Este trabalho foi financiado pelo National Institutes of Health EUA conceder NIH / NICHD # HD051998 (para CAM).

1. Preparação de colágeno Bead <ol><li> Antes de TEVs carregamento para 3-D de cultura de células, é preciso preparar as contas Cytodex-3 microcarregador: <ol><li> Pesar a quantidade adequada de Cytodex-3 contas necessárias para o experimento. Este protocolo é adaptado para o navio RCCs 10ml, em que 0,05 g de contas são necessários. Para que um navio RCCs 50ml, escala em conformidade. Em um tubo de 50 ml autoclavável cônica, misturar 250 mg Cytodex-3 contas com 12 ml de fosfato tamponada Dulbecco (DPBS). Este montante é suficiente para 5 navios CCRs. </li></ol></li><li> Assegurar um volume adequado está presente no tubo cônico, como o processo de autoclave irá resultar em evaporação. Vagamente tampa do tubo e autoclave por 10 minutos a 110 ° C. </li><li> Remover o tubo cônico na conclusão do ciclo de autoclave e deixar a solução Cytodex-3 talão para esfriar. </li><li> Após o tubo cônico esfriou a temperatura ambiente, use uma técnica estéril para trazer o volume total de 12.5mL usando 1X DPBS. </li><li> Tampa e guarde o preparado Cytodex-3 contas à temperatura ambiente. Permitir que as contas preparados para inchar e esfriar a temperatura ambiente antes do uso. A preparação acima de Cytodex-3 contas irá fornecer para cinco navios 10ml CCRs. Temos observado que o armazenamento prolongado de Cytodex-3 contas à temperatura ambiente irá resultar em desempenho reduzido, como o colágeno vai desestabilizar após aproximadamente um mês. Cytodex-3 contas variam em tamanho 133-215 mM. </li></ol> 2. Preparação da mídia <ol><li> Prepare 1L de CCRs otimizado GTSF media-2 (adaptado de 22), que consiste em 40% MEM alfa, além de suplementos e 60% ​​L-15 Leibovitz de mídia (adaptado de 22). Primeiro, prepare 400mL no volume total de alfa MEM suplementado com: Bicarbonato de sódio 21.2mm Peptona 0,06% Frutose 0,7 mm Galactose 1,4 mm Glucose 5.6mm HEPES 1% 1% L-glutamina 0,5% ITS F 10%BS </li><li> Para preparar uma solução estoque de ITS, dissolver em 5 mL estéreis acidificados H 2 O preparado por adição de ácido acético glacial (cerca de 0,05 ml). Agite para dissolver, siga com água estéril 45ml. </li><li> Pesar o suficiente L-15 em pó Leibovitz para 600ml de meio de dissolvendo em cultura de tecidos de grau H 2 O. </li><li> Trazer o volume total de meio de cultura celular para 1L com L-15 de médio Leibovitz. Filtro-esterilizar e armazenar a 4 ° C no escuro. Adicione 1% Penicilina-estreptomicina individualmente para cada alíquota de meio utilizado. </li><li> Muitas formulações de outros meios de mídia GTSF-2 ter sido testado com sucesso nos CCRs. Laboratórios indivíduo deve decidir por si quais médio é o ideal para o experimento que está sendo executada. </li></ol> 3. Células e Incubação Bead <ol><li> Propagar a TEVs para ~ 80% confluência, trypsinize e contar com práticas aceitas cultura de células. </li><li> Suspender 1x10 6 TEVs em 4mL de mornoed mídia. </li><li> Misture delicadamente preparado Cytodex-3 esferas. Usando técnicas estéreis, retire 2,5 ml de contas preparada com a ponta de largura, 10 ml sorológicos pipeta e transferir para um tubo cônico 15mL não utilizados. Note-se que pode haver uma pequena perda de contas que atribuem à pipeta. </li><li> Permitir que o Cytodex-3 contas para resolver sobre o fundo do tubo. Após a sedimentação, use uma pipeta para remover a camada superior da DPBS sem perturbar as contas Cytodex-3. </li><li> Misture a 1x10 6 TEVs preparadas em media com o preparado Cytodex-3 esferas. </li><li> Incubar a mistura de células-grânulo em temperatura ambiente por 30 min. Periodicamente, misture delicadamente. Incubar durante 30 min ainda a 37 ° C e 5% CO 2. Periodicamente, misture delicadamente. </li></ol> 4. Carregando o RWV <ol><li> Em uma câmara de fluxo laminar, remover um RCCs 10 ml da embalagem e colocá-lo em uma placa de cultura estéril, 6-bem para a estabilidade. Consulte a Figura 1 para ver um diagrama do rótulo do RCCS. </li><li> Remova a tampa do porta grande. </li><li> Trazer o volume total da mistura de incubação de células-talão até 10 ml com a mídia aquecido. </li><li> Carregar o celular / mistura-grânulo no RCCs através da porta grande. Os CCRs deve ser inclinado em um ângulo de 45 ° (até grande porto) ao carregar para ajudar na remoção de bolhas de ar em potencial. Evitar acúmulo de pressão positiva através do preenchimento da embarcação lenta e progressivamente. </li><li> Substituir a tampa do porta grande. </li><li> Remover os pistões do seringas de 3 ml e coloque as seringas vazias para os portos de pequeno porte. Adicionar 1-3 mL de mídia para cada seringa. Abrir lentamente as duas válvulas. Substitua os pistões seringa na seringas suavemente. Adicionar mídia a partir de uma seringa até todas as bolhas são removidos do interior da câmara. </li><li> Pegue o CCRs e bata suavemente de lado enquanto gira-lo na frente de você para verificar se há bolhas. Se houver bolhas, você deve levá-los para fora da câmara, como bolhas de ar irá interferir com a formação of agregados celulares e introduzir forças de cisalhamento. Remova as bolhas girando o RCCs até que as bolhas estão sob o pequeno porto. Depois, empurre para baixo sobre a seringa no lado oposto à força as bolhas na porta e fora da câmara. </li><li> Fechar uma porta lateral. Gentilmente empurre para baixo o pistão outra seringa para introduzir uma pequena quantidade de pressão positiva dentro do vaso, o que impede a formação de bolhas. Feche a válvula segundo. </li><li> Carregar os CCRs para o rotor. Iniciar a rotação em 19rpm a 37 ° C incubadora de CO 2. </li></ol> 5. Alterando a mídia <ol><li> Troque a mídia todos os dias durante os primeiros três dias, então todos os dias depois. </li><li> Desligue o rotor e remova o CCRs. Puxe os pistões para criar alguma sucção, retire as seringas de cada pequeno porto e coloque o CCRs em um ângulo de modo que as contas resolver oposto do grande porto. </li><li> Após as contas têm tudo acertado, abra uma das válvulas pequenas epermitir que os meios de comunicação a fluir para fora do CCRs e em um recipiente de resíduos. Retire 2 / 3 dos meios de comunicação desta forma. Certifique-se de não perturbar as contas e para não descartar qualquer agregados. </li><li> Feche a válvula de pequenas e abrir a porta grande. Adicionar mídia de volta para o RCCs através da porta grande. Substituir a tampa do porta grande. </li><li> Repita os passos de 4,6-4,9. </li><li> Como os agregados aumentam de tamanho, você precisa aumentar a velocidade de rotação para mantê-los em suspensão em todos os momentos, de preferência em um padrão circulatório pequeno na velocidade ideal. Geral, aumentar a velocidade entre 0,3-0,7 rpm após cada mamada, uma vez agregados começar a crescer visivelmente. </li></ol> 6. Coleta de células propagadas <ol><li> Remover o CCRs do rotor. Remove as seringas de cada pequeno porto e coloque o CCRs em um ângulo de modo que as contas poderá resolver oposto do grande porto. </li><li> Após as contas têm tudo acertado, abra uma das válvulas de pequenos e permitir que os meios de comunicação a fluir para forados CCRs e em um recipiente de resíduos. Retire 1 / 3 dos meios de comunicação desta forma. </li><li> Feche a válvula de pequenas e abrir a porta grande. Agite suavemente o navio para dispersar os agregados na solução. Mistura líquida vazia em um tubo cônico estéril 50ml. Permitir que os agregados coletados para se estabelecer no tubo cônico antes de usar o sobrenadante para lavar o vaso cultura para maximizar a recuperação de agregados. Os agregados podem ser usados ​​para ensaios de downstream imediatamente, como a citometria de fluxo, ensaios de invasão, imunofluorescência e outros. </li></ol> 7. Resultados representante Um exemplo de como as células-EVT (SGHPL-4 linha de células do trofoblasto) cultivadas no CCRs em Cytodex-3 contas é mostrado na Figura 2. A linhagem celular EVT-like exibe projeções estende para longe do principal grupo e anexando a clusters vizinhos. Muitas das contas são completamente cobertas com células propagação. Uma vez retirado do CCRs e plated em uma matriz extracelular, a EVT-3-D como células cultivadas de forma agressiva invadir e / ou migrar (Figura 3). RT-PCR dados confirmam o aumento da expressão de MMPs visto em 3-D agregados ao contrário de monocamadas de células tradicionais de cultura (Figura 4). Curiosamente, os genes não associados com a invasão também são upregulated na CCRs (Tabela 1) e pode ajudar no delineamento de áreas tais como interações imune com células invadindo trofoblasto. <img alt="Figura 1" src="/files/ftp_upload/3367/3367fig1.jpg" /> Figura 1. Representação dos desenhos animados do Sistema de Rotação de Cultura Celular (CCRs). <img alt="Figura 2" src="/files/ftp_upload/3367/3367fig2.jpg" /> Figura 2. Micrografia de contraste de fase de um agregado representante após 5 dias de crescimento em um CCRs. Setas indicam células invasoras e * denota as contas Cytodex-3 microcarregador. <imgalt = &quot;Figura 3&quot; src = &quot;files/ftp_upload/3367/3367fig3.jpg /&quot; /&gt; Agregados figura 3. RCCs Grown foram incorporados em géis de fibrina. Invasão através do gel de fibrina foi observada já em (A) 24 horas após a incorporação de fibrina e (B) continuou até 48 horas. Setas indicam células invasoras e * denota as contas Cytodex-3 microcarregador (Adaptado, com permissão, de Ref. 7). <img alt="Figura 4" src="/files/ftp_upload/3367/3367fig4.jpg" /> Figura 4. Zymogram gelatina comparativo de monocamada e CCRs propagada SGHPL-4 EVT células semelhantes. Formas pró-ativa e de MMP-2 e MMP-9 foram secretado pelas células trofoblásticas cultivadas CCRs (Adaptado, com permissão, de Ref. 7). <img alt="Tabela 1" src="/files/ftp_upload/3367/3367table1.jpg" /> Tabela 1. Resumo dos resultados de microarray SGHPL-4 células cultivadas em CCRs (Adaptado, com permissão, de Ref. 7). <ol class="lalpha"><li&gt; Dobre indução nos agregados 3-D foi calculado da seguinte forma: 3-D normalizado gene valor / valor gene normalizado monlayer. </li><li> Indica genes que estavam abaixo do limite de detecção em monocamadas. </li></ol>

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Não temos nada a revelar.

A técnica de cultura aqui apresentada fornece aos investigadores altamente invasivo EVT células semelhantes. Ele já foi reconhecido que a perda de diferenciação ocorre em monocamadas devido à inibição da resposta celular a agentes químicos e sinais moleculares em três dimensões (superfícies celulares apical, basal e lateral) 10,13. Esta técnica reflete características observou in utero invadir células EVT. Como o procedimento imita convencionais monocamada tecido cinética de tempo da ...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Contas Cytodex microcarregador </td><td> Sigma-Aldrich </td><td> C3275 </td><td></td></tr><tr><td> Sistema de Cultura rotativa Cell (CCRs) </td><td> Synthecon </td><td> RCCs-D </td><td> Inclui rotor base de fornecimento de alimentação, 4 unidades descartáveis ​​CCRs </td></tr><tr><td> Unidades RCCs descartáveis </td><td> Synthecon </td><td> Fale Synthecon </td><td></td></tr><tr><td> 3ml da seringa Luer-Lock </td><td> BD </td><td> 309585 </td><td></td></tr><tr><td> 10ml de grande ponta de pipeta sorológica </td><td> BD </td><td> 357504 </td><td></td></tr><tr><td> MEM Alpha </td><td> Invitrogen </td><td> 12561-072 </td><td></td></tr><tr><td> Leibovitz L-15 de médio pó, </td><td> Invitrogen </td><td> 41300-039 </td><td></td></tr><tr><td> H 2 O, de endotoxina livre </td><td> Pescador </td><td> MT-25-055-CM </td><td></td></tr><tr><td> Bicarbonato de sódio </td><td> Sigma-Aldrich </td><td> S-7795 </td><td></td></tr><tr><td> Peptona </td><td> Fisher Scientific </td><td> BP1420-100 </td><td></td></tr><tr><td> Frutose </td><td> Sigma-Aldrich </td><td> F3510-100 </td><td></td></tr><tr><td> Galactose </td><td> Sigma-Aldrich </td><td> G5388-100 </td><td></td></tr><tr><td> Glicose </td><td> Sigma-Aldrich </td><td> G7528-250 </td><td></td></tr><tr><td> HEPES </td><td> Invitrogen </td><td> 15630-080 </td><td></td></tr><tr><td> L-Glutamina </td><td> Invitrogen </td><td> 25030 </td><td></td></tr><tr><td> Insulina-transferrina-selenito de sódio (ITS) </td><td> Sigma-Aldrich </td><td> I1884 </td><td></td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 10437 </td><td></td></tr><tr><td> Penicilina-estreptomicina </td><td> Invitrogen </td><td> 15140 </td><td></td></tr></tbody></table>

rotating cell culture systems for human cell culture: human trophoblast cells as a model

O campo de pesquisa trofoblasto humano ajuda na compreensão do ambiente complexo estabelecido durante placentação. Devido à natureza destes estudos, humanos em experimentação in vivo é impossível. A combinação de culturas primárias, as culturas de explante e linhas de células do trofoblasto um auxiliar para a compreensão de invasão da parede uterina 2 e remodelação das artérias uterinas espiral 3,4 por células do trofoblasto extravilosas (TEVs), que é necessário para o estabelecimento de sucesso da gravidez. Apesar da riqueza de conhecimentos provenientes de tais modelos, aceita-se que em modelos de células in vitro utilizando linhas cultura EVT-como célula de exibição alterado propriedades celular quando comparados com os seus homólogos em 5,6 vivo. Células cultivadas em sistema de rodízio de cultura de células (CCRs) exibir as propriedades morfológicas, fenotípicas e funcionais de linhas de EVT-como célula que mais perto imitar diferenciar em uTEVs Tero, com aumento da expressão de genes de mediação invasão (por exemplo metaloproteinases de matriz (MMPs)) e diferenciação do trofoblasto 7,8,9. O Hospital Saint Georges de células placentária Linha-4 (SGHPL-4) (gentilmente doado pelo Dr. Guy Whitley e Judith Dr. Cartwright) é uma linha de células EVT-like que foi usado para testes no CCRs. O design do navio cultura RCCs é baseado no princípio de que órgãos e tecidos em função de uma imagem tridimensional do ambiente (3-D). Devido às condições de cultura dinâmica no reservatório, incluindo as condições de cisalhamento fisiologicamente relevantes, as células cultivadas em três dimensões formam agregados com base em afinidades naturais celular e se diferenciar em tecido organotípicas-like assembléias 10,11,12. A manutenção de uma órbita de fluido proporciona um baixo cisalhamento, o ambiente de turbulência de baixa semelhante às condições encontradas in vivo. Sedimentação das células cultivadas for anulada, ajustando a rotaçãovelocidade do CCRs para assegurar uma constante queda livre de células. Troca de gases ocorre através de uma membrana permeável hidrofóbica localizado na parte traseira do biorreator. Como seus pais tecido in vivo, CCRs de produção de células são capazes de responder a química molecular e gradientes em três dimensões (ou seja, suas superfícies apical, basal e lateral), porque eles são cultivadas na superfície dos grânulos microcarregador poroso. Quando cultivada como bidimensional monocamadas em superfícies impermeáveis, como o plástico, as células são privados dessa comunicação importante em sua superfície basal. Conseqüentemente, as restrições espaciais impostas pelo ambiente afetam profundamente sentido como células e decodificar sinais do microambiente em torno, o que implica um papel importante para o meio 3-D 13. Temos usado a CCRs para engenheiro biologicamente significativas modelos 3-D de vários tecidos epiteliais humanas 7,14,15,16. De fato, muitos relatórios anteriores demtrada que as células cultivadas em CCRs pode assumir fenótipos fisiologicamente relevantes que não foram possíveis com outros modelos 10,17-21. Em resumo, a cultura na RCCs representa uma tarefa fácil, reprodutível, plataforma de alto rendimento que proporciona um grande número de células diferenciadas que são passíveis de uma variedade de manipulações experimentais. No protocolo a seguir, usando TEVs como exemplo, que descrevem claramente os passos necessários para as células de forma tridimensional de cultura aderente na CCRs.

Watch this Scientific Journal Video about Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model at JoVE.com

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

Tradicionais, duas técnicas de cultura de células dimensional muitas vezes resultam em características alteradas com relação a marcadores de diferenciação, citocinas e fatores de crescimento. Tridimensional de cultura de células no sistema de rotação de cultura de células (CCRs) restabelece a expressão de muitos destes fatores, como mostrado aqui com uma linha extravilosas trofoblasto celular.

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 ...

rotating-cell-culture-systems-for-human-cell-culture-human

Research

JoVE Journal

Bioengineering

16.4K visualizações.  Tulane University Medical School. Tradicionais, duas técnicas de cultura de células dimensional muitas vezes resultam em características alteradas com relação a marcadores de diferenciação, citocinas e fatores de crescimento. Tridimensional de cultura de células no sistema de rotação de cultura de células (CCRs) restabelece a expressão de muitos destes fatores, como mostrado aqui com uma linha extravilosas trofoblasto celular.

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

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

Plataformas microfabricated para cultura de células mecanicamente dinâmico

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Bioengenharia

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

Forma Polímeros Memória da Cultura célula ativa

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Microcápsulas de alginato como uma plataforma 3D para Propagação e Diferenciação de Células-Tronco Embrionárias (hESC) para diferentes linhagens

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

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

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

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 ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Padronização de células co-cultura Usando duas fases aquosas Sistemas

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

A cultura in vitro modelo celular para Toxic Chemical Testing Inhaled

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

Cultura de células de hidrogel ultrafinos Porated elástico como uma membrana do porão biomimético para Dual

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

Cultura de células-tronco pluripotentes humanas em polivinil álcool-Co-itacónico ácido hidrogel com rigidez diferentes condições Xeno-livre

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

Um modelo de cultura de matriz-adipócito extracelular humano para estudar crosstalk metabólico de matriz e células

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Medição potencial de ação óptica unicelular em cardiomiócitos derivados de células-tronco pluripotentes induzidos por humanos

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...