1. Ensaio Set-up <ol><li> Descongelar uma pequena alíquota (&lt;uL 500) de Matrigel em gelo a 4 ° C (cerca de 2 h). </li><li> Preparar receita gel (ver volumes exemplo na Tabela em baixo): PBS 10x (1x em volume total), hidróxido de sódio 1N (equivalente a 0,023 volumes de colagénio adicionado, ou segundo as recomendações do fabricante de colagénio, como apropriado), Matrigel e colagénio do tipo I para concentrações finais de 1 mg / ml e 1,3 mg / ml, respectivamente (formulações de matriz podem ser utilizados outros dependendo do tipo de célula e da experiência). </li></ol> Receita gel exemplo <table border="1"><tbody><tr><td> Componentes </td><td> Concentração estoque </td><td> Concentração Final </td><td> Adicionar o volume </td></tr><tr><td> PBS 10X </td><td> 10X </td><td> 1X </td><td> 0,090 ml </td></tr><tr><td> Água estéril </td><td></td><td></td><td> 0,346 ml </td></tr><tr><td> NaOH 1N </td><td></td><td></td><td> 0,008 ml </td></tr><tr><td> Matrigel </td><td> 9,90 mg / ml </td><td> 1 mg / ml </td><td> 0,101 ml </td></tr><tr><td> Rat cauda colágeno tipo I </td><td> 3,66 mg / ml </td><td> 1,3 mg / ml </td><td> 0,355 ml </td></tr></tbody></table><ol start="3"><li> Misturar os componentes do gel em gelo na mesma sequência como acima e solução final incubar durante 1 hr a 4 ° C. Na nossa experiência, uma incubação de 1 hora antes da semeadura das células resultados em uma gelificação mais uniforme do colagénio. Certifique-se manter Matrigel e colágeno no gelo em todos os momentos e trabalhar o mais rápido possível para evitar gelificação. </li><li> Coloque 12 mm de diâmetro 8 mM insere celulares poros cultura em uma placa de 12 poços usando uma pinça esterilizada. </li><li> Contar as células e ressuspender em meio isento de soro a 5 x 10 6 células / ml (volume total deve ser de 10% da solução de gel). </li><li> Adicionar 100 ul de su célulaspension a 900 ml de solução-gel (concentração celular final de 5 x 10 5 células / ml) e misture bem por pipetagem suavemente para cima e para baixo. </li><li> Adicionar 150 ul da mistura final para cada inserção e transferência para uma 37 ° C, 5% de CO 2 incubadora durante 30 minutos até gel polimeriza. </li><li> Usando meios isentos de soro, </li></ol><ul><li> Adicionar 100 uL no topo do gel e 1200 uL sob o inserto para a condição estática. Os níveis de fluido no interior do inserto e fora do poço deve ser aproximadamente igual, resultando em diferença de pressão mínima através do gel e sem fluxo intersticial. </li><li> Adicionar 100 ul sob a inserção e 650 uL acima gel para a condição de fluxo. Tenha cuidado para evitar bolhas de ar abaixo da inserção, como eles vão evitar que as células migrem através da membrana a esses locais. A diferença de pressão gerado por estes volumes é de aproximadamente 1,3 centímetros de H2O (ou 1 mm Hg). </li></ul><ol start="9"><li> Coloque em uma placa de 37° C, 5% de CO 2 incubadora durante 24 horas. </li></ol> 2. Células coradas e Contagem <ol><li> Adicionar 500 ul de PBS 1X por poço em placas de 24 poços novo, o que irá ser usada para lavar as inserções. </li><li> Remover o meio restante na porção superior dos transwells de fluxo e determinar o volume. Calcular o volume total de fluido, subtraindo o volume remanescente a partir do volume total originalmente adicionado, 650 uL (isto será usado para os cálculos de taxa de fluxo, ver abaixo). Use cotonetes para remover o gel a partir dos insertos e para limpar a superfície superior da membrana para remover as células não-invadidas. Colocar as inserções na placa de 24 poços contendo 1X PBS durante 15 s para lavar inserções. </li><li> Remover PBS e adicionar 500 uL de paraformaldeído a 4% (PFA) por baixo de cada inserto e incubar durante 30 minutos à temperatura ambiente, para fixar as células transmigradas. </li><li> Remover o PFA e enxaguar uma vez com 500 ul 1X PBS para remover fixador residual. </li><li> Adicionar 500 o ulf 0,5% de Triton X-100 solução sob as inserções e incubar durante 10 minutos à temperatura ambiente para permeabilizar as células. </li><li> Cortar as membranas para fora da inserção usando uma lâmina de barbear e coloque em 100 uL de 2 ug / ml em 1X DAPI solução de PBS, tendo o cuidado de a colocar o lado de baixo da face da membrana para baixo (células transmigradas viradas para baixo). </li><li> Placa de envoltório em folha de alumínio e local num agitador a 150 rpm durante 30 minutos à temperatura ambiente. </li><li> Lave as membranas em 500 uL de PBS 1X em agitador (repetição 3 vezes durante 10 minutos cada) para remover DAPI livre. </li><li> Membranas Coloque as lâminas de vidro com células transmigradas voltadas para cima, adicione solução de montagem e lamínula. </li></ol> 3. Análise de Dados <ol><li> Conte DAPI manchado de núcleos em 5 locais selecionados aleatoriamente de cada membrana (ficar longe das bordas e use um 10x ou 20x lente objetiva). </li><li> Calcula-se a contagem média de células e obter a invasão por cento usando a seguinte fórmula: Invasão por cento x = 100% (média contagem de células x área de superfície de membrana) / (área de superfície do número x microscópio imagem de células semeadas) A invasão por cento pode ser normalizado a uma condição de controlo (geralmente a condição estática) para permitir a comparação entre experiências independentes. </li><li> Calcular a taxa de fluxo médio: dividir o volume total eluído na condição de fluxo pelo tempo de incubação (por exemplo, 24 hr). </li><li> Calcula-se a velocidade de fluxo médio: dividir a taxa de fluxo médio pela área da secção transversal do gel / membrana (neste caso 60 milímetros 2). </li></ol> 4. Os resultados representativos Para medir a invasão de células tumorais sob fluxo intersticial, foi realizada a nossa 3-D ensaio de invasão de fluxo, utilizando MDA-MB-435S células de melanoma metastático. Estas células têm sido anteriormente demonstrado que invadem em resposta ao fluxo de fluido intersticial 13-14. As células foram incorporados em uma matriz composta de 1,3 mg / rato ml tail colagénio do tipo I e do tendão de 1 mg / ml Matrigel matriz da membrana basal, a uma concentração celular final de 5 x 10 5 células / ml. Duas condições diferentes foram comparados: (1) o fluxo intersticial média = 0,1 uM s -1 e (2) condição estática = não caudal mensurável (Figura 1). Após 24 horas, as células que invadiram através dos poros da membrana foram coradas com DAPI para facilitar a contagem das células. A Figura 2 mostra uma imagem representativa das células invadidas. Sob brightfield, apenas os poros da membrana são visíveis. Usando de fluorescência, os núcleos DAPI manchado foram utilizados para a contagem de célula e os faloidina coradas F-actina estruturas foram utilizados para visualizar o corpo da célula (opcional). Usando um teste t de Student-assumindo variâncias iguais, mostrámos que o fluxo intersticial aumenta significativamente MDA-MB-435S invasão de células de 2,3 vezes em relação a condições estáticas (p = 0,003) (Figura 3). Esta corroboraçãotes resultados semelhantes (mas com as condições da matriz diferentes e, portanto, velocidades de fluxo), utilizando esta linha de células de 13-14. <img alt="A Figura 1" src="/files/ftp_upload/4159/4159fig1.jpg" /> Figura 1. Esquemático do intersticial ensaio de fluido 3-D invasão de fluxo. Primeiro preparar solução de gel utilizando concentrações apropriadas e volumes. Em seguida, adicionar as células a solução de gel e transferir para inserções de cultura de células. Finalmente adicionar volume adequado de meios de comunicação para cada condição e incubar. Fluxo de fluido intersticial é accionado por uma cabeça de pressão de fluido. <img alt="A Figura 2" src="/files/ftp_upload/4159/4159fig2.jpg" /> Figura 2. Transmigradas MDA-MB-435S células em membrana. Células invadidas foram fixados após a nossa intersticial ensaio de invasão do fluxo do fluido e corados com DAPI e Alexa Fluor 488-conjugados faloidina para facilitar a contagem de células invadidas; A Picture) da membrana em campo claro; B) DAPI stanúcleos INED (em azul); C) Alexa Fluor 488-faloidina manchado F-actina (em verde). Barra de escala representa 50 um. <img alt="A Figura 3" src="/files/ftp_upload/4159/4159fig3.jpg" /> Figura 3. Da invasão de MDA-MB-435S células sob fluxo. Invasão celular após 24 hr significativamente melhorada pelo fluxo intersticial (p = 0,003). Os resultados são normalizados para a condição média estático e os valores representam a média ± SEM de 6 inserções de cultura de células.

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Aqui temos descrito um método para quantificar o efeito do fluxo intersticial na invasão de células de tumor, utilizando células embebidas numa matriz de 3-D dentro de uma inserção de cultura de célula. Isto e métodos semelhantes têm sido utilizados para estudar o efeito de fluxo intersticial em uma variedade de tipos de células 13-15, 17. A nossa abordagem parcialmente imita o microambiente matriz do tumor usando colagénio tipo I e Matrigel que contêm proteínas encontradas na membrana basal de tecido epitelial e do estroma circundante 21-22. Este sistema é relativamente fácil de configurar, fácil e mais rentável do que a maioria dos dispositivos microfluídicos que são usados ​​para estudar o fluxo de fluido intersticial in vitro 23-24. Ele não requerem bombas ou equipamentos especializados e permite a várias condições a serem testadas simultaneamente. Além disso, as medições são comparáveis ​​às dos ensaios de câmara existente Boyden comumente utilizado para testar invasão e migraçãoção das células cancerosas. Ao alterar o tipo de célula e da composição da matriz, diferentes sistemas biológicos podem ser modelados, tais como o cancro da mama 13, os vasos sanguíneos 15, e tráfico de células dendríticas 17. Alterando as propriedades da matriz e da composição e da cabeça da pressão modulando também irá alterar a velocidade de fluxo de fluido intersticial, permitindo assim a flexibilidade nos parâmetros de ensaio dependente do sistema biológico de interesse. Este sistema pode também ser utilizado em ensaios de co-cultura. 14, 17 Além de medir a invasão, o ensaio de fluxo de fluido intersticial descrito aqui pode ser expandido para medir as alterações no comportamento de células, tais como a expressão da proteína, a proliferação celular e as diferenças de eventos de sinalização celular. As células podem ser facilmente isolados directamente a partir do gel para o RNA, DNA, e extracção da proteína e usado para ensaios subsequentes de biologia molecular, tais como PCR e ocidentalapagar. Este é um ensaio altamente flexível que pode ser facilmente configurado e adaptado para investigar os efeitos de fluxo intersticial sobre uma série de processos celulares, em um número de sistemas de células. Uma das principais desvantagens deste ensaio é o facto de que as velocidades de fluxo são muito dependentes da concentração de matriz. Velocidade de fluxo aumenta à medida que a concentração de Matrigel diminui. Isto significa que uma matriz constituída exclusivamente por colagénio, que pode ser usado no estudo de células do estroma associadas, terá maiores velocidades de fluxo (&gt; 1 mícron s -1) do que uma que consiste principalmente de Matrigel (menos de 0,05 uM s -1) para um diferencial de pressão determinada. Se uma velocidade de fluxo específico é necessário, pode-se primeiro realizar um teste inicial para identificar a composição da matriz que proporciona a velocidade de fluxo desejada. A espessura do gel pode também ser variada para ajustar a velocidade de fluxo. O ensaio também não permite imagens de células vivas, onde o comportamento de células individuais umd alterações na sua velocidade e direccionalidade pode ser medido. Em vez disso, este ensaio proporciona uma medição de ponto de extremidade de alterações na invasão através de uma população de células.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Colágeno (cauda de rato) </td><td> BD </td><td> 354236 </td><td> Mantenha estéril </td></tr><tr><td> Millicell inserção de cultura de célula </td><td> Millipore </td><td> PI8P01250 </td><td> 8 diâmetro de poro iM, policarbonato membrana </td></tr><tr><td> Matrigel </td><td> BD </td><td> 354234 </td><td> Mantenha estéril </td></tr><tr><td> PBS </td><td> Sigma Aldrich </td><td> 100M-8202 </td><td> 10x para a preparação de solução de gel, 1x para a lavagem passos </td></tr><tr><td> Hidróxido de sódio em solução 1,0 N </td><td> Sigma Aldrich </td><td> S2770 </td><td> Mantenha estéril </td></tr><tr><td> DMEM 1X </td><td> CellGro </td><td> 10-013-CV </td><td> Mantenha estéril </td></tr><tr><td> Soro Fetal Bovino </td><td> Atlanta Biologicals </td><td> 511150 </td><td> Mantenha estéril </td></tr><tr><td> Estreptomicina penicilina </td><td> CellGro </td><td> 30002CI </td><td> Mantenha estéril </td></tr><tr><td> Triton X-100 </td><td> Sigma Aldrich </td><td> X100-500 ml </td><td> 0,5% em PBS </td></tr><tr><td> Paraformaldeído </td><td> Fisher Scientific </td><td> 04042-500 </td><td> 4% em PBS </td></tr><tr><td> Água deionizada </td><td></td><td></td><td> Mantenha estéril </td></tr><tr><td> 4 &#39;,6-diaminido-2-fenilindol (DAPI) </td><td> Biomedicals MP </td><td> 0215757401 </td><td> 1 mg / ml de solução stock </td></tr><tr><td> Solução de montagem </td><td> Thermo Scientific </td><td> TA-030-FM </td><td></td></tr><tr><td> Tripsina-EDTA </td><td> CellGro </td><td> 25-052-CI </td><td> Mantenha estéril </td></tr></tbody> &lt;/ Table&gt;

three-dimensional cell culture model for measuring the effects of interstitial fluid flow on tumor cell invasion

O crescimento ea progressão da maioria dos tumores sólidos dependem da transformação inicial das células cancerosas e da sua resposta ao estroma associado à sinalização no microambiente do tumor 1. Anteriormente, a investigação sobre o microambiente do tumor tem se concentrado principalmente em tumores estromais interações 1-2. No entanto, o microambiente do tumor também inclui uma variedade de forças biofísicas, cujos efeitos permanecem pouco compreendidas. Estas forças são conseqüências biomecânicas do crescimento do tumor que levam a mudanças na expressão gênica, a divisão celular, diferenciação e invasão 3. Matriz de densidade 4, rigidez 5-6, 6-7 e estrutura, a pressão do fluido intersticial 8, e fluxo de fluido intersticial 8 são todos alterada durante a progressão do cancro. Fluxo de fluido intersticial em particular, é maior em tumores em comparação com 8-10 normais tecidos. O fl fluido intersticial estimadoow velocidades foram medidos e verificou-se ser na gama de 0,1-3 uM s -1, dependendo do tamanho do tumor e diferenciação 9, 11. Isto é devido à pressão do fluido intersticial elevada causada por induzida por tumor angiogénese e aumento da permeabilidade vascular 12. Fluxo de fluido intersticial foi mostrado para aumentar a invasão de células de cancro 13-14, fibroblastos vasculares e células de músculo liso 15. Esta invasão pode ser devido aos gradientes de quimiotáticas autólogas criados em torno de células em 3-D 16 ou aumentada metaloproteinase de matriz (MMP) expressão 15, a secreção de quimiocinas e células expressão da molécula de adesão 17. No entanto, o mecanismo pelo qual as células sentir o fluxo de fluido não é bem compreendida. Além de alterar o comportamento das células do tumor, o fluxo de fluido intersticial modula a actividade de outras células no microambiente do tumor. Ele está associado (a) diferenciação de condução de fibroblastos em tumor-promoção myofibroblasts 18, (b) transporte de antigénios e outros factores solúveis para linfonodos 19, e (c) modulando linfático morfogénese de células endoteliais 20. A técnica aqui apresentada impõe fluxo de fluido intersticial em células in vitro e quantifica os seus efeitos sobre invasão (Figura 1). Este método tem sido publicado em vários estudos para medir os efeitos de fluxo de fluido sobre estroma e invasão de células do cancro 13-15, 17. Ao alterar a composição da matriz, tipo de célula, e concentração de células, este método pode ser aplicado a outras doenças e sistemas fisiológicos para estudar os efeitos de fluxo intersticial em processos celulares, tais como invasão, diferenciação, proliferação e expressão de genes.

Watch this Scientific Journal Video about Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion at JoVE.com

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

Fluxo de fluido intersticial é elevada em tumores sólidos e pode modular a invasão de células tumorais. Descrevemos aqui uma técnica para aplicar o fluxo de fluido intersticial para as células embebidas numa matriz e, em seguida medir os seus efeitos sobre a invasão celular. Esta técnica pode ser facilmente adaptado para estudar outros sistemas.

Um modelo tridimensional de Cultura Celular para avaliar os efeitos de fluxo de fluido intersticial na invasão da célula tumoral

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

three-dimensional-cell-culture-model-for-measuring-effects

Nanyang Technological University

Research

JoVE Journal

Bioengineering

16.5K visualizações.  Drexel University. Fluxo de fluido intersticial é elevada em tumores sólidos e pode modular a invasão de células tumorais. Descrevemos aqui uma técnica para aplicar o fluxo de fluido intersticial para as células embebidas numa matriz e, em seguida medir os seus efeitos sobre a invasão celular. Esta técnica pode ser facilmente adaptado para estudar outros sistemas.

Vídeo: Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Hidrogéis alginato para Tridimensional Cultura Órgão de ovários e tubas uterinas

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Bioengenharia

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Impressão de planar e tridimensional de tintas condutoras

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

Sistemas Célula de rotação de cultura para cultura de células humanas: células trofoblásticas Humanos como um modelo

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

Investigando a separação de fluxo tridimensional induzida por um polip de dobra vocal modelo

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

Engenharia de Tecidos de tumor estromal Microenvironment com Aplicação a invasão Cancer Cell

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

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 ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

A transfecção Gene direção Células Spheroid em placas micropadronadas Cultura para Transplante de Células geneticamente modificados

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

Fabricação de Inverted coloidal de cristal poli (etileno-glicol) Andaime: Uma Plataforma Cultura celular tridimensional para Liver Engenharia de Tecidos

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

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 ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Avaliação da proliferação queratinócito em duas e três dimensões tipo I colágeno substratos

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...