Os autores agradecem W. Sun e K. Jansen para as discussões críticas, e reconhecer o apoio pelo Nano Laboratório de Biomecânica da Universidade Nacional de Cingapura. NAK reconhece o apoio por um Marie Curie IIF Fellowship.

Colheita 1. celular <ol><li> Obter MD-MBA-231 a partir do 37 ° C, 5% de CO2. Separar as células do tecido placa de cultura utilizando-se 0,5% de solução de Tripsina-EDTA. Use 1 ml de solução de tripsina-EDTA para células cultivadas num frasco T25. </li><li> Células de pellets em um tubo de 15 ml por centrifugação a 200 x g durante 4 min, aspirar o sobrenadante e ressuspender as células em 5 ml de meio de cultura. </li><li> Contagem de densidade celular, ρ, utilizando um hemocitómetro. Nota: Para preparar o gel interior da célula-semeado, a suspensão de células vai ser, mais tarde, diluiu-se 10 × para atingir a densidade final de células de semeadura. Portanto, 10 × suspensão concentrada de célula é necessário. </li><li> Calcula-se o volume de meio necessário para atingir uma concentração de 10 × celular: <img alt="Equação 1" fo:content-width="2in" src="/files/ftp_upload/52735/52735eq1.jpg" width="175" /> Nota: A densidade final semeadura de células, c final, de cerca de 215; 10 6 células / ml é recomendado para MD-MBA-231 e é utilizado no presente protocolo. Outros densidades de sementeira podem também ser explorado para outros tipos de células. </li><li> Células de pellets mais uma vez em um tubo de 15 ml por centrifugação a 200 × g por 4 min, e aspirar o sobrenadante. </li><li> Ressuspender as células na quantidade necessária (V médio calculadas no passo 1.3) de meio de cultura celular livre de soro cuidadosamente para minimizar a aglutinação celular. Nota: O fenol vermelho é auto-fluorescente, e pode interferir com a imagiologia por fluorescência / reflectância. O uso de meio isento de vermelho de fenol pode ser considerado para alcançar melhor qualidade de imagem. </li></ol> 2. Preparação de Soluções de colágeno <ol><li> Obter a solução de colagénio de estoque, 10 × tampão PBS, Milli-Q H 2 O, NaOH a 0,1 M, e vários tubos de microcentrífuga. Mantenha tudo no gelo para evitar a polimerização prematura de colágeno, e manter a condição estéril. </li><li> Equilibrar uma Glass- estérilprato inferior por pré-aquecendo-a no 37 ° C incubadora. Nota: Todos os volumes neste protocolo foram otimizados para um prato fundo de vidro de 12 mm assim. Se outro tipo prato é usado, ajustar os volumes proporcionalmente. </li><li> Calcular o volume requerido necessário para preparar 50 ul de solução de colagénio a 2,4 mg / ml para o gel interior (solução I) com base na concentração de estoque de colagénio. Nota: Outras concentrações de colagénio para o interior de gel pode também ser usado. </li><li> Em um ambiente estéril (tipicamente um capuz biosegurança), adiciona-se lentamente 5 mL de 10 × tampão PBS para a quantidade necessária de solução-mãe de colagénio (calculado no passo 2.3), com agitação suave. Tome cuidado para evitar a formação de bolhas de ar. </li><li> Ajustar o pH da mistura para 7,4 utilizando NaOH 0,1 M calibrado usando medidor de pH. Como orientação geral, utilizar cerca de 5 ul para trazer o pH para perto de 7,4 (a quantidade varia dependendo da concentração estoque e pH). Nota: Observe que os volumes envolvidos nestepasso é demasiado pequeno para o uso do medidor de pH padrão. Use um dos seguintes truques: <ol><li> Preparar soluções de colágeno para várias amostras. Ajustar o pH em grandes quantidades usando um medidor de pH padrão e distribuir as soluções de colagénio entre as amostras. </li><li> Em alternativa, ajustar o pH de uma solução de colagénio em maior volume (i. E., O volume que permitem o uso de medidor de pH padrão). Note-se a quantidade de NaOH necessária para trazer o pH para o valor final de pH. Reduza os volumes e usar a quantidade apropriada de NaOH para o experimento. Confirme o valor pH utilizando papel Litmus. </li><li> Caso contrário, utilizar um eléctrodo de pH de micro para ajustar de forma mais precisa o pH de pequenas quantidades. </li></ol></li><li> Levar a solução para um volume de 45 ul usando H 2 O. Execute todas as etapas no gelo para evitar a polimerização prematura de colágeno. </li></ol> 3. Formação de Concentric Cultura Gel <ol><li> Pegue o prato com fundo de vidro pré-aquecido (veja o passo 2.2) a partir de the incubadora. </li><li> Adiciona-se 5 ul da suspensão de células de 10 x concentrado (preparado no passo 1.5) a solução I. Ressuspender completamente. Tome cuidado para evitar a formação de bolhas de ar. A mistura agora tem um volume de 50 ul e contém a concentração final de colagénio (2,4 mg / ml) e a densidade celular (final c = 2 × 10 6 células / ml). </li><li> Adicionar 20 ul da solução contendo as células que se lentamente para o centro do poço, de modo a formar uma gotícula (Figura 1A) em forma de cúpula. Tome cuidado para evitar a formação de bolhas nesta etapa. Caso se forme um bolha, com cuidado, mas rapidamente quer tentar rompê-lo ou sugá-lo para fora usando uma ponteira. Suavemente colocar o prato de volta na incubadora para permitir que o gel interior polimerizar durante 45 minutos. </li><li> Preparar a solução de O (para o, gel de colagénio acelular exterior) cerca de 15 minutos antes do final deste passo de incubação. Nota: A condição exterior de gel pode ser variada em termos de concentração de colagénio e ao pH de polimerizaçãoobter redes com diferentes microestruturas 10. Neste protocolo, o foco sobre um 1,5 - gel de colagénio a 4,0 mg / ml polimerizada a um pH de 7,4. <ol><li> Com base na concentração de estoque de colagénio, calcular o volume requerido necessário para preparar 200 mL de solução de colagénio O na concentração final. </li></ol></li><li> Adicionar 20 ul de tampão PBS 10 × lentamente para a quantidade necessária de solução-mãe de colagénio (calculado no passo 3.4), com agitação suave. Ajustar o pH da mistura para o valor final de pH utilizando NaOH 0,1 M e o uso de medidor de pH calibrado. Veja a nota para o passo 2.5 relativamente ao ajuste de pH. </li><li> Levar a solução para um volume final de 200 ul usando H 2 O. Execute todas as etapas no gelo para evitar a polimerização prematura de colágeno. </li><li> Leve o prato da incubadora após 45 min de polimerização do gel interior (veja o passo 3.3). Adicionar cuidadosamente 180 ul de solução de S no topo do gel interior, de modo que a solução de cobre completamente o interiorgel e enche-se a cavidade (Figura 1B). <ol><li> Executar este passo cuidadosamente sem agitar a solução, o que pode levar a orientações de fibras não-uniformes no exterior de gel. Tome cuidado para evitar tocar o gel interno com uma ponteira, e para evitar a formação de bolhas ou bolsas de ar. Caso se forme um bolha, com cuidado, mas rapidamente quer tentar rompê-lo ou sugá-lo para fora usando uma ponteira. Suavemente colocar o prato de volta na incubadora para permitir que a polimerizar exterior de gel. </li></ol></li><li> Leve o prato da incubadora após 45 min de polimerização. O gel deve ser já bastante solidificado, neste ponto, embora ainda pode destacar da superfície inferior, se manuseada com cuidado. <ol><li> Suavemente para 2 ml de meio de cultura celular aquecida ao prato (Figura 1C). Certifique-se de que o gel é completamente submergido no meio. Atualize a médio a cada 2-3 dias ao longo do período de cultura. </li></ol></li></ol> 4. Imagens ao vivo de células <ol><li> Realizar imagem usando um microscópio confocal invertido equipado com capacidade de geração de imagens de células vivas de longo prazo. Incluir um embutido câmara de incubação com uma temperatura (37 ° C) e CO2 (5%) de controlo. Ligue o microscópio e aquecer o palco, pelo menos, 1 hora antes do início do experimento. Nota: Use lente objetiva com longa distância de trabalho para otimizar a observação e localização das células nos géis 3D. </li><li> Incubar o gel em meio contendo 5 ul de corante de rastreador de células fluorescentes durante 30 min para permitir uma localização precisa das células no sistema 3D. Posteriormente, remover o corante não ligado por lavagem três vezes com 1 × PBS. Em seguida, adicionar meio de cultura de células para o prato. </li><li> Leve o prato da incubadora e colocá-lo no palco microscópio (Figura 1D). Nota: imagens de células vivas pode começar em princípio, logo após a polimerização do gel exterior. No entanto, neste momento, as células in O gel interno não se espalharam ainda. Para examinar a migração das primeiras células que invadiram o exterior de gel, de imagem de células vivas pode começar 24 horas (dependendo do tipo de células) após o inicio da cultura. Como um guia, em torno de 12 - são necessárias mais 14 dias de cultura para a maioria das células no interior do gel para entrar no exterior de gel. </li><li> Selecionar volumes de Vista (VOV de) em regiões do gel exterior em torno do gel interior. Após 24 h de incubação, a população de células terá difundido, atravessou a interface entre os geles de interior e exterior, e começou a invadir o exterior de gel. <ol><li> Para o VOV de, incluem as regiões de gel imediatamente ao lado da interface de gel, regiões intermédias, e as regiões perto da periferia da gel exterior 7. Excluir regiões mais próximas do que 50 mm a partir das superfícies de fundo e laterais, bem como a partir do topo do gel, a fim de evitar possíveis efeitos de borda. Cada VOV tipicamente mede 647 × 647 × 100 mm 3 (em x, y, z e indicações, respectivamente), com intervalo de 5 mm na -stack z. </li></ol></li><li> Assegurar modos de imagem, canais / filtros, tempos de exposição, e resoluções de imagem estão corretamente selecionado. Para imagens de livre-label da rede de colágeno, utilize microscopia confocal de reflectância simultaneamente durante o lapso de tempo de imagens ao vivo de células. </li><li> Tome imagens de exemplo, traçar os histogramas de intensidade, e ajustar os ganhos e compensações para observar sinal suficiente e evitar a saturação, assegurando que o histograma fica entre zero e a intensidade máxima. Não altere estas configurações mais durante todo o período do experimento. </li><li> Tome imagens de lapso de tempo dos VOV de selecionados, com um intervalo de tempo, Δ t, de 10 min para 8 horas (ou mais, se necessário). </li></ol> 5. Acompanhamento de celular e Análise de Dados <ol><li> Realizar imagem quantitativa pós-processamento das imagens -stack z utilizando appropria<html> software de processamento de imagem te. <ol><li> Segmento as imagens de lapso de tempo para selecionar automaticamente as posições celulares 3D em (x, y, z, t). </li><li> Para cada quadro, verificar manualmente a precisão de localização e remover falsos positivos devido a restos de células e saliências celulares que podem ter sido confundido com células. Remover as células de proliferação a partir da análise e as células que se sobrepõem ou ligados divididas em objetos distintos. </li></ol></li><li> Gerar faixas de células de lapso de tempo em 3D a partir de células (as coordenadas x, y, z, t) obtidos no passo anterior, ligando a localização de cada célula na sequência de tempo. </li><li> Elimine o ruído aleatório e sistema removendo faixas mais curtos do que um comprimento de rasto limiar (tipicamente 20 minutos). </li><li> Corrija para a amostra deriva subtraindo os deslocamentos líquidos globais dos trilhos, se necessário. </li><li> Calcular o deslocamento de células,iles / ftp_upload / 52735 / 52735eq2.jpg &quot;width =&quot; 80 &quot;/&gt;, e distância de migração celular, <img alt="Equação 3" fo:content-width="1in" src="/files/ftp_upload/52735/52735eq3.jpg" width="100" /> , A partir de células observadas as trajectórias, em que é um vector que representa a localização de uma célula de 3D em tempo e é o número total de pontos de tempo. <ol><li> Calcular a velocidade célula como S = d / (n • Δ t), onde Δ t é o intervalo de tempo entre os quadros. Calcule direcionalidade migração celular (ou persistência) usando P = Δd / d. Esta simples medida de persistência implica que, para P = 0 o deslocamento líquido é zero e para P = 1 a trajetória é uma linha reta direcional. </li></ol></li></ol>

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In this protocol we describe an in vitro assay to study the 3D migrational behavior of cells in matrix environments that topologically resemble ECMs encountered in vivo. There are three main strengths of this assay as compared to other currently available methods. First, this assay allows one to simultaneously examine the cell migration mechanisms at both population level and individual cell level. This opens up possibilities of studying collective cell migration13, which has to date been lar...

A migração de células desempenha um papel chave em várias respostas fisiológicas, tais como o desenvolvimento embrionário, hemostase, e resposta imunitária, bem como em processos patológicos tais como doenças vasculares, inflamação e cancro 1. Dissecando os factores bioquímicos e biofísicos subjacentes a migração celular é portanto fundamentalmente importante não só para compreender os princípios básicos de funções celulares, mas também para fazer avançar várias aplicações biomédicas, tais como em engenharia de tecidos, anti-metástases e desenvolvimento de drogas anti-inflamatórias. Desde observação in vivo é tecnicamente desafiador, uma série de esforços tem sido focada em in vitro recapitulação da migração celular. Os métodos in vitro para estudar a migração de células têm sido amplamente concebido para ensaios em duas superfícies (2D) dimensionais, mais notavelmente o arranhão ou ferimento ensaio 2 de cura. Tais ensaios oferecem configuração experimental simples, fácil live-imagens de células, e fornecer informações úteis sobre vários mecanismos bioquímicos subjacentes migração celular. No entanto, estes ensaios não têm em conta a arquitetura da matriz extracelular (ECM) e remodelação, que são aspectos críticos na compreensão da migração vivo. Recentemente, tem sido cada vez mais apreciado que um modelo de cultura 3D, muitas vezes em matrizes à base de colagénio 3, fornece uma plataforma que melhor se assemelha à situação in vivo. De fato, as células apresentam dinâmicas migratórias que são distintas daquelas em superfícies 2D, especialmente devido à diferente dimensionalidade do meio ambiente 4. Além disso, as propriedades biofísicas e mecânicas da matriz afecta sensivelmente a migração celular 5, nomeadamente no contexto de invasão de células tumorais 6. Aqui, apresentamos um método para estudar o comportamento de migração celular 3D em ECM com propriedades biofísicas que podem ser facilmente variado, com condições de preparação. As células sãosemeadas em um &quot;gel interior&quot; e estão autorizados a escapar para dentro e invadir o &quot;gel exterior&quot; inicialmente acelular. O método baseia-se na capacidade da célula para reconhecer a presença de, e a propensão para migrar para dentro, regiões livres de células no exterior de gel, que está intimamente ligado à célula mechanosensing 7. Neste estudo, utilizamos redes de colágeno que a ECMs invadidas por células de câncer de mama altamente invasivos, MD-MBA-231. As propriedades mecânicas e microestrutura de ambos os géis de interiores e exteriores pode ser sintonizado 8 e 9 caracterizado para atingir as condições f isiologicamente relevantes. Reconstrução e análise das faixas de células permitir o exame quantitativa detalhada do comportamento de migração espaço-temporais, tanto a nível da população e nível de célula individual. É importante notar que a configuração do sistema de gel concêntrico imita a topologia de tecido in vivo enfrentado pelas células migrantes, especialmente células cancerosas invasoras, oferecendo, assim, conhecimentos importantespara os mecanismos físicos de migração celular e metástase.

<table><tbody><tr><td>Cell culture incubator</td><td>Fisher Scientific Pte Ltd</td><td>Model: 371, S/No 318854-6055</td><td></td></tr><tr><td>Confocal microscope</td><td>Nikon A1R</td><td></td><td>Inverted confocal laser scanning microscope equipped with incubator chamber</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Life Technologies</td><td>11965-092</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>10082-147</td><td></td></tr><tr><td>Fluorescent CellTracker dye CMTMR</td><td>Life Technologies</td><td>C2927</td><td></td></tr><tr><td>Glass-bottom dish</td><td>IWAKI Cell Biology</td><td>3931-035</td><td>35 mm diameter dish with 12 mm diameter glass-bottom well</td></tr><tr><td>Hemocytometer</td><td>iN CYTO</td><td>DHC-N01 (Neubauer Improved)</td><td></td></tr><tr><td>Microprocessor pH meter</td><td>Hanna Instruments</td><td>pH 211</td><td></td></tr><tr><td>Nutragen Collagen</td><td>Advanced BioMatrix</td><td>#5010-D</td><td>Acid-solubilized bovine collagen type I (stock pH ~ 2)</td></tr><tr><td>Objective lens</td><td>Nikon</td><td></td><td>CFI Super Plan Fluor ELWD ADM 20XC, W.D. 8.2-6.9mm, NA 0.45.</td></tr><tr><td>Penicillin-Streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>pH meter</td><td>Sartorius</td><td>S/No 29153352</td><td>Basic pH Meter PB-11</td></tr><tr><td>Trypsin-EDTA</td><td>Life Technologies</td><td>15400-054</td><td></td></tr></tbody></table>

concentric gel system to study the biophysical role of matrix microenvironment on 3d cell migration

A capacidade das células para migrar é crucial numa grande variedade de funções celulares ao longo da vida do desenvolvimento embrionário e na cicatrização de tumor e a metástase do cancro. Apesar dos esforços intensos de investigação, os princípios bioquímicos e biofísicos básicos de migração de células ainda não são totalmente compreendidos, especialmente nas três dimensões (3D) microambientes fisiologicamente relevantes. Aqui, nós descrevemos um ensaio in vitro concebido para permitir a análise quantitativa de 3D ​​comportamentos de migração celular. O método explora mechanosensing capacidade da célula e propensão para migrar para dentro da matriz extracelular previamente desocupado (ECM). Usamos a invasão de células do cancro da mama altamente invasivos, MDA-MB-231, em géis de colagénio, tal como um sistema modelo. A disseminação da população de células e as dinâmicas migratórias de células individuais ao longo de semanas de cultura pode ser controlada por meio de imagens ao vivo de células e analisados ​​para extrair dados espaço-temporalmente-resolvidos. Além Do Mais, O método é facilmente adaptável para diversas matrizes extracelulares, oferecendo assim uma maneira simples e poderosa para investigar o papel dos factores biofísicos no microambiente sobre a migração celular.

O ensaio de gel concêntrico aqui apresentada foi realizada utilizando células de cancro da mama altamente invasivos, MDA-MB-231, com 2,4 mg / ml de gel de colagénio interior e uma densidade de sementeira de células de = 2 x 10 6 células / ml, como um exemplo. Como mostrado na Figura 2, tipicamente depois de alguns dias de cultura, as células violou a interface gel interior-exterior e começou a invadir o exterior de gel. A população de células predominantemente espalhar radialmente p...

Watch this Scientific Journal Video about Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration at JoVE.com

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

As propriedades mecânicas e a micro estrutura da matriz extracelular afectar fortemente a migração de células 3D. Um método in vitro para estudar o comportamento de migração celular no espaço-temporal ambientes biofísico variáveis, tanto na população e os níveis de células individuais, é descrito.

Sistema de Gel Concentric para estudar o papel Biofísica da Matrix Microenvironment sobre a migração celular 3D

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

concentric-gel-system-to-study-biophysical-role-matrix

Research

JoVE Journal

Bioengineering

8.5K visualizações.  FOM Institute AMOLF.  As propriedades mecânicas e a micro estrutura da matriz extracelular afectar fortemente a migração de células 3D. Um método in vitro para estudar o comportamento de migração celular no espaço-temporal ambientes biofísico variáveis, tanto na população e os níveis de células individuais, é descrito. 

Vídeo: Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

Em células vivas, imagens de células que expressam proteínas fluorescentes Migrando-marcado em uma matriz tridimensional

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Bioengenharia

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 ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Análise da migração celular dentro de uma matriz de colagénio tridimensional

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration in more appropriate 3D environments, which better resemble the dimensionality of the physiological processes in question. Migratory cells can substantially differ in their morphology and mode of migration depending on whether they are moving on 2D or 3D substrates. Due to technical difficulties and incompatibilities with most standard protocols, structural and functional analysis of cells embedded within 3D matrices still remains uncommon. This article describes methods for preparation and imaging of 3D cancer cell cultures, either as single cells or spheroids. As an appropriate ECM substrate for cancer cell migration, we use nonpepsinized rat tail collagen I polymerized at room-temperature and fluorescently labeled to facilitate visualization using standard confocal microscopes. This work also includes a protocol for 3D immunofluorescent labeling of endogenous cell cytoskeleton. Using these protocols we hope to contribute to a better description of the molecular composition, localization, and functions of cellular structures in 3D.

This article presents a method to fluorescently label collagen that can be further used for both fix and live imaging of 3D cell cultures. We also provide an optimized protocol to visualize endogenous cytoskeletal proteins of cells cultured in 3D environments.

Revelando a Organização do citoesqueleto das células invasoras câncer em 3D

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration ...

Medicina

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

Gradiente planar do sistema de difusão para investigar Quimiotaxia numa Matriz de Colagénio 3D

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microambientes sanduíche-como aproveitarar Telemóveis / interações materiais

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparação de 3D colágeno Géis e microcanais para o estudo das interações 3D<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Caracterizando a migração celular no tridimensional em Vitro ferida ambientes

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Análise 3D de respostas multicelulares a gradientes Quimioatrativos

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial 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 ...