Os autores reconhecem Programa criativo Inquérito da Universidade de Clemson e NSF CBET1254609 por fornecer financiamento para este projeto.

1. Produzir uma Câmara MicroChannel criar um gradiente de concentração <ol><li> Cortando uma peça de molde de acrílico <ol><li> Obter um pedaço de acrílico com a largura desejada. Normalmente, utilizam 1 placas de acrílico /-16 polegadas de espessura. folhas mais espessas são difíceis de cortar bem e muito finas folhas não têm a resistência mecânica, causando-lhes a quebrar ou deformar durante o processo. </li><li> Criar um ficheiro CAD com a forma desejada da peça de acrílico para produzir uma cavidade no interior do polidimetilsiloxano (PDMS). Ajustar a largura do canal e comprimento para atingir o desejado gradiente relevantes para protocolos experimentais individuais. <ol><li> Aqui, utilizar uma peça de acrílico com as seguintes dimensões: dois quadrados (0,254 cm x 0,254 centímetros) ligada por um canal de largura 0,127 cm (0,254 cm de comprimento) (Figura 1). </li></ol></li><li> Importar o arquivo CAD para dentro do aparelho de corte a laser e coloque a peça de acrílico em um cortador de laser de acordo com a manuo protocolo do cante. NOTA: Como alternativa, 3D-imprimir a peça a partir do desenho CAD. A vantagem deste método é que os materiais alternativos podem ser usadas para o molde; No entanto, a resolução e a reprodutibilidade pode ser diminuída com impressão em 3D em comparação com o corte a laser. </li></ol></li></ol><img alt="figura 1" src="/files/ftp_upload/55264/55264fig1.jpg" /> Figura 1: (CAD) Representação Computer-Aided Design de microcanais Câmara. Esta imagem descreve o desenho CAD da inserção acrílico necessário para criar a câmara de microcanais. 1 A) Vista de cima do desenho CAD, 1B) Vista lateral do desenho CAD com dimensões em polegadas, 1C) Vista superior de desenho CAD com dimensões em polegadas <a href="http://ecsource.jove.com/files/ftp_upload/55264/55264fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <ol start="2"><li&gt; Verter o polidimetilsiloxano (PDMS) <ol><li> Em um barco de pesagem, misturar 10 partes em peso de base de elastómero comercial (por exemplo, Sylgard 184) com 1 parte em peso de um elastómero comercial Agente de cura (por exemplo, Sylgard 194). Tipicamente, misturar 10 g de Elastómero de base para 1 g de Elastómero Agente de cura. </li><li> Agita-se para combinar a base e o agente de cura com uma micropipeta durante 5 min. </li><li> Defina-se um sino de vácuo e coloque o barco pesar com PDMS no vácuo durante 30 min para remover as bolhas de ar aprisionadas. </li><li> Desligue o vácuo e remover o barco pesa. Despeje o PDMS em cima do acrílico cut-out em uma pequena placa de Petri. Certifique-se de que os PDMS cobre completamente a inserção. </li><li> Permitir que o PDMS a curar durante a noite (pelo menos 18 h) à temperatura ambiente. </li></ol></li><li> Removendo a inserção de PDMS <ol><li> Após a cura do PDMS, remova a inserção cortando cuidadosamente as PDMS ao redor do pedaço de acrílico com um bisturi. </li></ol&gt; </li></ol> 2. plaqueamento das células em uma Câmara MicroChannel <ol><li> Esterilizar a câmara de células de plaqueamento. NOTA: Enquanto PDMS pode ser esterilizado usando óxido de etileno ou outras técnicas, um tratamento rápido de UV é rápido, barato, e suficiente para estudos de culturas celulares. A duração do tratamento UV curto não prejudicar a estrutura mecânica ou a integridade da peça de PDMS. <ol><li> Em um armário de cultura celular padrão, cobrir completamente a câmara com 70% de etanol. </li><li> Coloque a placa de Petri na capela de fluxo laminar com as tampas fora. </li><li> Feche a tampa e ligar a luz UV. Expor à luz UV durante 1-2 h. </li><li> Abra a câmara de fluxo laminar e aguarde 15 minutos para restabelecer o fluxo. </li><li> Remover o etanol a 70%. </li><li> Lavam-se as câmaras duas vezes com solução salina estéril tamponada com fosfato (PBS). Use 1 ml de PBS para cada lavagem. Remover PBS e permitir câmaras para secar durante a noite com as tampas fora. NOTA: Como alternativa, em vezda utilização de uma câmara de fluxo laminar, use uma caixa de câmara de UV para esterilização se estiver disponível. Adicione uma caixa de UV colocando uma fonte de luz UV na parte de trás de uma caixa de cartão revestida com folha de alumínio. A folha de alumínio reflecte a luz para permitir a iluminação uniforme da amostra. </li></ol></li><li> Chapeamento de células em câmara <ol><li> Contagem de células utilizando azul tripano e um hemocitómetro. <ol><li> Adiciona-se 100 uL da suspensão de células de 400 uL de 0,4% de azul de Tripano e misturar suavemente. Adicionar 100 ul desta solução para o hemocitómetro preenchendo suavemente ambas as câmaras sob a lamela de vidro. </li><li> Use um microscópio com uma objetiva de 10X para se concentrar na grelha no hemocitômetro. Use um contador de contagem mão para contar as células não coradas ao vivo dentro de um conjunto de 16 quadrados no hemocitômetro. </li><li> Contagem de células em todas as 4 séries de 16 quadrados. Tome a contagem média de células a partir dos 4 conjuntos de 16 quadrados e multiplicar por 5x10 4. Este número final é o number de células / ml na suspensão de células. </li></ol></li><li> Adicionar 2500 células a uma cavidade em cada uma das câmaras. Isto traduz-se para uma densidade quase confluentes de células de ~ 30000 / cm 2. </li><li> Permitir que as células para se sentar na câmara durante 60 minutos antes de adicionar mídia (de Eagle Modificado por Dulbecco Médio, 10% de soro fetal bovino, 1% de penicilina-estreptomicina). </li></ol></li></ol> 3. Blocos de agarose Dextran Soaked <ol><li> Criando um bloco de agarose <ol><li> Pour 3% de agarose em molde 3D impressa (2 mm x 2 mm x 2 mm). </li><li> Permitir que a agarose solidificar em uma câmara de vácuo durante 30 min para remover quaisquer bolhas. </li><li> Remover o bloco de agarose a partir do molde. NOTA: Como alternativa, derrame 3% de agarose em pequena placa de Petri com uma espessura de 2 mm. Corte um bloco de 2 mm x 2 mm x 2 mm, utilizando um bisturi ou outra ferramenta de corte. Este não é tão preciso como o sistema de molde mas pode ser um pouco mais fáceis de fazer. </li></ol></li><li> completamente submerge o bloco de agarose numa solução com a concentração desejada do factor quimiotáctico. Para avaliar a formação de gradiente de concentração, lavar a câmara pela primeira vez com dextrano fluorescente. A concentração e peso molecular do dextrano deve coincidir com a de um factor solúvel ou droga de interesse. </li><li> Mergulhe a noite bloco de agarose. </li></ol> 4. Time-lapse of a difusão Dextran para avaliar o gradiente de concentração Solúvel Fator <ol><li> Coloque o bloco de agarose dextrano embebido em um pequeno poço quadrado da câmara de microcanais. </li><li> Coloque a câmara de microcanais em um sistema de imagens de células ou utilizar outro microscópio de fluorescência com uma capacidade de lapso de tempo. Comece o lapso de tempo de acordo com as instruções do fabricante. Nota: Os resultados apresentados são tomadas com filtro GFP, 50% de luz, 4/10 pH, e uma ampliação de 4X. </li></ol> 5. Time-lapse do crescimento celular <ol><li> células placa na câmara de microcanais em uma extremidade docâmara. Aqui, usar fibroblastos 3T3. Adicionar 2500 células a uma cavidade na câmara de microcanais. Contagem de células como descrito em 2.2.1. <ol><li> Permitir que as células para se sentar na câmara durante 60 minutos antes de adicionar mídia (de Eagle Modificado por Dulbecco Médio, 10% de soro fetal bovino, 1% de penicilina-estreptomicina). Manter a câmara de microcanais com células plaqueadas a 37 ° C em 5% de CO 2. A migração celular através do canal pode ser vista com um microscópio. </li><li> Adicionar um marcador ou utilização de um defeito microscópico na câmara como um marcador, a fim de servir como um ponto de partida para quantificar a distância que a célula move-se. </li></ol></li><li> Estabelecer um gradiente por colocação de um gel de agarose (8 mm3 bloco) que contém o factor de crescimento concentrado, quimio-atractor, droga, ou outro factor a ser ensaiado (aqui, 40% de FBS está a ser utilizada como um exemplo), a uma extremidade do microcanal câmara. </li><li> Tome imagens de lapso de tempo de frente da célula em movimento. Aqui, os resultados mostram imagens da célula front que foram tomadas a cada 12 h, utilizando um sistema de imagem. </li><li> Use imagens de frente da célula para quantificar a taxa de crescimento celular. Calcula-se a taxa de crescimento utilizando primeiro a função polyfit MATLAB (roipoly) para criar uma máscara poligonal definido pela frente da célula, as paredes do canal, e o marcador de referência. As dimensões desta máscara se obter a distância de migração da frente a partir do qual a velocidade média frente célula é calculada. Outros estudos têm utilizado um método semelhante 8. NOTA: Comparar a borda da frente de crescimento da célula em cada quadro para a concentração do factor solúvel naquela mesma distância. O gradiente de concentração factor solúvel é calculado utilizando um modelo de difusão 1D aproximação. </li><li> Tome imagens fluorescentes das células utilizando faloidina e DAPI coloração. <ol><li> Fix células antes da coloração com faloidina. <ol><li> Quente paraformaldeído a 4% a 37 ° C. </li><li> Adicionar suficiente paraformaldeído a 4% para cobrir as células. Manter as células naparaformaldeído durante 10 min. </li><li> Lavar duas vezes com PBS durante 15 min de cada vez. Use PBS suficiente para cobrir as células. </li></ol></li><li> Remover PBS a partir de células e adicionar solução de permeabilização suficiente (0,1% de Triton X-100 em PBS) para cobrir as células. Deixar solução durante 10 min. </li><li> Lavar duas vezes com PBS durante 5 min de cada vez. Use PBS suficiente para cobrir as células. </li><li> Remover PBS a partir de células e adicionar solução (1% de albumina de soro de bovino em PBS) de bloqueio durante 20 minutos. </li><li> Remoção da solução de bloqueio e lavar 2 vezes com PBS durante 5 min de cada vez. Use PBS suficiente para cobrir as células. </li><li> A partir deste ponto, a proteger as células da luz com folha de alumínio, quando não estiver sendo usado. Remover PBS a partir de células e adicionar 488 faloidina diluídas a 1:50 em PBS durante 1 h. </li><li> Lavar 2 vezes em PBS durante 5 minutos para cada lavagem. Use PBS suficiente para cobrir as células. Em seguida, retire PBS a partir de células. </li><li> Adicionar DAPI (4 &#39;, 6-diamidino-2-fenilindol) diluído 1: 1000 em PBS às células durante 15 min. </li><li> Remover DAPI elavar as células duas vezes com PBS durante 5 minutos para cada lavagem. </li><li> Remover última lavagem e adicione o suficiente PBS para cobrir as células. As células podem agora ser visualizados com um microscópio de fluorescência. </li></ol></li></ol>

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<li>Chen, H. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/15576901">Boyden Chamber Assay.</a> Methods Molec Biol. 2, 15-22 (2005).</li>
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<li>Wells, C. M., Ridley, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+cell+migration+using+the+Dunn+chemotaxis+chamber+and+time-lapse+microscopy%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Analysis of cell migration using the Dunn chemotaxis chamber and time-lapse microscopy.</a> Methods Mol Biol. , 31-41 (2005).</li>
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</ol>

Os autores não têm nada a revelar.

A nossa câmara de microcanal pode ser utilizado para uma variedade de fins, incluindo a determinação da taxa de migração celular em resposta a factores de crescimento e quimioatractores e medindo a taxa de difusão de um fármaco a partir de uma matriz de polímero. É possível utilizar a câmara de microcanais para crescer as células e colocar um quimioatractor numa extremidade da câmara. As células crescem em resposta ao quimioatraente, e a migração de células pode ser quantificada tomando imagens de lapso...

In order for vital processes such as wound healing, immune responses, and embryonic development to occur, cell migration must take place. Cell migration involves the interaction between cells and neighboring cells, the extracellular matrix, and soluble chemical cues (attractants or repellants). As an example, in the process of wound healing, fibroblasts play an integral role in fibrogenesis and wound contraction, where the cells are recruited to the site of injury to synthesize collagen in order to form the extracellular matrix3. Numerous mechanisms behind the migration of fibroblasts to a wound site have been studied, and they include different mechanical, physical, electrical, and chemotactic factors4. Fibroblasts respond especially well to different concentration gradients of growth factors. These different growth factors work together to optimize tissue regeneration5. While observing the chemotactic response of fibroblasts to growth factor concentrations, one can study the pattern of directional migration of fibroblasts and how they orient themselves around physical obstacles in order to reach their destination. Therefore, the goals of this study were to first develop a system in which fibroblast growth could be tracked under guidance by physical barriers and to secondly model the growth of fibroblasts as they navigate through the system.
Currently, the Boyden chamber assay is the most widely used system to measure the migration of cells6. The Boyden chamber consists of a two-chamber multi-well plate where each well may contain medium with or without chemoattractants7. A filter membrane provides a porous interface between the two chambers in each well; this creates a barrier so that cells cannot pass through unless it is by active migration. Typically, for the Boyden chamber, a chemoattractant is added to the lower chamber, and the system is allowed to equilibrate to form a gradient between the upper and lower wells4. One problem with the Boyden chamber assay is that steep gradients end up forming along a single axis perpendicular with the surface of the membrane. This causes the difference in the chemoattractant concentration between the upper and lower wells to be a lower than what was originally expected. Due to this constraint, the Boyden chamber assay makes it hard to correlate specific cell responses with particular gradient characteristics, such as the slope and the concentration difference. Without these measurements, it is hard to study multi-gradient signal integration.
To address some of the constraints of the traditional Boyden chamber assay, microfluidic assays have been developed to form customizable concentration gradients1. Standard methods for creating microfluidic systems require clean rooms for lithographic techniques. These techniques can be difficult to learn especially in a standard classroom setting. Thus, we have designed a chamber system for measuring cell migration that can be made without using a clean room. Using our system, the wells of the assay can be adjusted to a preferred size, and a linear concentration gradient of customized slope can be produced. This allows for accurate measurement of chemotaxis from random movement. The design is an inexpensive and easy-to-use system to model cell growth in response to different chemical stimuli.

<table><tbody><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Sigma-Aldrich</td><td>761036</td><td>poly(dimethylsiloxane) 2-part kit including silicone elastomer base and silicone elastomer curing agent</td></tr><tr><td>Acrylic Sheets</td><td>US Plastic</td><td>44200</td><td></td></tr><tr><td>Disposable Petri Dishes&amp;nbsp;</td><td>Falcon&amp;nbsp;</td><td>25373-041</td><td></td></tr><tr><td>Fluorescein isothiocyanate&amp;ndash;dextran</td><td>Sigma-Aldrich</td><td>FD20s-100MG</td><td></td></tr><tr><td>Agarose, Type I, Low EEO</td><td>Sigma-Aldrich</td><td>A6013-100G</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium</td><td>Fisher Scientific</td><td>11965092</td><td>Cell media components</td></tr><tr><td>Fetal Bovine Serium</td><td>Fisher Scientific</td><td>16000036</td><td>Cell media components</td></tr><tr><td>Penicillin-streptomycin</td><td>Fisher Scientific</td><td>15140148</td><td>Cell media components</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Fisher Scientific</td><td>BP24384</td><td></td></tr><tr><td>EVOS XL Cell Imaging System</td><td>Thermo Fisher Scientific</td><td>AME3300</td><td>Instrument used for taking time-lapse images</td></tr><tr><td>Versa Laser</td><td>Universal Laser Systems, Inc.&amp;nbsp;</td><td>Model number VLS2.30</td><td>Laser cutter used for cutting plastic</td></tr></tbody></table>

a customizable chamber for measuring cell migration

A migração celular é uma parte vital da resposta imune, crescimento e cicatrização de feridas. A migração celular é um processo complexo que envolve interacções entre células, a matriz extracelular e factores químicos solúveis e não solúveis (por exemplo, quimioatractores). Métodos padrão para medir a migração de células, tais como o ensaio de câmara de Boyden, trabalho por contagem de células em ambos os lados de uma divisória. Estas técnicas são fáceis de utilizar; no entanto, eles oferecem pouca modificação geométrica para diferentes aplicações. Em contraste, os dispositivos microfluidicos podem ser utilizados para observar a migração de células com gradientes de concentração personalização de factores solúveis 1, 2. No entanto, os métodos para fazer os ensaios com base microfluidos pode ser difícil de aprender. Aqui, nós descrevemos um método fácil para criar câmaras de cultura de células para medir a migração celular em resposta a gradientes de concentração química. Nossos mi celularesmétodo câmara gração pode criar diferentes gradientes de concentração linear de modo a estudar a migração de células para uma variedade de aplicações. Este método é relativamente fácil de usar e é normalmente realizada por alunos de graduação. A câmara de microcanais foi criado por colocação de uma inserção de acrílico na forma da câmara de microcanais finais bem em uma placa de Petri. Depois disso, o poli (dimetilsiloxano) (PDMS) foi deitada no topo da inserção. O PDMS foi deixada endurecer e, em seguida, a inserção foi removida. Isto permitiu a criação de cavidades em qualquer forma ou tamanho desejado. As células podem ser posteriormente adicionado à câmara de microcanais, e agentes solúveis podem ser adicionados a um dos poços por imersão de um bloco de agarose, o agente desejado. O bloco de agarose é adicionado a um dos poços, e as imagens de lapso de tempo pode ser feita da câmara de microcanal, a fim de quantificar a migração celular. Variações deste método pode ser feita para uma dada aplicação, tornando este método highly personalizável.

A Figura 2 mostra o movimento da parte dianteira da célula através do canal em resposta a um gradiente de soro fetal de bovino colocado na extremidade oposta do canal, de onde as células são plaqueadas. A parte dianteira da célula é mostrada em 48 h (Figura 2A), 72 h (Figura 2B), 96 h (Figura 2C), e 120 h (Figura 2D) pós chapeamento. O movimento da frente de celular foi rastreado com estas imagens...

Watch this Scientific Journal Video about A Customizable Chamber for Measuring Cell Migration at JoVE.com

A Customizable Chamber for Measuring Cell Migration

Este protocolo detalha um método personalizada para medir a migração celular em resposta a quimioatractores, que também podem ser utilizados para determinar a taxa de difusão de um fármaco a partir de uma matriz de polímero.

A Câmara customizáveis ​​para a migração celular de medição

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between ...

a-customizable-chamber-for-measuring-cell-migration

Research

JoVE Journal

Biology

10.6K visualizações.  Clemson University.  Este protocolo detalha um método personalizada para medir a migração celular em resposta a quimioatractores, que também podem ser utilizados para determinar a taxa de difusão de um fármaco a partir de uma matriz de polímero. 

Vídeo: A Customizable Chamber for Measuring Cell Migration

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels &#8211; either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention.
We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

Avaliação da migração de células endoteliais após a exposição a produtos químicos tóxicos

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

Biologia do Desenvolvimento

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an &#34;in-incubator&#34; video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

Avaliação do processo de migração e invasão celular: uma comparação da risque baseado no microscópio a ferida do ensaio e o ensaio de câmara Boyden

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

Pesquisa do câncer

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

Avaliação do Câncer de Migração Stem Cell Usando compartimentar dispositivos microfluídicos e imagens de células vivas

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

Medicina

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Criando Gradientes adesivas e solúvel para a migração celular Imaging com Microscopia de Fluorescência

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Bioengenharia

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

Um fluxo de trabalho simples migração/invasão usando um automatizado Imager viver-pilha

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

Estudo da migração celular em Canais microfabricados

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

Real-Time Quantitative Measurement of Tumor Cell Migration and Invasion Following Synthetic mRNA Transfection

Tumor cells are highly motile and invasive and display altered gene expression patterns. Knowledge of how changes in gene expression regulate tumor cell migration and invasion is essential for understanding the mechanisms of tumor cell infiltration into neighboring healthy tissues and metastasis. Previously, it was demonstrated that gene knockdown followed by the impedance-based real-time measurement of tumor cell migration and invasion enables the identification of the genes required for tumor cell migration and invasion. Recently, the mRNA vaccines against SARS-CoV-2 have increased interest in using synthetic mRNA for therapeutic purposes. Here, the method using synthetic mRNA was revised to study the effect of gene overexpression on tumor cell migration and invasion. This study demonstrates that elevated gene expression with synthetic mRNA transfection followed by impedance-based real-time measurement may help identify the genes that stimulate tumor cell migration and invasion. This method paper provides important details on the procedures for examining the effect of altered gene expression on tumor cell migration and invasion.

Many upregulated genes stimulate tumor cell migration and invasion, leading to poor prognosis. Determining which genes regulate tumor cell migration and invasion is critical. This protocol presents a method for investigating the effects of the increased expression of a gene on the migration and invasion of tumor cells in real time.

Mensuração Quantitativa em Tempo Real da Migração e Invasão de Células Tumorais Após Transfecção de RNAm Sintético

Tumor cells are highly motile and invasive and display altered gene expression patterns. Knowledge of how changes in gene expression regulate tumor cell ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

Na migração celular in vitro e invasão Ensaios

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Biologia

Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells

The physiological electric field serves specific biological functions, such as directing cell migration in embryo development, neuronal outgrowth and epithelial wound healing. Applying a direct current electric field to cultured cells in vitro induces directional cell migration, or galvanotaxis. The 2-dimensional galvanotaxis method we demonstrate here is modified with custom-made poly(vinyl chloride) (PVC) chambers, glass surface, platinum electrodes and the use of a motorized stage on which the cells are imaged. The PVC chambers and platinum electrodes exhibit low cytotoxicity and are affordable and re-useable. The glass surface and the motorized microscope stage improve quality of images and allow possible modifications to the glass surface and treatments to the cells. We filmed the galvanotaxis of two non-tumorigenic, SV40-immortalized prostate cell lines, pRNS-1-1 and PNT2. These two cell lines show similar migration speeds and both migrate toward the cathode, but they do show a different degree of directionality in galvanotaxis. The results obtained via this protocol suggest that the pRNS-1-1 and the PNT2 cell lines may have different intrinsic features that govern their directional migratory responses.

We present a method to apply a physiological electric field to migrating, immortalized prostate cells in a custom-made galvanotaxis chamber. Using this method, we demonstrate that 2 lines of non-tumorigenic prostate cells demonstrate different degrees of migration directionality in the field.

Utilizando a pedido de clientes Galvanotaxis Chambers para Estudar Direcional migração das células da próstata

The physiological electric field serves specific biological functions, such as directing cell migration in embryo development, neuronal outgrowth and ...

Quantifying Three-Dimensional Cell Migration Within and Into Granular Hydrogel Biomaterials

Granular hydrogel scaffolds hold significant potential in regenerative medicine, functioning either as carriers for cell delivery or as interfaces for tissue integration. This article introduces two novel approaches for quantifying cell migration within and into granular hydrogels, highlighting the distinct applications of these scaffolds. First, a cell monolayer interface assay that simulates tissue growth into granular hydrogels for integration purposes is presented. Second, a spheroid-based assay is described, designed to track cell movement within the hydrogel matrix, specifically suited for applications involving cell delivery. Both methods enable precise and controlled measurements of cell migration, providing a comprehensive toolkit for researchers utilizing granular hydrogel scaffolds. The motivation for these methods stems from the need for tailored control over cell migration within the scaffold to align with specific applications. By optimizing and standardizing these quantification techniques, researchers can iteratively refine granular hydrogel properties, ensuring their effectiveness in diverse regenerative medicine contexts. This robust set of quantitative tools offers new opportunities to enhance granular hydrogel scaffolds, advancing their use in both cell delivery and tissue integration applications.

A protocol for the quantitative evaluation of 3D cell migration within and into the interface of granular hydrogels is presented here.

Quantificando a migração celular tridimensional dentro e dentro de biomateriais de hidrogel granular

Granular hydrogel scaffolds hold significant potential in regenerative medicine, functioning either as carriers for cell delivery or as interfaces for ...

A Câmara customizáveis para a migração celular de medição

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