Este trabalho foi financiado em parte pelo National Science Foundation (CBET 0.846.453) e os Institutos Nacionais de Saúde (1R01GM089999).

1. Fabricação de mestres de silício usando fotolitografia SU-8 padrão 1 (não é mostrado neste vídeo). <ol><li> Uso padrão SU-8 métodos de fotolitografia para criar um SU-8 &quot;mestres&quot; (SU-8 2050, Microchem, Newton, MA) para a fabricação de diferentes componentes (membranas PDMS de diferentes espessuras, co-cultura câmara) em uma sala limpa. Moldes como mestre pode ser fabricado em qualquer instalação de microfabricação (por exemplo, Stanford microfluídica Fundição; <a href="http://thebigone.stanford.edu/foundry/">http://thebigone.stanford.edu/foundry/</a> ). </li><li> Antes da replicação do PDMS, exponha o SU-8 mestres de vapor fluorosilane em um dessecador conectado a uma fonte de vácuo para facilitar a liberação fácil de PDMS do mestre. Adicionar uma gota de fluorosilane para uma toalha de papel e aplicar vácuo para uma min. Remova a vácuo e aguarde 30 min para a deposição de fluorosilane. Mantenha o mestre SU-8 em um recipiente fechado para uso futuro. </li></ol> 2. Moldagem réplica de PDMS de SU-8 master 2 <ol><li> A camada fluídica ea camada de controle são feitos por moldagem réplica de PDMS de SU-8 master. </li><li> Misture o PDMS pré-polímero e cross-linker em um wt 10:01. relação e desgaseificar em secador durante uma hora (ou até que as bolhas de ar são removidos da mistura de PDMS). </li><li> Coloque o mestre-8 SU numa placa de Petri e cuidadosamente despeje a mistura PDMS em cima do mestre SU-8. Degas do PDMS novamente. </li><li> Girar o molde a 1200 rpm por um minuto para obter uma espessura uniforme de cerca de PDMS 100μm sobre o molde. </li><li> Aqueça a placa de petri contendo o PDMS eo SU-8 molde mestre em 80 ° C por uma hora para curar o PDMS. </li><li> Remover o curado PDMS coberto SU-8 mestre do fogão. </li><li> Descasque o molde PDMS do mestre SU-8. </li><li> Faça quatro furos no molde PDMS usando uma agulha de calibre 20 blunt-end para duas entradas bactérias, uma saída e uma porta pneumática. </li></ol> 3. Colagem de Multilayer PDMS dispositivos: Este dispositivo é montado com três camadas, uma camada superior que contém o canal principal, uma camada pneumática meio, e uma camada inferior de bactérias que contém os canais para as bactérias. As três camadas são montados conforme descrito abaixo. <ol><li> Molhar as membranas PDMS com metanol e cuidadosamente descascá-los de seus SU-8 molde mestre usando uma pinça limpa. Membrana lugar em uma folha de teflon. </li><li> Coloque a membrana pneumática ea camada fluídica em uma câmara de plasma de oxigênio. Ligue plasma por 40 segundos (pressão de oxigênio 10 sccm, RF potência de 100W). </li><li> Após a exposição ao plasma, alinhar (10 minutos) da membrana na parte inferior PDMS canal usando o fórceps. Adição de metanol na membrana e as camadas de PDMS ajuda a livre circulação das camadas, alinhamento e torna mais fácil. Neste caso, o dispositivo é colocado em um pedaço de gaze para permitir que os vapores de metanol para escapar. </li><li> Cure as camadas alinhadas PDMS numa placa de aquecimento a 80 ° C por 8 h. </li><li> Após a colagem da membrana pneumática para a camada inferior, repita os passos de 3,2-3,4 para unir o canal superior para o dispositivo montado PDMS composto. </li></ol> 4. A montagem do dispositivo de PDMS 3,4 <ol><li> Faça furos nos portos de entrada e saída usado para semeadura células eucarióticas e bactérias. </li><li> Conecte a tubulação de Tygon (0,01 polegadas OD) para os portos e operam na camada pneumática, puxando com uma seringa de 20 mL. </li><li> Coloque o modelo PDMS e uma lâmina de vidro pré-limpeza em uma câmara de plasma de oxigênio. Ligue plasma por 20 segundos (pressão de oxigênio 20 sccm, RF potência de 300W) sem aplicar o vácuo. </li><li> , A fim de evitar o contacto entre a parede ilha de ligação (PDMS) para a lâmina de vidro, imediatamente se conectar uma seringa para o dispositivo e aplicar vácuo puxando a seringa e mantê-lo no lugar. </li><li> Pressione suavemente o modelo PDMS no vidro (2 minutos). </li><li> Cure o dispositivo PDMS a 80 ° C por 10 minutos. </li></ol> 5. Tratar a superfície de vidro com fibronectina <ol><li> Esterilizar dispositivo sob UV por 15 minutos depois de conectar tubos Tygon para cada porta. </li><li> Preenchimento e fluxo da PBS esterilizado para o canal para remover quaisquer detritos no canal. Aplicar o fluxo por 10 minutos para remover bolhas de ar. As bolhas de ar podem facilmente escapar do PDMS por causa de sua permeabilidade ao gás. </li><li> Preencher uma solução corante de cor no canal pneumático para prevenir a formação de bolhas de ar no canal. </li><li> Para facilitar a semeadura célula eucariótica no vidro, introduzir uma solução a 1 mg / mL de fibronectina no dispositivo e incubar por 40 min a 37 ° C. </li><li> Lavar o excesso de fibronectina com 1 mL de meio de crescimento. </li></ol> 6. Formação e medição de comensais E. biofilme em Escherichiailhas <ol><li> Diluir as bactérias comensais (eg, E. coli BW25113/pCM18) em media M9 mínimos para OD 600 ~ 1 para a formação de um biofilme comensais nas câmaras fechadas / ilhas. </li><li> Feche a parede ilha, aplicando-líquido positivo de pressão. Um conversor de pressão caseira ar-líquido é usado para prevenir a formação de bolha de ar no canal. </li><li> Introduzir o E. comensais coli nas ilhas com a válvula fechada, e permitir que as bactérias para anexar por 1 hora a 32 ° C. </li><li> Lave as bactérias solto e perfundir uma suspensão bacteriana diluída (OD 600 ~ 0,05) em 0.25mL/hr por 12 horas. </li><li> Lavar o biofilme com a mídia crescimento fresco DMEM para remover bactérias fracamente ligados e se preparar para fixação das células eucarióticas. </li><li> A imagem do biofilme usando uma Leica TCS SP5 Confocal Laser Scanning Microscope (Bannockburn, IL), e visualizar a arquitetura do biofilme usando o software IMARIS 5. </li></ol> 7. Semeadura células eucarióticas em torno de ilhas bacteriana 6-8 <ol><li> Trypsinize células HeLa do frasco de cultura de tecidos e ressuspender em DMEM mídia para uma densidade de semeadura de 5 x 10 5 células / mL. </li><li> Introduzir células HeLa no dispositivo a uma vazão de 2 mL / min usando uma bomba de seringa Picoplus (Harvard Apparattus, MA). Certifique-se que a parede ilha é reduzida para que o biofilme bacteriano é separada da região de células HeLa. Esta etapa deve ser realizada no palco de um microscópio de luz para que a uniformidade de semeadura pode ser determinada. </li><li> Grampo da tomada de célula para reduzir o movimento da suspensão de células no canal e incubar por 6 horas a 37 ° C em 5% incubadora de CO 2. </li><li> Perfundir o dispositivo com o meio de crescimento de 0,5 mL / hr para remover as células acopladas. </li><li> Atualizar os meios de comunicação DMEM para HeLa e E. comensais coli a cada 6 horas. </li></ol> 8. Introdução de bactérias patogênicas na ilha e exposição a células eucarióticas <ol><li> Prepare E. coli O157: H7 (EHEC) para infectar as células HeLa em uma multiplicidade de infecção (moi) de 100:1 200:1 (número de EHEC por célula HeLa). </li><li> Enxaguar e lavar fracamente ligados bactérias comensais. </li><li> Introduzir uma suspensão diluída EHEC (OD 600 ~ 0,1) para as ilhas. </li><li> Incubar dispositivo a 37 ° C em 5% incubadora de CO 2 por 6 horas sem fluxo para permitir a EHEC ser exposto a sinais presentes no biofilme comensais. </li><li> Levantar o muro PDMS para expor EHEC nas ilhas comensais às células HeLa por 6 horas. </li><li> Lave o canal com PBS e mancha para as células vivas e mortas utilizando o kit Live / Dead (L3224, Invitrogen, CA). </li><li> Locais de várias imagens em um microscópio Zeiss 200M Axiovert fluorescência e estimar o número de células vivas e mortas. </li></ol> 9. Resultados representativos 9 A seqüência de eventos em infecções do trato GI foi imitado pelo desenvolvimento de uma monocamada de células HeLa e um comensal E. biofilme coli e expondo EHEC para o biofilme comensais antes da infecção de células HeLa. As etapas envolvidas na fabricação do modelo de co-cultura microfluídicos são mostrados na Figura 1. Figura 2A mostra uma renderização 3-dimensional do dispositivo co-cultura. As duas regiões no dispositivo de co-cultura da câmara de cultura de células eucarióticas e da ilha de bactérias são mostrados na Figura 2B com tintas de cores diferentes (roxo para as regiões eucarióticas e verde para a ilha de bactérias). A viabilidade de isolar as regiões cultura bacteriana da região envolvente eucarióticas é mostrado na Figura 2C usando corantes. Figura 3 mostra os resultados representante da co-cultura de células epiteliais e bactérias. Figura 3A mostra a colonização do biofilme comensais (verde) por ilha EHEC (vermelho) Figura. 3B mostra a justaposição das células epiteliais e E. comensais coli com EHEC na ilha bacteriana. O RFP expressando GFP EHEC e expressando bactérias comensais estão localizadas na ilha quando a parede PDMS é reduzida, ilustrando a viabilidade de isolar a ilha de bactérias a partir de células epiteliais. <img src="/files/ftp_upload/1749/1749fig1.jpg" alt="Figura 1" /> Figura 1: esquema de celular semeadura no modelo de co-cultura. (1) A parede PDMS é reduzido para formar uma ilha, bactérias comensais são introduzidos na ilha, e fibronectina é corria ao redor da ilha. (2) células HeLa são semeados nas regiões que cercam a ilha. (3) Depois de células HeLa atingir confluência e do biofilme comensais se desenvolveu, EHEC é introduzido na ilha. (4) A parede PDMS é levantadaaté expor as células HeLa em torno da ilha para EHEC. Inset mostra detalhes do funcionamento da válvula. <img src="/files/ftp_upload/1749/1749fig2a.jpg" alt="Figura 2a" /> <img src="/files/ftp_upload/1749/1749fig2bc.jpg" alt="Figura 2BC" /> Figura 2: Modelo microfluídicos para co-cultura de células epiteliais e bactérias. (A) versão tridimensional do dispositivo co-cultura mostrando-pneumaticamente acionada regiões armadilhas para a formação de ilhas de bactérias entre as células epiteliais. Cada ilha bacteriana (1200 mm e diâmetro de 1.000 M de distância) tem uma entrada separada e saída para fornecer meios de crescimento e remoção de resíduos da ilha. (B) Micrografia do dispositivo co-cultura com tintas de cores mostrando as diferentes regiões (zona de células epiteliais, ilhas bacteriana). (C) A fidelidade do sistema de captura pneumático é mostrado através da redução da parede PDMS (painel esquerdo) usando um canal de pneumaticamente ativada (azul), introduzindo a tintura roxa nas ilhas fechadas ilha, e fluindo corante amarelo em torno dele por 48 h. Quando a parede é levantada PDMS (painel direito), a região insular é exposta ao corante amarelo ao redor. Barra de escala representa 500 mm. <img src="/files/ftp_upload/1749/1749fig3.jpg" alt="Figura 3" /> Figura 3: Co-cultura de células HeLa e bactérias. (A) imagem de fluorescência de RFP-expressando GFP-EHEC e expressando E. coli BW25113 na ilha. (B) Sobreposição de transmissão, verde e vermelho imagens de fluorescência no dispositivo. Barra de escala representa 200 mM.

<ol>
	<li>McDonald, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prototyping+of+microfluidic+devices+in+poly(dimethylsiloxane)+using+solid-object+printing.">Prototyping of microfluidic devices in poly(dimethylsiloxane) using solid-object printing.</a> Anal Chem. 74, 1537-1545 (2002).</li><li>Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+fabrication+of+integrated+passive+valves+and+pumps+for+flexible+polymer+3-dimensional+microfluidic+systems.">Design and fabrication of integrated passive valves and pumps for flexible polymer 3-dimensional microfluidic systems.</a> Biomed Microdevices. 4, 117-121 (2002).</li><li>Baek, J. Y., Park, J. Y., Ju, J. I., Lee, T. S., Lee, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pneumatically+controllable+flexible+and+polymeric+microfluidic+valve+fabricated+via+in+situ+development.">A pneumatically controllable flexible and polymeric microfluidic valve fabricated via in situ development.</a> J Micromech Microeng. 15, 1015-1020 (2005).</li><li>Grover, W. H., Ivester, R. H., Jensen, E. C., Mathies, R. A., A, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+multiplexed+control+of+latching+pneumatic+valves+using+microfluidic+logical+structures.">Development and multiplexed control of latching pneumatic valves using microfluidic logical structures.</a> Lab Chip. 6, 623-631 (2006).</li><li>Lee, J., Jayaraman, A., Wood, T. K., K, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indole+is+an+inter-species+biofilm+signal+mediated+by+SdiA.">Indole is an inter-species biofilm signal mediated by SdiA.</a> BMC Microbiol. 7, 42-42 (2007).</li><li>Hsu, C. H., Folch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+with+tunable+topographies.">Microfluidic devices with tunable topographies.</a> Appl Phys Lett. 86, 023508-023508 (2005).</li><li>Hui, E. E., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromechanical+control+of+cell-cell+interactions.">Micromechanical control of cell-cell interactions.</a> Proc Natl Acad Sci USA. 104, 5722-5726 (2007).</li><li>Lee, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+sensing+hydrogel+microstructures+into+micropatterned+hepatocellular+cocultures.">Integrating sensing hydrogel microstructures into micropatterned hepatocellular cocultures.</a> Langmuir. 25, 3880-3886 (2009).</li><li>Kim, J., Hegde, M., Jayaraman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-culture+of+epithelial+cells+and+bacteria+for+investigating+host-pathogen+interactions.">Co-culture of epithelial cells and bacteria for investigating host-pathogen interactions.</a> Lab-on-Chip. , (2009).</li></ol>

Ensaios convencionais para fixação e colonização de patógenos utilizam uma monocamada de células eucarióticas em placas de cultura de tecido em que os patógenos são adicionados. Estes modelos não são fisiologicamente relevantes como eles não incorporam um biofilme bacteriano comensais desenvolvida em células eucarióticas. Simples adição de uma cultura pré-grown bacteriana às células eucarióticas é improvável que levam a essa conformação como biofilmes são estruturas altamente organizadas que se ...

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		<th>Type</th>
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		<th>Catalogue Number</th>
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<td>SU-8 2050</td>
<td>&nbsp;</td>
<td>Microchem Corp, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>high-resolution (16,256 dpi) photolithography mask</td>
<td>&nbsp;</td>
<td>Fineline-Imaging Inc, CO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Trichloro(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)silane</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>77279</td>
<td>&nbsp;</td>
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<td>PDMS</td>
<td>&nbsp;</td>
<td>Dow Corning, WI</td>
<td>184 SIL ELAST KIT 0.5KG</td>
<td>&nbsp;</td>
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<td>DMEM</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>SH30002.02</td>
<td>&nbsp;</td>
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<td>Programmable spin coater</td>
<td>&nbsp;</td>
<td>Laurell Tech Corp</td>
<td>WS0650S</td>
<td>&nbsp;</td>
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<td>Mask aligner</td>
<td>&nbsp;</td>
<td>Neutronix-Quintel, PA</td>
<td>Q4000</td>
<td>&nbsp;</td>
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<td>Oxygen plasma etcher</td>
<td>&nbsp;</td>
<td>March Plasma System, CA</td>
<td>CS-1701</td>
<td>&nbsp;</td>
<tr>
<td>Syringe pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Live/Dead Viability/Cytotoxicity Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>L-3224</td>
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microfluidic co-culture of epithelial cells and bacteria for investigating soluble signal-mediated interactions

O ser humano no trato gastrointestinal (GI) é um ambiente único em que células do epitélio intestinal e não-patogênicos (comensais) bactérias coexistem. Tem sido proposto que o microambiente que o patógeno encontra na camada de comensais é importante para determinar a extensão da colonização. Métodos de cultura atual para investigar a colonização de patógenos não são adequados para investigar esta hipótese, pois não permitem co-cultura de bactérias e células epiteliais de uma forma que imita o microambiente do trato GI. Descrevemos aqui um modelo de co-cultura microfluídicos que permite que a cultura independente de células eucarióticas e bactérias, e testar o efeito do microambiente comensais sobre a colonização do patógeno. O modelo de co-cultura é demonstrada através do desenvolvimento de um comensal<em&gt; Escherichia coli</em&gt; Biofilme entre as células HeLa, seguido pela introdução de entero<em&gt; E. coli</em&gt; (EHEC) na ilha comensais, em uma seqüência que imita a seqüência de eventos em infecção do trato GI.

Watch this Scientific Journal Video about Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions at JoVE.com

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

Este protocolo descreve um modelo de co-cultura microfluídicos para a cultura simultânea e localizada de células epiteliais e bactérias. Este modelo pode ser usado para investigar o papel dos diferentes sinais moleculares solúveis na patogênese, bem como tela a eficácia do suposto probiótico cepas bacterianas.

Microfluídicos Co-cultura de células epiteliais e bactérias para a Investigação Solúvel sinal mediada por interações

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

microfluidic-co-culture-epithelial-cells-bacteria-for-investigating

Research

JoVE Journal

Biology

14.0K visualizações.  Texas A&M University. Este protocolo descreve um modelo de co-cultura microfluídicos para a cultura simultânea e localizada de células epiteliais e bactérias. Este modelo pode ser usado para investigar o papel dos diferentes sinais moleculares solúveis na patogênese, bem como tela a eficácia do suposto probiótico cepas bacterianas.

Vídeo: Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

A Neuronal e astrocitária Ensaio Co-Cultura de Análise de Conteúdo de Alta de neurotoxicidade

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biologia

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

Um compartimento Multi-glia do SNC Neuron-Co-cultura plataforma microfluídica

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Isolamento de células epiteliais mamárias de tridimensional Mixed-célula esferoidal co-cultura

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Indução e Análise de epitelial para mesenquimal Transição

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Investigando interações proteína-proteína em células vivas usando a transferência de Bioluminescência Resonance Energy

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Medidas cálcio citosólico em células epiteliais renais por citometria de fluxo

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

Método cryosectioning para Microdissection de Murino Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

Usando dispositivos microfluídicos medir Longevidade e Celular fenótipos em células individuais levedura de brotamento

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

Monitoração in situ de montagens moleculars formadas Transientemente do chaperone nas bactérias, no fermento, e nas pilhas humanas

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor &#945;) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.

Modelo Alveolar Humano Multicelular Composto por Células Epiteliais e Células Imunes Primárias para Avaliação de Riscos

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...