Os autores agradecem Ronald F. Renzi e Michael S. Bartsch por suas contribuições para a concepção e desenvolvimento de dispositivos de DMF eo hub DMF. Esta pesquisa foi totalmente suportado pelo Laboratório Direção de Pesquisa e Programa de Desenvolvimento do Sandia National Laboratories. Sandia é um laboratório multi-programa gerenciado e operado pela Sandia Corporation, uma subsidiária integral da Lockheed Martin Corporation, para o Departamento de Administração de Segurança Nacional de Energia Nuclear dos EUA sob contrato DE-AC04-94AL85000.

Este protocolo geral é concebida para suportar a aplicação da plataforma para uma ampla variedade de estudos, aspectos específicos do estudo representativo descrito neste relatório são separados em suportes Figura 2 ilustra um estudo representativo realizado utilizando o protocolo.. Note-se que no protocolo, todos os &quot;instruir&quot; os comandos são automatizados através do uso de pré-determinados scripts. Note também que o Passo 2 pode ser efectuado em paralelo com o Passo 1 (por exemplo, durante os Passos 1.7 e 1.8), e que é muitas vezes Passo 2.1 contornado (porque as placas do dispositivo modelado DMF / revestido pode ser lavado e reutilizado, ver Passo 5.2) . 1. Montar e preencher as Câmaras de Perfusão celulares <ol><li> Revestir as superfícies interiores das esterilizado (autoclavado) microcapilares (sílica fundida; od 679 uM, 534 uM ID; 15 cm de comprimento), com a fibronectina, por instruir a bomba de seringa para estabelecer uma solução estéril de fibronectina (30 ul de 50 ug / ml) em cadamicrocapilar, incubação (37 ° C durante 2 horas), instruindo a bomba de seringa para retirar a solução, e deixando o microcapilares seco (temperatura ambiente (RT), durante 6 h). </li><li> Conecte cada fibronectina revestido microcapilar (câmara de perfusão) para tubos de policarbonato (500 mM od, ID 100 mm) e da tubulação para a bomba de seringa multi-porta, usando acessórios CapTite (6 câmaras de perfusão, de 8 portas bomba de seringa). </li><li> Utilizando fita, proteger o corpo de cada câmara de perfusão para um bloco de aquecimento até à temperatura definida desejada (37 ° C). </li><li> Mergulhar as extremidades abertas das câmaras de perfusão em um reservatório (microcentifuge tubo ou placa de microtitulação) células contendo P388D.1 (macrófagos murinos) suspensas em meio de crescimento fresco (RPMI-1640, 10% FBS, 500 unidades / ml de penicilina, 500 ug / mL de estreptomicina) a concentração desejada (10 6 células / ml). </li><li> Instruir a bomba de seringa para extrair células (10 uL, 10 4 culas) em cada câmara de perfusão seguido por ai suficienter para mover o tampão líquido (~ 4,5 cm) para a secção fixada ao bloco de calor. </li><li> Permitir que as células a aderir às superfícies internas revestidas com fibronectina das câmaras de perfusão (37 ° C durante 1 - 2 h). Ao mesmo tempo, transferir as extremidades abertas das câmaras de perfusão a partir do reservatório de células para um novo reservatório contendo meio de crescimento fresco. </li><li> Após o período de adesão, instruir a bomba de seringa para enviar as fichas líquidos (meios condicionados e células acopladas) para o lixo, retirar meio fresco (10 ml), e seguir com ar suficiente para posicionar os novos plugues líquidos (meio fresco) sobre o aderido células. </li><li> Continuar a realizar trocas médio (ou seja, repita o Passo 1.7) em intervalos regulares (a cada 2 horas) até as populações de células são suficientemente equilibrada e expandido (16 - 24 horas de micro-escala da cultura de populações gerados ~ 1.000 células / Câmara). </li></ol> 2. Fabricar o dispositivo de DMF e montar o Hub Architecture <ol><li&gt; Conforme descrito anteriormente 14,29, padrão da placa inferior do dispositivo DMF com quarenta e seis óxido de índio e estanho (ITO) eletrodos (condutores de gotículas de atuação) por fotolitografia e corrosão, e casaco com 5 mm de Parylene-C e 50 nm de Teflon AF. Forma da placa de topo do dispositivo de DMF por revestimento de um substrato de vidro ITO unpatterned com Teflon AF, como acima. </li><li> Uso de quadros de metal de compressão, fixar as placas de DMF em um molde de polímero 14,30 com recessos que mantêm o espaçamento de 400 um entre as chapas, o espaçamento, combinada com o tamanho do eléctrodo de accionamento (2,5 mm 2), define o volume de gotas (2,5 ul por eletrodo). Note-se que a montagem de DMF pode ser conseguida por meios mais simples 24. </li><li> Inserir revestida de Teflon transferência microcapilares (360 mM OD, 100 uM ID; 3,5-4,0 cm de comprimento) para o espaço entre as placas de DMF, posicionando cada um para o rebordo do seu eléctrodo de accionamento cognato. </li><li> Para o en opostod de cada transferência apor microcapilar um encaixe CapTite. </li><li> Envolver os conectores elétricos para tensão de alimentação para os eletrodos de ITO. Seqüências de ativação de eletrodos são automatizados através do uso de uma interface eletrônica controlada por computador funcionando pré-determinados scripts. </li><li> Opcional: Mova o hub DMF montada no palco de translação de um microscópio (SZ-6145TR (Olympus, Japão)) equipado com um dispositivo de carga acoplada (CCD) da câmera (DCR-HC96 (Sony, Japão)) para facilitar o rastreamento de gotículas 31. </li></ol> 3. Ligue as câmaras de perfusão para o Hub DMF e estimular a populações de células <ol><li> Remover as extremidades abertas das câmaras de perfusão do reservatório de meio (ver Passo 1.6) e inseri-los nos encaixes CapTite apostos nas extremidades da transferência microcapilares do cubo DMF (ver Passo 2.4). </li><li> Instrua a bomba de seringa para enviar as fichas líquidos (meios condicionados) nas câmaras de perfusão para o lixo. </li><li> Instruct o cubo DMF para desenhar estímulo (E. coli biopartículas a 100 ug / ml em meio fresco com 0,1% de Pluronic F127 w / v) a partir de um reservatório de bordo e proporcionar uma gota de dimensão adequada (10 ul) para cada transferência microcapilar . Os reservatórios de bordo são constituídos por compartimentos em forma cilíndrica (1,5 x 1,5 cm cada) e ligado ao cubo por meio da tubagem de DMF. </li><li> Instruir a bomba de seringa para extrair o estímulo conecta à transferência de microcapilares, e seguir com ar suficiente para posicionar os tampões sobre as populações de células nas câmaras de perfusão. </li><li> Incubar as células com estímulo (37 ° C durante 1-4 horas). Opcional: Troca de estímulo fresco (ou seja, repita os passos 3,2-3,4) em intervalos regulares. </li></ol> 4. Terminar Estimulação e recuperar lisados ​​celulares para Análise <ol><li> Opcional: Se é necessário um período de pós-estimulação, trocar as fichas contendo estímulo líquido por meio fresco (ou seja, repita os passos 3,2-30,4, substituindo meio fresco para o estímulo), e continuar a incubar e trocar, se necessário. </li><li> Troca as fichas líquidos nas câmaras de perfusão para tampão de lavagem (Prelude Lise direto Módulo tampão B) (ou seja, repita os passos 3,2-3,4, substituindo o tampão de lavagem de estímulo), e incubar conforme necessário (5 min). </li><li> Troca as fichas líquidos (tampão de lavagem) para a solução de lise (Prelude direto Lise módulo A, com 0,1% Pluronic F127 w / v) (ou seja, repita os passos 3,2-3,4, substituindo solução de lise de estímulo), e incubar conforme necessário (5 min) . </li><li> Instruir a bomba de seringa para enviar os tampões líquidos (lisados ​​de células) para o centro de DMF. </li><li> Desmontar o cubo DMF, remover a placa de topo do dispositivo de DMF (tomando cuidado para não inclinar a placa de lado, como o que pode resultar em gotículas de mistura), e recolher os lisados ​​usando um Pipetman ou seringa para análise off-plataforma (perfil de expressão do gene através qPCR). </li></ol> 5. Limpo Plataforma de Hardware paraRe-use <ol><li> Imergir a transferência microcapilares e acessórios CapTite em lixívia a 10% (v / v em água) durante 10 min à TA, lave bem com água desionizada, e seco usando gás azoto. </li><li> Mergulhar as placas do dispositivo de DMF em lixívia a 10% durante 10 min à RT, enxaguar bem com isopropanol e depois com água desionizada, e utilizando gás de azoto seco, seguido por cozedura a 160 ° C durante 10 min. </li><li> Policarbonato tubulação da bomba de seringa, e elenco polímero do dispositivo DMF e quadro de compressão, pode ser reutilizado sem limpeza. A câmara de perfusão microcapilares são descartados após cada experimento. </li></ol>

<ol>
	<li>Young, E. W. K., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+microfluidic+cell+culture+in+controlled+microenvironments.">Fundamentals of microfluidic cell culture in controlled microenvironments.</a> Chemical Society Reviews. 39, 1036-1048 (2010).</li><li>Yeo, L. Y., Chang, H. C., Chan, P. P. Y., Friend, J. R. <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+for+bioapplications.">Microfluidic devices for bioapplications.</a> Small. 7, 12-48 (2011).</li><li>Zhang, B., Kim, M. C., Thorsen, T., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+self-contained+microfluidic+cell+culture+system.">A self-contained microfluidic cell culture system.</a> Biomedical Microdevices. 11, 1233-1237 (2009).</li><li>Skafte-Pedersen, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+self-contained,+programmable+microfluidic+cell+culture+system+with+real-time+microscopy+access.">A self-contained, programmable microfluidic cell culture system with real-time microscopy access.</a> Biomedical Microdevices. 14, 385-399 (2012).</li><li>Barbulovic-Nad, I., Au, S. H., Wheeler, 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=A+microfluidic+platform+for+complete+mammalian+cell+culture.">A microfluidic platform for complete mammalian cell culture.</a> Lab Chip. 10, 1536-1542 (2010).</li><li>Zhou, Y., Pang, Y., Huang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Openly+Accessible+Microfluidic+Liquid+Handlers+for+Automated+High-Throughput+Nanoliter+Cell+Culture.">Openly Accessible Microfluidic Liquid Handlers for Automated High-Throughput Nanoliter Cell Culture.</a> Analytical Chemistry. 84, 2576-2584 (2012).</li><li>Wang, H. Y., Bao, N., Lu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+cell+array+with+individually+addressable+culture+chambers.">A microfluidic cell array with individually addressable culture chambers.</a> Biosensors and Bioelectronics. 24, 613-617 (2008).</li><li>Cimetta, E., Cagnin, S., Volpatti, A., Lanfranchi, G., Elvassore, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+culture+of+droplet-confined+cell+arrays.">Dynamic culture of droplet-confined cell arrays.</a> Biotechnology Progress. 26, 220-231 (2010).</li><li>Hung, P. J., Lee, P. J., Sabounchi, P., Lin, R., Lee, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+perfusion+microfluidic+cell+culture+array+for+high-throughput+cell-based+assays.">Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays.</a> Biotechnology and Bioengineering. 89, 1-8 (2005).</li><li>King, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+microfluidic+real-time+gene+expression+living+cell+array.">A high-throughput microfluidic real-time gene expression living cell array.</a> Lab on a Chip. 7, 77-85 (2007).</li><li>G&#243;mez-Sj&#246;berg, R., Leyrat, A. A., Pirone, D. M., Chen, C. S., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile,+fully+automated,+microfluidic+cell+culture+system.">Versatile, fully automated, microfluidic cell culture system.</a> Analytical Chemistry. 79, 8557-8563 (2007).</li><li>Wheeler, 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=Chemistry+-+Putting+electrowetting+to+work.">Chemistry - Putting electrowetting to work.</a> Science. 322, 539-540 (2008).</li><li>Gong, J., Kim, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-electronic+droplet+generation+on-chip+with+real-time+feedback+control+for+EWOD+digital+microfluidics.">All-electronic droplet generation on-chip with real-time feedback control for EWOD digital microfluidics.</a> Lab Chip. 8, 898-906 (2008).</li><li>Choi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+field+effect+transistor-based+biosensors+with+a+digital+microfluidic+device+for+a+lab-on-a-chip+application.">Integration of field effect transistor-based biosensors with a digital microfluidic device for a lab-on-a-chip application.</a> Lab Chip. 12, 1533-1536 (2012).</li><li>Barbulovic-Nad, I., Yang, H., Park, P. S., Wheeler, 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=Digital+microfluidics+for+cell-based+assays.">Digital microfluidics for cell-based assays.</a> Lab Chip. 8, 519-526 (2008).</li><li>Gentilucci, L., de Marco, R., Cerisoli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+modifications+designed+to+improve+peptide+stability:+incorporation+of+non-natural+amino+acids,+pseudo-peptide.">Chemical modifications designed to improve peptide stability: incorporation of non-natural amino acids, pseudo-peptide.</a> 16, 3185-3203 (2010).</li><li>Miller, E. M., Wheeler, 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=A+digital+microfluidic+approach+to+homogeneous+enzyme+assays.">A digital microfluidic approach to homogeneous enzyme assays.</a> Anal. Chem. 80, 1614-1619 (2008).</li><li>Jebrail, M. J., Bartsch, M. S., Patel, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+microfluidics:+a+versatile+tool+for+applications+in+chemistry,+biology.">Digital microfluidics: a versatile tool for applications in chemistry, biology.</a> 12, 2452-2463 (2012).</li><li>Ng, A. H. C., Choi, K., Luoma, R. P., Robinson, J. M., Wheeler, 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=Digital+Microfluidic+Magnetic+Separation+for+Particle-Based+Immunoassays.">Digital Microfluidic Magnetic Separation for Particle-Based Immunoassays.</a> Analytical Chemistry. 84, 8805-8812 (2012).</li><li>Sista, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+immunoassays+using+magnetic+beads+on+a+digital+microfluidic+platform.">Heterogeneous immunoassays using magnetic beads on a digital microfluidic platform.</a> Lab Chip. 8, 2188-2196 (2008).</li><li>Malic, L., Veres, T., Tabrizian, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochip+functionalization+using+electrowetting-on-dielectric+digital+microfluidics+for+surface+plasmon+resonance+imaging+detection+of+DNA+hybridization.">Biochip functionalization using electrowetting-on-dielectric digital microfluidics for surface plasmon resonance imaging detection of DNA hybridization.</a> Biosens. Bioelectron. 24, 2218-2224 (2009).</li><li>Malic, L., Veres, T., Tabrizian, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanostructured+digital+microfluidics+for+enhanced+surface+plasmon+resonance+imaging.">Nanostructured digital microfluidics for enhanced surface plasmon resonance imaging.</a> Biosens. Bioelectron. 26, 2053-2059 (2011).</li><li>Jebrail, M. J., Wheeler, 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=Digital+microfluidic+method+for+protein+extraction+by+precipitation.">Digital microfluidic method for protein extraction by precipitation.</a> Anal. Chem. 81, 330-335 (2009).</li><li>Nelson, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incubated+protein+reduction+and+digestion+on+an+electrowetting-on-dielectric+digital+microfluidic+chip+for+MALDI-MS.">Incubated protein reduction and digestion on an electrowetting-on-dielectric digital microfluidic chip for MALDI-MS.</a> Anal. Chem. 82, 9932-9937 (2010).</li><li>Mousa, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet-scale+estrogen+assays+in+breast+tissue,+blood,+and+serum.">Droplet-scale estrogen assays in breast tissue, blood, and serum.</a> Sci. Transl. Med. 1, 1ra2 (2009).</li><li>Jebrail, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+digital+microfluidic+method+for+dried+blood+spot+analysis.">A digital microfluidic method for dried blood spot analysis.</a> Lab Chip. 11, 3218-3224 (2011).</li><li>Choi, K., Ng, A. H. C., Fobel, R., Wheeler, 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=Digital+microfluidics.">Digital microfluidics.</a> Annual Review of Analytical Chemistry. 5, 413-440 (2012).</li><li>Jebrail, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+Microfluidics+for+Automated+Proteomic+Processing.">Digital Microfluidics for Automated Proteomic Processing.</a> J. Vis. Exp. (33), e1603 (2009).</li><li>Thaitrong, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+Control+of+Next+Generation+Sequencing+Library+through+an+Integrative+Digital+Microfluidic+Platform.">Quality Control of Next Generation Sequencing Library through an Integrative Digital Microfluidic Platform.</a> , (2012).</li><li>Shin, Y. J., Lee, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Machine+vision+for+digital+microfluidics.">Machine vision for digital microfluidics.</a> Review of Scientific Instruments. 81, (2010).</li><li>Thornton, M., Eward, K. L., Helmstetter, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+minimally+disturbed+synchronous+cultures+of+hematopoietic+cells.">Production of minimally disturbed synchronous cultures of hematopoietic cells.</a> BioTechniques. 32, 1098-1105 (2002).</li><li>Huang, X., Dai, W., Darzynkiewicz, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enforced+adhesion+of+hematopoietic+cells+to+culture+dish+induces+endomitosis+and+polyploidy.">Enforced adhesion of hematopoietic cells to culture dish induces endomitosis and polyploidy.</a> Cell Cycle. 4, 801-805 (2005).</li><li>Raje, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charged+nylon+membrane+substrate+for+convenient+and+versatile+high+resolution+microscopic+analysis+of+Escherichia+coli+&amp;+mammalian+cells+in+suspension+culture.">Charged nylon membrane substrate for convenient and versatile high resolution microscopic analysis of Escherichia coli &amp; mammalian cells in suspension culture.</a> Cytotechnology. 51, 111-117 (2006).</li><li>Chatterjee, D., Hetayothin, B., Wheeler, A. R., King, D. J., Garrell, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet-based+microfluidics+with+nonaqueous+solvents+and+solutions.">Droplet-based microfluidics with nonaqueous solvents and solutions.</a> Lab Chip. 6, 199-206 (2006).</li><li>Perroud, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic-based+cell+sorting+of+Francisella+tularensis+infected+macrophages+using+optical+forces.">Microfluidic-based cell sorting of Francisella tularensis infected macrophages using optical forces.</a> Analytical Chemistry. 80, 6365-6372 (2008).</li><li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+A+revolutionary+tool+for+transcriptomics.">RNA-Seq: A revolutionary tool for transcriptomics.</a> Nature Reviews Genetics. 10, 57-63 (2009).</li><li>Malone, J. H., Oliver, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarrays,+deep+sequencing+and+the+true+measure+of+the+transcriptome.">Microarrays, deep sequencing and the true measure of the transcriptome.</a> BMC Biology. 9, (2011).</li><li>Wu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidically-unified+cell+culture,+sample+preparation,+imaging+and+flow+cytometry+for+measurement+of+cell+signaling+pathways+with+single+cell+resolution.">Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution.</a> Lab on a Chip. 12, 2823-2831 (2012).</li><li>Srivastava, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+integrated+microfluidic+platform+enabling+automated+phosphoprofiling+of+macrophage+response.">Fully integrated microfluidic platform enabling automated phosphoprofiling of macrophage response.</a> Analytical Chemistry. 81, 3261-3269 (2009).</li><li>Herr, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+immunoassays+as+rapid+saliva-based+clinical+diagnostics.">Microfluidic immunoassays as rapid saliva-based clinical diagnostics.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 5268-5273 (2007).</li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

Nós desenvolvemos uma plataforma automatizada simples e versátil para a cultura de células micro-escala e experiências de estimulação. A plataforma permite-nos trabalhar com pequenos volumes de cultura e populações celulares (1-20 mL e 100 - 2000 células, por câmara); tamanhos cultura pode ser ainda mais reduzida com o uso de microcapilares de menor diâmetro. Trabalhando em ambas as escalas reduz o custo de estudos de rotina e faz estudos viáveis, que requerem o uso de reagentes preciosos e / ou de ...

O estudo de células mantidas em cultura (análise in vitro) forneceu uma visão valiosa sobre os princípios fundamentais e os mecanismos moleculares que regem os sistemas biológicos complexos ea saúde humana. Os métodos convencionais de cultura, estimulação e coleta de células para análise, que utilizam placas de Petri e placas de poliestireno, foram projetados para o estudo de grandes populações de células (≥ 10 5) em volumes de cultura mililitro escala (≥ 0,1 ml). No entanto, há muitos casos em que apenas quantidades limitadas de células estão disponíveis (por exemplo, as células primárias), ou de pequenas populações de células são desejáveis ​​(por exemplo, para reduzir a variabilidade de-célula através da população), ou os reagentes necessários são de difícil obtenção ou proibitivamente caro (por exemplo, células purificadas fatores secretados). Tais questões podem ser abordadas com sucesso pelo redimensionamento tamanho cultura, que tem a vantagem de reduzir o consumo de todos osOs reagentes necessários para a análise in vitro, em 1,2. Infelizmente, equipamentos e métodos convencionais não suportam manipulação precisa e reprodutível de culturas micro-escala e os sistemas automatizados baseados em microfluídica atualmente disponíveis 3-11 são muito complexas e especializadas para uso de rotina pela maioria dos laboratórios. Neste relatório, nós descrevemos a montagem e utilização de uma plataforma de tecnologia simples e versátil para a cultura automatizada, estimulação e recuperação de pequenas populações de células (100 - 2000 células) em volumes micro-escala (1-20 mL). A arquitetura da plataforma (Figura 1) é um projeto modular: um conjunto de fibronectina revestido microcapilares (&quot;câmaras de perfusão de células&quot; Módulo), que serve como local para o estabelecimento, manutenção e estimulação das culturas micro-escala, e uma microfluídica digital (DMF ) 12,13 dispositivo equipado com &quot;transfer&quot; microcapilares (módulo central &quot;hub&quot;) 14,15 células rotase reagentes de e para as câmaras de perfusão. DMF permite ao usuário resolver individualmente várias gotas em simultâneo e de alterar ou re-ordenar as manipulações (ou seja, os trens de processamento de amostras reconfigurar) sem alterar o hardware do dispositivo. A sua grande flexibilidade é evidente em seu surgimento recente como uma tecnologia chave numa vasta gama de aplicações, incluindo a cultura da célula 16,17 18,19 ensaios enzimáticos, os imunoensaios 20,21, 22,23 A análise do DNA, o processamento de proteínas, 24,25 e processamento de amostras clínicas. 26,27 Nossa cubo central aproveita a flexibilidade inerente aos dispositivos de DMF, e aumenta ainda mais através da adição de interfaces de microcapilares, que proporciona oportunidade para a realização de um subconjunto de manipulações (por exemplo, cultura de células) em periférico especializado módulos, em vez de no próprio dispositivo de DMF. A compartimentalização dos comboios de processamento deste modo também simplifica a concepção do plaarquitetura TForm (não é necessário para construir um dispositivo DMF que pode realizar todas as etapas de processamento) e facilita a sua evolução à medida que novas funções são necessárias (simplesmente integrar novos módulos periféricos como necessário). Transporte de células e reagentes dentro do cubo central é accionado por electrowetting forças geradas pela activação sequencial dos eléctrodos no interior do dispositivo de DMF 13,28; transporte para, de e no interior das câmaras de perfusão é alimentado por alterações de pressão geradas por uma seringa de alta precisão bomba. Todos esses movimentos fluidos são controlados através de uma interface eletrônica simples e automatizado através da utilização de pré-determinados scripts. Como um exemplo representativo, que demonstram a utilização da plataforma para o estudo das respostas da transcrição induzida em células do sistema imune após o desafio com bactérias (Figura 2). Realizar esses experimentos na plataforma nos permitiu trabalhar com um pequeno número de células (~ 1.000 por experimentaçãotal condição), para minimizar a variabilidade experiência-to-experimento, reagentes conservar e re-direta hands-on de trabalho. Dadas as vantagens que ela confere, assim como a sua acessibilidade e versatilidade, esta plataforma deve encontrar utilização numa ampla variedade de aplicações e de laboratórios e ser especialmente útil para facilitar a análise de células e os estímulos que estão disponíveis em quantidades limitadas.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Prelude Direct Lysis Module</td>
 <td>NuGEN</td>
 <td>1400-24</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypan Blue (0.4% w/v)</td>
 <td>GIBCO</td>
 <td>15250-061</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell Stripper</td>
 <td>Cellgro</td>
 <td>25-056-C1</td>
 <td></td>
 </tr>
 <tr>
 <td>Ovation PicoSL WTA</td>
 <td>NuGEN</td>
 <td>3310-048</td>
 <td></td>
 </tr>
 <tr>
 <td>Agencourt RNAClean XP</td>
 <td>Beckman Coulter Genomics</td>
 <td>A63987</td>
 <td></td>
 </tr>
 <tr>
 <td>pHrodo BioParticles</td>
 <td>Invitrogen</td>
 <td>P35361</td>
 <td></td>
 </tr>
 <tr>
 <td>CCL4 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00443111_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>CCL5 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm01302428_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>PTGS2 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00478374_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>TNF TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00443258_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>GAPDH TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm99999915_g1</td>
 <td></td>
 </tr>
 <tr>
 <td>Pluronic F127</td>
 <td>Sigma Chemical</td>
 <td>2594628</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluorinert FC-40</td>
 <td>Sigma Chemical</td>
 <td>51142-49-5</td>
 <td></td>
 </tr>
 <tr>
 <td>Parylene C dimer</td>
 <td>Specialty Coating Systems</td>
 <td>28804-46-8</td>
 <td></td>
 </tr>
 <tr>
 <td>Teflon-AF</td>
 <td>DuPont</td>
 <td>AF1600</td>
 <td></td>
 </tr>
 <tr>
 <td>Polyimide tape</td>
 <td>ULINE</td>
 <td>S-11928</td>
 <td></td>
 </tr>
 <tr>
 <td>Indium tin oxide (ITO) coated glass substrates </td>
 <td>Delta Technologies </td>
 <td>CB-40IN-1107</td>
 <td></td>
 </tr>
 <tr>
 <td>DMF hub Teflon-coated fused-silica microcapillaries</td>
 <td>Polymicro Technologies</td>
 <td>TSU100375</td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber microcapillaries</td>
 <td>Polymicro Technologies</td>
 <td>TSP530700</td>
 <td></td>
 </tr>
 <tr>
 <td>Tubing and microcapillary fittings</td>
 <td>Sandia National Laboratories</td>
 <td>&nbsp;</td>
 <td></td>
 </tr>
 <tr>
 <td>Polycarbonate tubing</td>
 <td>Paradigm Optics</td>
 <td>CTPC100-500-5</td>
 <td></td>
 </tr>
 <tr>
 <td>8-port precision syringe pump equipped with 30 mm (500 &mu;l capacity) syringes</td>
 <td>Hamilton Company</td>
 <td>54848-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Parylene-C vapor deposition instrument</td>
 <td>Specialty Coating Systems</td>
 <td>PDS 2010 Labcoter 2</td>
 <td></td>
 </tr>
 <tr>
 <td>High-voltage function generator</td>
 <td>Trek</td>
 <td>615A-1 615-3</td>
 <td></td>
 </tr>
 <tr>
 <td>MVX10 microscope</td>
 <td>Olympus</td>
 <td></td>
 <td>Optional (facilitates tracking of droplets on DMF hub)</td>
 </tr>
 <tr>
 <td>QIClick digital CCD camera</td>
 <td>QImaging</td>
 <td>QIClick-F-CLR-12</td>
 <td>Optional (facilitates tracking of droplets on DMF hub)</td>
 </tr>
</tbody>
</table>

a versatile automated platform for micro-scale cell stimulation experiments

Estudo de células em cultura (análise in vitro) forneceu importantes insights sobre sistemas biológicos complexos. Os métodos convencionais e equipamentos para a análise in vitro são bem adequados ao estudo de um grande número de células (≥ 10 5) em volumes mililitro escala ≥ (0,1 ml). No entanto, há muitos casos em que é necessário ou desejável reduzir a dimensão de tamanho para reduzir o consumo de cultura das células de interesse e / ou os reagentes necessários para a sua cultura, a estimulação ou processamento. Infelizmente, as abordagens convencionais não suportam manipulação precisa e reprodutível de culturas micro-escala, e os sistemas automatizados baseados em microfluídica atualmente disponíveis são muito complexos e especializados para uso de rotina pela maioria dos laboratórios. Para resolver este problema, foram desenvolvidos uma plataforma de tecnologia simples e versátil para a cultura automatizado, estimulação e recuperação de pequenas populações de células (100 - 2000 células) no volume de micro-escalas (1 - 20 ul). A plataforma é constituído por um conjunto de microcapilares revestidos com fibronectina (&quot;câmaras de perfusão de células&quot;), no qual as culturas de micro-escala são estabelecidos, mantida e estimulada, um dispositivo digital de microfluidos (DMF) equipado com &quot;transfer&quot; microcapilares (&quot;hub central &quot;), que as células e reagentes rotas de e para as câmaras de perfusão, uma bomba de seringa de alta precisão, que o transporte de materiais de poderes entre as câmaras de perfusão eo hub central, e uma interface eletrônica que fornece controle sobre o transporte de materiais, o que é coordenado e automatizados via pré-determinados scripts. Como um exemplo, utilizou-se a plataforma para facilitar o estudo de respostas eliciadas transcrição em células imunes ao desafio com bactérias. A utilização da plataforma permitiu-nos a reduzir o consumo de células e reagentes, minimizar a variabilidade experiência para experiência e reorientar as mãos sobre o trabalho. Dadas as vantagens que ela confere, assim como a sua acessibilidade e versatilidade, oplataforma ur deve encontrar o uso em uma ampla variedade de laboratórios e aplicações, e revelar-se especialmente útil para facilitar a análise de células e os estímulos que estão disponíveis apenas em quantidades limitadas.

Como uma demonstração da plataforma automatizada, que utilizado para levar a cabo um estudo em que as pequenas populações de células imunitárias foram cultivados em culturas em micro-escala, desafiado com bactérias, e lisadas para análise off-plataforma de respostas pró-inflamatórias (Figura 2 ). Cada uma das seis câmaras de perfusão de células foi semeada com 10 4 P388D.1 células imunitárias (macrófagos murinos) foram ressuspensas em 10 ul de meio...

Watch this Scientific Journal Video about A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments at JoVE.com

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Nós desenvolvemos uma cultura de células automatizado e uma plataforma de interrogação para experiências de estimulação de células micro-escala. A plataforma oferece um controle simples, versátil e preciso para cultivar e estimular pequenas populações de células e recuperar lisados ​​para análises moleculares. A plataforma é bem adequado para os estudos que utilizam células preciosos e / ou reagentes.

A plataforma automatizada versátil para experiências de estimulação Micro escala celulares

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

a-versatile-automated-platform-for-micro-scale-cell-stimulation

Research

JoVE Journal

Bioengineering

10.5K visualizações.  Sandia National Laboratories. Nós desenvolvemos uma cultura de células automatizado e uma plataforma de interrogação para experiências de estimulação de células micro-escala. A plataforma oferece um controle simples, versátil e preciso para cultivar e estimular pequenas populações de células e recuperar lisados ​​para análises moleculares. A plataforma é bem adequado para os estudos que utilizam células preciosos e / ou reagentes.

Vídeo: A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Organotípica Colágeno Ensaio I: Uma Plataforma maleável para Avaliar Comportamento celular em um contexto de 3-Dimensional

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Bioengenharia

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
<div id="myEventWatcherDiv">
	 </div>

GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.

GENPLAT: uma plataforma automatizada para Discovery Biomassa Enzima e Otimização Cocktail

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Espremendo celular como uma plataforma de entrega intracelular Robust Microfluidic

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

Uma plataforma Novel Alongamento para Aplicações em Celular e Tecidual mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Uma plataforma microfluídica para a isolação de alta taxa de transferência de célula única e Cultura

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Em camadas de alginato Constrói: Uma Plataforma para Co-cultura de populações de células heterogéneas

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

Um de alta capacidade: Plataforma celular Microarray para Análise correlativa da diferenciação celular e forças de tração

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Um método versátil de padronização proteínas e células

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Nanotubos de DNA como uma ferramenta versátil para estudar semiflexível polímeros

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

Uma plataforma Microfluídico multicamada para a condução de expressão gênica sem células prolongada

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...