (aproximadamente 100 l) alrededor de la base del tubo de un puerto de entrada. Retire con cuidado la tubería usada para cebar el dispositivo. La gota de agua evitará que entre aire en el dispositivo. </li><li> Preparar una jeringa de 1 ml cargado con avidina a una concentración de 20 mg / ml. Conectar la jeringa a un 24 T de la aguja y tubo de acero inoxidable. Inserte la tubería a uno de los puertos de entrada del dispositivo. Sujete el puerto de entrada que no se utiliza con una pinza mandíbula. </li><li> Inyectar avidina a un caudal de 1 l / min durante 10 min para permitir la perfusión completa del dispositivo. Al final del tiempo de flujo, sujetar el tubo con la abrazadera de la mandíbula y colocar el dispositivo a 4 ° C durante la noche. </li></ol> 3. Transmitir el Biotinylated Partículas los estudios de adhesión <ol><li> Deje que el dispositivollegar a temperatura ambiente. Coloque el dispositivo en un microscopio de fluorescencia invertido equipado con una platina motorizada y una cámara de alto rendimiento. </li><li> Preparar una solución de 2 micras partículas biotinilados a una concentración de 5 x 10 6 partículas / ml en tampón fosfato salino (PBS). Cargue las partículas en una jeringa de 1 ml. Preparar una segunda jeringa de 1 ml de PBS solo. Cargue cada jeringa en una bomba de jeringa y conectar con la aguja y el tubo. </li><li> Con una pipeta, coloque una gota de agua (aproximadamente 100 l) alrededor de la base del tubo de orificio de entrada. Retire con cuidado el tubo utilizado para recubrir el dispositivo. La gota de agua evitará que entre aire en el dispositivo. </li><li> Inserte con cuidado el tubo de partículas con biotina y PBS de la etapa 3.2 en cada uno de los puertos de entrada. Figura 2A muestra la imagen de la puesta a punto. </li><li> Iniciar la inyección de partículas biotiniladas a un caudal de 2,5 l / min. Monitorear el puerto de entrada en el microscopio. A la primera señalde partículas, iniciar el temporizador y continuar flujo durante 3 min. </li><li> Al final de los 3 minutos, detener el flujo de partículas biotiniladas, mientras que mirando simultáneamente el flujo de PBS a un caudal de 2,5 l / min. Permita PBS fluya en el dispositivo durante 3 min para lavar las partículas no unidas. </li></ol> 4. Adquisición de imágenes y realización de su área de interés (AOI) Mediciones Usando software de imágenes (NIKON Elementos) <ol><li> Utilice la función &quot;imagen grande de exploración&quot; en el software de imágenes para adquirir la imagen de todo el dispositivo. </li><li> Número secuencialmente las bifurcaciones en el dispositivo y crear un AOI circular con dos veces el diámetro de los canales. En este caso, ajuste el diámetro AOI a 200 m desde el diámetro del canal es de 100 micras. </li><li> Utilice la función de conteo automatizado en el software de imagen para exportar el número de partículas en cada AOI a una hoja de MS Excel. </li><li> Del mismo modo, utilizar la función de conteo automatizado para exportar el número de partículas en todo eldispositivo. </li></ol> 5. Análisis de partículas de fundente Usando Dinámica de Fluidos Computacional (CFD) Modelos <ol><li> Simulaciones CFD se ejecutan mediante un software comercial (CFD-ACE +, ESI Inc.) para la topología de dispositivo SynVivo-SMN. Los resultados se almacenan en una base de datos para el análisis de observaciones experimentales. La información del almacén resultados de la simulación en las tasas de cizallamiento, velocidad, flujo de partículas, y la adhesión en el dispositivo. </li><li> Los resultados de la simulación se utilizan para determinar el número de partículas que entran en cada AOI basado en una concentración de partículas de entrada dada. </li></ol> 6. Generación de cizallamiento Adhesión Mapa <ol><li> Calcular el% de adherencia dividiendo las partículas adheridas en la bifurcación por las partículas que fluyen en la bifurcación como se muestra en la ecuación 6.1. <img alt="Ecuación 1" fo:content-width="3in" src="/files/ftp_upload/51025/51025eq1.jpg" /> donde el número de partículas adheridas y las partículas que fluyen se obtienen a partir de protocol los pasos 4.3 y 5.2, respectivamente. </li><li> Trazar el mapa de adhesión por cizalladura usando la velocidad de cizallamiento en cada bifurcación de las redes obtenidas a partir de la base de datos en el paso (5.1) y los valores de adhesión% obtenidos a partir de la ecuación 6.1. </li></ol>

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Tasa de publicación de este artículo, patrocinado por CFD Research Corporation.

Cámaras de flujo de placas paralelas, mientras que proporciona una mejor aproximación a las interacciones célula-célula y célula-partículas, sufren de varias limitaciones tales como el alto consumo de reactivos y la necesidad de múltiples corridas experimentales para generar un mapa de adhesión por cizalladura. El uso de redes microvasculares SynVivo-sintéticos (SynVivo-las SNM) permite la generación de un mapa de adhesión al cizallamiento de un solo experimento en condiciones que imitan las condiciones i...

Los ensayos actuales para estudiar a la célula-célula y las interacciones célula-partícula implican típicamente formato de placa de estática en la que las partículas o células se incuban en matrices de proteínas o células adherentes. Al final del tiempo de incubación especificado, los números de partículas adherentes o células se cuantifican mediante microscopía de 1. A pesar de que estos ensayos proporcionan una información valiosa sobre los procesos bioquímicos detrás de estas interacciones, una limitación clave es la falta de flujo de fluido fisiológico (típico de la microcirculación) y su impacto en la adherencia de partículas. Para superar esta limitación, en cámaras de flujo in vitro se han desarrollado en los últimos años. Un elemento común de estas cámaras de flujo es un aparato transparente perfundido a bajos números de Reynolds para que coincida con velocidades de cizallamiento de pared observadas en los vasos sanguíneos in vivo 2. La pared del vaso se modela por cualquiera de recubrimiento de biomoléculas o crecimiento de células en una superficie del flujo Chamber 3. Las partículas de 4-7 o 8-16 células son luego fluyeron en al intervalo deseado de velocidades de flujo para cuantificar el número de adherirse partículas bajo diversas velocidades de cizallamiento. Sin embargo, el uso de cámaras de flujo de placas paralelas para estudiar y validar los fenómenos bioquímicos es bastante caro y consume mucho tiempo. Esto se debe principalmente al hecho de que múltiples experimentos deben llevarse a cabo para generar un mapa de la cizalla de fluidos vs el número de partículas / células adheridas. Además, las cámaras de flujo placa requieren grandes volúmenes de reactivos debido a su gran tamaño (altura&gt; 250 micras y la anchura&gt; 1 mm). Finalmente, estos dispositivos no modelan con precisión las características geométricas (por ejemplo, bifurcaciones) y las condiciones de flujo (por ejemplo, convergentes vs flujos divergentes) que están presentes in vivo. Los recientes avances en la litografía basada microfabricación 17-19 han acelerado el campo de lab-on-a-chipdispositivos 20-21. Estos dispositivos han sido fundamentales en el desarrollo de una versión en miniatura de la cámara de flujo de placas paralelas con las dimensiones en el régimen micrómetro. La reducción en la dimensión también produce beneficios significativos en términos de volúmenes de reactivos, células o de partícula requerido para los experimentos. Sin embargo, una limitación clave de los dispositivos actualmente disponibles es el uso de canales lineales para modelar microvasos, que no imitan la microvasculatura complejo observado in vivo. Recientemente hemos desarrollado una nueva metodología para recrear las redes microvasculares sobre sustratos de plástico desechables resultantes en representación sintética de las condiciones in vivo. Estos dispositivos denominados redes microvasculares SynVivo-sintéticos (SMN) se desarrollan utilizando PDMS basado proceso de soft-litografía. Dispositivos SynVivo-SMN se pueden utilizar para obtener cizallamiento mapa de adhesión de la adhesión celular / de partículas 22, estudio dirigido de suministro de fármacos 23 y HAVE ha validado contra datos in vivo 24-25. En este trabajo, presentamos un protocolo que permite la generación del mapa de adhesión de cizallamiento de un solo experimento en volúmenes tan pequeños como 5.1 l que resulta en un ahorro significativo de recursos y tiempo.

<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

Ensayos de adhesión celular / partículas son fundamentales para la comprensión de las interacciones bioquímicas involucradas en la fisiopatología de la enfermedad y tienen importantes aplicaciones en la búsqueda del desarrollo de nuevas terapias. Los ensayos con condiciones estáticas no logran captar la dependencia de adherencia en cizalla, lo que limita su correlación con el medio ambiente en vivo. Cámaras de flujo de placas paralelas que cuantifican la adhesión bajo un flujo de fluido fisiológico necesitan múltiples experimentos para la generación de un mapa de adhesión por cizalladura. Además, no representan la escala in vivo y la morfología y requieren volúmenes grandes (~ ml) de reactivos para los experimentos. En este estudio, hemos demostrado la generación de cizalla mapa adhesión de un solo experimento utilizando una red microvascular basado en un dispositivo de microfluidos, SynVivo-SMN. Este dispositivo recrea el complejo en la vasculatura vivo incluyendo una escala geométrica, elementos morfológicos, las características de flujo y las interacciones celulares en unFormato in vitro, proporcionando de esta manera un ambiente biológicamente realista para la investigación básica y aplicada en el comportamiento celular, la administración de fármacos, y el descubrimiento de fármacos. El ensayo se demostró mediante el estudio de la interacción de las partículas de 2 micras de biotina-revestido con superficies recubiertas con avidina del microchip. Se obtiene toda la gama de cizallamiento observado en la microvasculatura en un único ensayo que permite la adhesión vs mapa de cizallamiento para las partículas en condiciones fisiológicas.

La Figura 1A muestra una imagen de un campo brillante y esquemática de dispositivo de SynVivo-SMN. Figura 1B muestra el dispositivo SynVivo-SMN montado en un portaobjetos de vidrio. Figura 1C muestra el dispositivo con un tubo siguiente cebado con agua en un desecador de vacío. La Figura 2A muestra una imagen de la-montaje experimental. Figura 2B muestra un típico dispositivo SynVivo-SMN avidina-revestido...

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.

Generación de Shear Adhesión Mapa Utilizando SynVivo redes microvasculares sintético

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

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8.8K vistas.  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.

La litografía de plasma superficial Patrones de Creación de redes de telefonía

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

Investigación

Bioingeniería

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.

Microfluídica endotelizado para estudiar las interacciones microvasculares en Enfermedades Hematológicas

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.

La inducción de señales de adhesión dependientes de Uso de ultrasonido de baja intensidad

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.

Utilizando Impedancia Cell-sustrato y imágenes de células vivas para medir los cambios en tiempo real en la adhesión celular y De-adhesión inducidas por Matrix Modificación

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.

El uso de la biología sintética para Ingeniero células vivas que interactúan con materiales programables

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.

Análisis de expresión génica de las células endoteliales expuestas a estrés mediante múltiples cámaras de placas paralelas flujo del esquileo

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.

Análisis de la célula de tensión de esquileo en vivo en Pseudomonas aeruginosa mediante un sistema de microfluidos superior rendimiento automatizado

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.

Generación de ratas transgénicas utilizando un enfoque vectorial lentiviral

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.

Preparación de células sintéticas productoras de proteínas mediante extractos bacterianos libres de células, liposomas y transferencia de emulsión

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.

Fabricación de redes de nanotubos lipídicos multicomponente utilizando el ensayo de motilidad de quinesina deslizante

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