Tecnologia SynVivo foi desenvolvido sob concessão # 2R44HL076034 do NHLBI.

1. Priming o dispositivo micro-SynVivo SMN <ol><li> Cada porta (entrada / saída) do dispositivo é constituída por duas portas paralelas - uma para fluir em porções de revestimento de superfície (moléculas de adesão, as matrizes de crescimento, etc) e / ou de células de sementeira e o outro para a execução do ensaio (Figura 1A ). </li><li> Completamente submergir o dispositivo de microfluidos SynVivo-SMN (Figura 1B) em uma placa de Petri contendo estéril desionizada (DI) e colocar o prato num exsicador de vácuo. Permitir que o exsicador para rodar até que todo o ar foi removido dos circuitos do dispositivo. Isso deve levar cerca de 15 min. </li><li> Antes de retirar o dispositivo da água, coloque tubos Tygon (OD de 0,06 &quot;e ID de 0,02&quot;) preparado com água em cada porta do dispositivo com uma pinça de ponta fina. O tubo deve ser de aproximadamente 1 cm em comprimento. O dispositivo pode agora ser removido a partir da água. Figura 1C mostra uma imagem do multenviar um e com o tubo. </li></ol> 2. Revestimento do dispositivo micro com proteína desejada (por exemplo, Avidin) <ol><li> Usando uma pipeta, colocar uma gota de água (cerca de 100 ul) à volta da base do tubo de uma porta de entrada. Remova cuidadosamente o tubo usado para preparar o dispositivo. A gota de água vai impedir que o ar que entra no dispositivo. </li><li> Prepara-se uma seringa de 1 ml carregada com avidina a uma concentração de 20 ug / ml. Conecte a seringa a uma agulha de aço inoxidável 24 G e tubos. Inserir o tubo a uma das portas de entrada do dispositivo. Prenda a porta de entrada não está sendo utilizada com uma braçadeira da mandíbula. </li><li> Injectar avidina a uma taxa de fluxo de 1 mL / min durante 10 min para permitir a perfusão completa do dispositivo. No final do tempo de escoamento, apertar o tubo com o grampo de mandíbula e colocar o dispositivo a 4 ° C durante a noite. </li></ol> 3. Fluindo o Biotinylated Partículas para experimentos de adesão <ol><li> Permitir que o dispositivovoltar à temperatura ambiente. Colocar o dispositivo sobre um microscópio invertido de fluorescência equipado com um estádio motorizado e uma câmara de alta performance. </li><li> Prepara-se uma solução de 2 mM partículas biotinilados numa concentração de 5 x 10 6 / ml, em partículas de Tampão Fosfato Salino (PBS). Carregar as partículas numa seringa de 1 ml. Prepara-se uma segunda seringa de 1 ml de PBS apenas. Coloque cada seringa em uma bomba de seringa e se conectar a agulha e tubos. </li><li> Usando uma pipeta, colocar uma gota de água (cerca de 100 ul) à volta da base do tubo da porta de entrada. Cuidadosamente remover a tubagem utilizada para o revestimento do dispositivo. A gota de água vai impedir que o ar que entra no dispositivo. </li><li> Insira com cuidado o tubo de partículas para biotinilados e PBS a partir do passo 3.2 em cada uma das portas de entrada. Figura 2A mostra imagem do set-up. </li><li> Começam a injectar partículas biotinilados a um caudal de 2,5 mL / min. Monitorar a porta de entrada no microscópio. Ao primeiro sinaldas partículas, começa a contagem regressiva e continuar o fluxo por 3 min. </li><li> No final dos 3 minutos, parar o fluxo de partículas biotinilados enquanto simultaneamente encarando o fluxo de PBS a uma taxa de fluxo de 2,5 mL / min. Permitir PBS a fluir no dispositivo por 3 min para lavar partículas livres. </li></ol> 4. Aquisição de Imagens e tornando a área de interesse (AOI) Medidas Usando Software Imaging (NIKON Elements) <ol><li> Use a função &quot;imagem grande varredura&quot; no software de imagem para obter a imagem de todo o dispositivo. </li><li> Número sequencialmente as bifurcações do dispositivo e criar uma AOI circular com duas vezes o diâmetro dos canais. Neste caso, definir o diâmetro AOI a 200 um, uma vez o diâmetro do canal é de 100 um. </li><li> Use a função de contagem automática no software de imagem para exportar o número de partículas em cada AOI para uma folha de MS Excel. </li><li> Da mesma forma, usar o recurso de contagem automática para exportar o número de partículas em toda adispositivo. </li></ol> 5. Partículas Análise de Fluxo Utilizando Dinâmica dos Fluidos Computacional (CFD) Modelos <ol><li> Simulações de CFD são executados através de software disponível comercialmente (CFD-ACE +, ESI Inc.) para a topologia dispositivo SynVivo-SMN. Os resultados são armazenados numa base de dados para a análise de observações experimentais. Os resultados da simulação armazenar informações sobre as taxas de cisalhamento de parede, velocidade, fluxo de partículas e de adesão no dispositivo. </li><li> Os resultados de simulação são usados ​​para determinar o número de partículas que entram cada AOI com base em uma dada concentração de partículas de entrada. </li></ol> 6. Geração de cisalhamento Adesão Mapa <ol><li> Calcular% de adesão através da divisão das partículas aderentes na bifurcação das partículas que fluem na bifurcação, como mostrado na equação 6.1. <img alt="Equação 1" fo:content-width="3in" src="/files/ftp_upload/51025/51025eq1.jpg" /> em que o número de partículas aderidas e as partículas fluem são obtidos a partir de protocolo os passos 4.3 e 5.2, respectivamente. </li><li> Traçar o mapa de adesão ao corte com base na taxa de cisalhamento em cada bifurcação das redes obtidas a partir da base de dados na etapa (5.1) e os valores de aderência% obtidos a partir da equação 6.1. </li></ol>

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Taxa de publicação deste artigo patrocinado pela CFD Research Corporation.

Câmaras de fluxo de placas paralelas, proporcionando insights significativos sobre as interacções célula-célula e célula-partículas, sofrem de várias limitações, tais como o elevado consumo de reagentes e a necessidade de vários ensaios experimentais para gerar um mapa de adesão de cisalhamento. O uso de redes microvasculares SynVivo-sintéticos (SynVivo-SMNs) permite a geração de um mapa de adesão ao cisalhamento de um único experimento em condições que imitam as condições in vivo. Além di...

Os ensaios correntes para estudar a célula-célula e as interacções célula-partículas tipicamente envolvem formato bem prato estático, em que as partículas ou as células são incubadas em matrizes de proteínas ou células aderentes. No final do tempo de incubação específico, o número de partículas aderentes ou células são quantificados usando microscopia 1. Mesmo que estes ensaios fornecem um panorama significativo para os processos bioquímicos por trás dessas interações, uma limitação importante é a falta de fluxo de fluido fisiológico (típico da microcirculação) e seu impacto sobre a adesão de partículas. Para superar esta limitação, na câmara de fluxo in vitro têm sido desenvolvidos nos últimos anos. Um elemento comum destas câmaras de fluxo é um aparelho transparente perfundidos a baixo número de Reynolds para combinar taxas de cisalhamento de parede observadas nos vasos sanguíneos in vivo 2. A parede do vaso é modelado por qualquer revestimento de biomoléculas ou crescimento de células numa superfície do fluxo cHamber 3. Partículas 4-7 ou células 8-16 são então fluiu na faixa desejada de taxas de fluxo para quantificar o número de aderir partículas sob várias taxas de cisalhamento. No entanto, a utilização de câmaras de fluxo de placa paralelas para estudar e validar os fenómenos bioquímicos é bastante dispendioso e moroso. Isto é principalmente devido ao fato de que as múltiplas experiências devem ser conduzidas para gerar um mapa de cisalhamento de fluidos em relação ao número de partículas / células aderidas. Além disso, as câmaras de fluxo de placa requerem grandes volumes de reagentes, devido ao seu grande tamanho (altura&gt; 250 um e largura de&gt; 1 mm),. Finalmente, estes dispositivos não modelar de forma precisa (por exemplo, características geométricas, bifurcações) e as condições de fluxo (por exemplo, contra os fluxos divergentes convergentes) que estão presentes in vivo. Os recentes avanços na litografia baseada microfabricação 17-19 aceleraram o campo de lab-on-a-chipdispositivos 20-21. Estes dispositivos têm sido fundamentais para o desenvolvimento de uma versão em miniatura da câmara de fluxo de placas paralelas com dimensões no regime micrômetro. A redução da dimensão também produz benefícios significativos em termos de volumes de reagentes, células ou partículas necessária para os experimentos. No entanto, uma limitação chave dos dispositivos actualmente disponíveis é a utilização de canais lineares para modelar microvasos, que não mimetizam a microvasculatura complexo observada in vivo. Recentemente, desenvolveu uma nova metodologia para a recriação de redes microvasculares sobre substratos de plástico descartáveis, resultando em representação sintética das condições in vivo. Estes dispositivos denominado Redes microvasculares SynVivo-sintéticos (SMN) são desenvolvidos utilizando PDMS processo soft-litografia baseia. Dispositivos SynVivo-SMN pode ser usado para obter mapa de adesão ao cisalhamento de celular / partícula adesão 22, o estudo direcionado a entrega da droga e 23 have foi validado contra dados in vivo 24-25. Neste artigo, apresentamos um protocolo que permite a geração do mapa de adesão ao cisalhamento de um único experimento em volumes tão pequenos quanto 1-5 mL, assim, resultando em significativa economia de recursos e tempo.

<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

Ensaios de adesão celular / partículas são fundamentais para a compreensão das interações bioquímicas envolvidas na fisiopatologia da doença e têm aplicações importantes na busca pelo desenvolvimento de novas terapias. Os ensaios com condições estáticas não conseguem captar a dependência da adesão em cisalhamento, limitando a sua correlação com o ambiente in vivo. Câmaras de fluxo de placas paralelas que quantificam a adesão sob fluxo de fluido fisiológico precisar de vários experimentos para a geração de um mapa de adesão ao cisalhamento. Além disso, elas não representam a escala in vivo e morfologia e requerem grandes volumes (~ ml) de reagentes para os experimentos. Neste estudo, demonstramos a geração de mapa de adesão ao cisalhamento de um único experimento usando uma rede microvascular baseado dispositivo micro, SynVivo-SMN. Este dispositivo recria o complexo na vasculatura vivo incluindo escala geométrica, elementos morfológicos, as características de fluxo e interações celulares em umin vitro formato, proporcionando assim um ambiente biologicamente realista para pesquisa básica e aplicada no comportamento celular, distribuição de medicamentos, e descoberta de drogas. O ensaio foi demonstrada pelo estudo da interacção das partículas de 2 um revestido com biotina com as superfícies revestidas com avidina da microplaca. A gama inteira de corte observada na microvasculatura é obtido com um único ensaio permitindo adesão versus mapa de cisalhamento para as partículas sob condições fisiológicas.

A Figura 1A mostra uma de uma imagem de campo brilhante e esquemática do dispositivo SynVivo-SMN. Figura 1B mostra o dispositivo SynVivo-SMN montado sobre uma lâmina de vidro. Figura 1C mostra o dispositivo com tubo seguinte priming com água num exsicador de vácuo. A Figura 2A mostra uma imagem do experimental-configurado. Figura 2B mostra um típico dispositivo revestido de avidina SynVivo-SMN após a l...

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.

Geração de cisalhamento Adesão Mapa Usando SynVivo Networks microvasculares sintético

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

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Bioengineering

8.8K visualizações.  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. 

Vídeo: 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.

Padronização de plasma de superfície litografia para a Criação de Redes de celular

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

Bioengenharia

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.

A microfluídica Endothelialized para estudo das interacções microvasculares em Doenças 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.

Indução de Sinais de adesão dependentes Usando ultra-som de baixa intensidade

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.

Usando impedância Cell-substrato e imagens de células vivas para medir as mudanças em tempo real no celular Adesão e De-adesão Induzidas por Matrix Modificação

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.

Usando biologia sintética para manipular células vivas que fazem interface com materiais programáveis

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.

Análise de expressão genética das células endoteliais expostos para Shear Stress usando múltiplas câmaras de fluxo paralelo-placa

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.

Ao vivo celular análise de tensão de cisalhamento na Pseudomonas aeruginosa , usando um sistema automatizado de Microfluidic superior-taxa de transferência

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.

Geração de ratos transgênicos usando uma abordagem vetorial 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.

Preparando proteínas que produzem células sintéticas usando extratos bacterianos livres de células, liposomos e transferência de emulsão

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.

Fabricando redes nanotubos lipídes de lipídicas multi-componentes usando o ensaio de motilidade de mobilidade de cinévola gliding

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