Células CHO-P foram um presente amável do Dr. Barbara Furie (Beth Israel Deaconess Medical Center, Harvard Medical School). Este trabalho foi financiado pelo Instituto Nacional de Saúde HL095722 concessão para JMK Este trabalho também foi apoiado em parte por um Movember-Prostate Cancer Foundation Prêmio Desafio para JMK

1. Cultura de Células <ol><li> Leucemia promielocítica humana (células HL-60) <ol><li> Culturas células HL-60 em 75 centímetros 2 frascos com 15 ml de Iscove Modified Médio (IMDM) de Dulbecco, suplementado com 20% (v / v) de soro fetal bovino (FBS), 1% (v / v), L-glutamina e 1 % (v / v) de Penicilina-Estreptomicina. </li><li> Mudança de mídia a cada 3 dias por aspiração metade do volume de suspensão celular e substituindo-o por meios IMDM completa. </li><li> Para diacetato de carboxifluoresceína, coloração de éster de succinimidilo (CFSE), centrífuga de HL-60 de suspensão de células (400 x g, 5 min), ressuspender em uma solução de 1 ^ M de CFSE (preparado em PBS pré-aquecido) e incubar durante 15 min a 37 ° C. Em seguida, as células de centrífuga, sobrenadante aspirado e as células ressuspender em meio pré-aquecido fresco por 30 min. Lave as células em PBS e depois usar para experimentos de rolamento (ver Figura 1B imagem representativa de CFSE corados células HL-60 em P-revestido de superfície para sel). </li></ol></li></ol><p class = &quot;jove_content&quot;&gt; Nota: coloração CFSE é opcional, e é apresentado aqui para demonstrar o fenômeno de rolamento no canal microfluídicos. Análise dos parâmetros de laminação apresentados neste manuscrito foi realizada em células não coradas usando imagens de campo claro padrão. <ol start="2"><li> Células endoteliais microvasculares Pulmonares (LMVECs) <ol><li> O revestimento 100 milímetros pratos de Petri com solução de gelatina a 0,1% (v / v em PBS) e incuba-se a 37 ° C durante pelo menos 30 min. </li><li> LMVECs cultura em placas de Petri de 100 milímetros revestido com gelatina em meio completo de crescimento endotelial (endotelial basal médio 2 (EBM-2)), suplementado com um kit de suplemento de crescimento específico, ver REAGENTES). Mudança de mídia a cada dois dias e as células sub-cultura ao atingir 80-90% de confluência. </li><li> Para a sub-cultura, lavar as células com PBS e, em seguida, separar as células com 4 mL de 1x tripsina-EDTA durante 3 min a 37 ° C e neutraliza em um volume igual de completos EBM-2 media. Transferência da suspensão de células para um tubo de 15 ml e centrifuge (400 xg, 5 min). Após a centrifugação, ressuspender o sedimento em 1 ml de meio completo endoteliais e contagem de células com um hemocitómetro. Não excesso de passagem das células, como isso afeta sua morfologia e função de usar apenas as células com menos de 7 passagem para todos os experimentos. </li></ol></li></ol><ol start="3"><li> Ovário de hamster chinês-P-selectina (CHO-P) Células <ol><li> Células CHO-P, que são células CHO transfectadas estavelmente para expressar humano P-sel, foram fornecidos por colaboradores (Beth Israel Deaconess Medical Center, Harvard Medical School) 11,12. </li><li> Células Cultura CHO-P no T175 cm 2 frascos de 25 ml de F-12 mídia. </li><li> Para passaging, lavar as células com 10 ml de PBS durante 4-5 segundos e depois trypsinize em 10 ml de 1x tripsina-EDTA durante 3 min a 37 ° C, seguido de neutralização em meios completos. </li><li> Centrifugar a suspensão de células (400 x g, 5 min), aspirar o sobrenadante cuidadosamente, ressuspender o sedimento de células em 1 ml de meio cheios e contagem das células comum hemocitômetro. </li></ol></li></ol> 2. Operação do Sistema Integrado Microfluidic Multi-bem placa <ol><li> Certifique-se que todo o equipamento está conectado corretamente e ligar os diferentes módulos: computador, controlador, microscópio invertido, e câmera CCD. </li><li> Abra o software de imagem, certifique-se o módulo de placa multi-bem eo módulo de imagem são devidamente apresentados na tela. </li><li> Conecte os tubos para a armadilha de vapor (ligado ao controlador) e também conectá-los para a interface de pressão. </li><li> Colocar a placa de multi-poços na placa de aquecimento / adaptador. Adicionar reagentes aos poços (a seguir descritos) e interface de prender em cima da placa. Coloque a placa para a imagem latente no palco automatizado. </li><li> A interface anexa à parte superior da placa e se aplica uma pressão pneumática a partir do controlador para o topo dos poços, a condução do fluido através dos canais de microfluidos para o caudal definido, facilmente controlados utilizando a placa de multi-poçostela do módulo no modo Manual. </li><li> Reagentes do canal de fluxo através de uma área de observação, localizado entre os poços. Dimensões do canal microfluídicos são 350 m de largura x 70 mM de altura. O comprimento do canal linear é de 1 mm e na parte inferior dos canais compreende uma lamela de vidro de 180 um, o que é compatível com o campo claro, de fase, de fluorescência e microscopia confocal. </li><li> Adquirir vídeos usando uma câmera CCD (aquisição de fluxo, 11 frames / seg) e analisar através de software compatível. </li></ol> 3. Revestimento de Canais Microfluidic com um substrato proteico ou uma monocamada de células <ol><li> Revestimento de canais microfluídicos com fibronectina ou P-/E-selectin <ol><li> Preparar 1 mL de 20 ug / ml de solução de fibronectina em PBS. Alterar o volume com base no número de canais a serem revestidas (usar 25-50 ul de fibronectina por canal). </li><li> Adicionar 25-50 mL de solução de fibronectina para cada entrada bem. Aplicar a força de cisalhamento de 2 dyn / cm 2durante 5 minutos para perfundir o canal. Por favor, note que a gota de líquido que aparece na saída do bem. Incubar durante 30-45 minutos à TA </li><li> Aspirar a solução de poços (não aspirar diretamente do círculo do meio que alimenta o canal) 1,13. Adicionar 200-500 ul de PBS a uma tomada bem e lavar com PBS canal por aplicação de fluxo de cisalhamento de 2 dines / cm 2 durante 5 min. O canal é agora devidamente revestidos com fibronectina e pronto para ser utilizado. </li><li> Para revestir com P-ou E-sel, preparar uma solução a 5 mg / ml de proteína recombinante humana desejada em PBS, e os canais de revestimento tal como descrito acima, com 1 h de incubação a 37 ° C para permitir que o revestimento de superfície. </li></ol></li><li> Criação de CHO-P ou LMVEC monocamada dentro do canal microfluídicos <ol><li> Suavemente trypsinize células de placas de cultura durante 3 min, têmpera usando um volume de 2 vezes de meios completos e centrifugação (5 min a 400 x g). Suspenda as células com 10 ml de meio completo e centrifugar (5 min a 400 xg) again. </li><li> Contar as células para determinar a concentração de células na suspensão. Para assegurar a formação de uma monocamada confluente LMVEC no interior do canal, trazer a concentração de células de 15-20 milhões de células / ml. Para confluente CHO-P monocamada de células, usar 50-60000000 células / ml. Número inicial de células utilizado para determinar a experiência em conformidade - Use 25-50 mL de suspensão de células para cada canal. </li><li> Adicionar 25-50 mL de suspensão de células na concentração apropriada para a entrada do poço. Coloque a placa no palco microscópio e introduzir células no canal (2 dyn / cm 2) até que as células são observadas na tela preenchendo os canais inteiros, e depois parar o fluxo. </li><li> Preencha tanto saída e de entrada com 200 mL de tanto LMVEC cheio ou meio CHO. Deixe as células assentar e aderir durante 3 horas na incubadora (37 ° C, 5% CO 2). </li><li> Após a incubação de 3 horas, lave o canal com total media (2 dyn / cm 2, 10-15 min) para remover desapegadocélulas. As células devem agora aparecer completamente confluentes eo canal já está pronto para uso. Dependendo da densidade de sementeira inicial de células, 2-3 hr adicional de tempo de estabilização pode ser necessário para assegurar a cobertura completa da superfície com as células. </li></ol></li></ol> 4. LMVEC Activation Pro-inflamatório e anticorpo bloqueador de P-/E-selectin <ol><li> Prepara-se uma solução de TNF-α (10 ng / ml) em meio basal LMVEC. </li><li> Para induzir a activação inflamatória do LMVEC nos canais, adicionar 100 ul da solução de TNF-α para a entrada e assim introduzir a solução para dentro do canal por aplicação de fluxo de cisalhamento de 2 dines / cm 2 durante 5 min. Para canais de controlo (células endoteliais não activado), adicionar 100 ul de LMVEC meio basal para a entrada e assim introduzir no interior do canal (2 dines / cm 2 a 5 min). Canal está pronto para um ensaio de rolamento. </li><li> Para bloquear P-sel e E-sel em LMVECs e células CHO-P, introduzir neutralizar P-sel (clone AK4, 5 mg / ml em bameios de sal) ou E-sel (P2H3 clone, 5 ug / ml em meio basal) anticorpos para o canal e incubar durante 1 hora a 37 ° C. Em seguida, lava-canais com meio basal (2 dines / cm 2 a 5 min). Canais estão prontos para um ensaio de rolamento. </li></ol> 5. HL-60 Rolando Ensaio sobre Substrato / Telemóveis Revestido-monocamada canais microfluídicos <ol><li> Examinar cuidadosamente os canais ao microscópio para confirmar que os canais são adequadamente revestidas (no caso do revestimento com células, uma monocamada confluente de células plenamente devem ser observados). </li><li> Para preparar a HL-60 de suspensão de células para as experiências de rolamento, centrífuga HL-60 de suspensão de células (5 min a 400 xg) e lavar uma vez com meio basal. Contar as células e ressuspender em IMDM (meios basais, contendo Ca 2 + e Mg 2 +) para criar uma suspensão de células HL-60 com 5 milhões de células / ml. Use 25-50 ul de suspensão celular para cada canal para realizar o ensaio de rolamento. </li><li> Adicionar 25-50 mL da suspe celularnsion para a saída bem, colocar a placa dentro placa titular o controlo de temperatura (37 ° C) e no palco microscópio. Em seguida, introduzir células no canal através da aplicação de força de cisalhamento de 2 dyn / cm 2 (células devem ser observados dentro de 10-15 seg fluindo da tomada de entrada). </li><li> Para examinar a resposta de rolamento como uma função da tensão de cisalhamento, para reduzir o cisalhamento 0,25 dines / cm 2 e 20-30 adquirem vídeos sec (utilizando a função &quot;aquisição de fluxo&quot;) em cada corte desejado (cisalhamento aumentam gradualmente a partir de 0,25 até 5 dines / cm 2. Também é possível a utilização de tesouras mais elevados). </li><li> Adquirir vídeos usando uma câmera CCD (aquisição de fluxo, 11 frames / seg) e analisar os caminhos de rolamento e rolamento velocidades através de um software compatível. </li></ol> 6. Citometria de fluxo para detectar a expressão de moléculas de superfície <ol><li> Após tripsinização, preparar uma suspensão de células (com 1-2 x 10 5 células / amostra) de tipo de célula desejada (HL-60, CHO-P ou LMVECs) em PBS (- / -), suplementado com 2% de FBS. Lave as células duas vezes e trazer o volume da amostra de 50 ul (utilizando o mesmo tampão). </li><li> Incubar cada amostra com o fluoróforo conjugado desejado Ab (ver tabela em anexo para obter informações detalhadas) a 4 ° C por 20 min (cubra com papel alumínio). </li><li> Lave as células duas vezes (mesmo tampão) e trazer o volume final da suspensão de células coradas com 200 ul. Analisar amostras usando um citómetro de fluxo para detectar a expressão de moléculas de superfície. </li></ol>

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A model for an antibody-based microfluidic screening system.</a> Microelectronic Eng. 98, 668-671 (2012).</li><li>Perozziello, G., 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+devices+modulate+tumor+cell+line+susceptibility+to+NK+cell+recognition.">Microfluidic devices modulate tumor cell line susceptibility to NK cell recognition.</a> Small. 8, 2886-2894 (2012).</li><li>Perozziello, G., 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+biofunctionalisation+protocols+to+form+multivalent+interactions+for+cell+rolling+and+phenotype+modification+investigations.">Microfluidic biofunctionalisation protocols to form multivalent interactions for cell rolling and phenotype modification investigations.</a> Electrophoresis. , (2013).</li><li>Simone, G., 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+facile+in+situ+microfluidic+method+for+creating+multivalent+surfaces:+toward+functional+glycomics.">A facile in situ microfluidic method for creating multivalent surfaces: toward functional glycomics.</a> Lab Chip. 12, 1500-1507 (2012).</li><li>Sarkar, 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=Engineered+cell+homing.">Engineered cell homing.</a> Blood. 118, e184-e191 (2011).</li><li>Cheng, Z., 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=Targeted+Migration+of+Mesenchymal+Stem+Cells+Modified+With+CXCR4+Gene+to+Infarcted+Myocardium+Improves+Cardiac+Performance.">Targeted Migration of Mesenchymal Stem Cells Modified With CXCR4 Gene to Infarcted Myocardium Improves Cardiac Performance.</a> Mol. Ther. 16, 571-579 (2008).</li><li>Enoki, 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=Enhanced+mesenchymal+cell+engraftment+by+IGF-1+improves+left+ventricular+function+in+rats+undergoing+myocardial+infarction.">Enhanced mesenchymal cell engraftment by IGF-1 improves left ventricular function in rats undergoing myocardial infarction.</a> Int. J. Cardiol. 138, 9-18 (2010).</li></ol>

Os autores declaram não haver conflito de interesses.

Um dos grandes desafios na tradução bem sucedida de terapia baseada em células exógena é a incapacidade de produzir com eficiência células para locais de lesão e inflamação com alta eficiência de enxerto 3. Laminagem celular representa um passo crítico no processo de homing célula, facilitando a desaceleração de células nas paredes de vasos sanguíneos, o que levou à sua adesão firme e transmigração através do endotélio para os tecidos 5. Para uma melhor compreensão do proces...

Um dos principais desafios na tradução clínica de sucesso da terapia baseada em células é a entrega ineficiente ou direcionamento de células sistemicamente infundidos aos locais desejados 3,4. Consequentemente, há uma busca constante de abordagens para melhorar homing celular e, especificamente, celular rolando, como uma estratégia para melhorar a terapia celular. Celular rolando sobre os vasos sanguíneos é um passo fundamental na cascata homing célula, classicamente definidos para leucócitos que são recrutados para sítios da doença 5. Este passo é governado por interacções específicas entre selectinas endoteliais, isto é, P-e E-selectina seus ligantes de contador na superfície de leucócitos 5,6 (P-e E-sel), e. Para uma melhor compreensão e melhoria da eficiência de homing celular e, especificamente, a etapa de rolamento, são de grande importância na busca de novas plataformas para melhorar a terapia baseada em células. Até agora isso tem sido conseguido através de câmaras de fluxo de placas paralelas (PPFCs), compreendendo dois plataforma planaes com uma junta entre eles, com uma porta de entrada e saída localizada sobre a placa superior, através da qual uma suspensão de células é perfundido através da utilização de uma bomba de seringa, 7,8, 9. A superfície da placa de fundo pode ser revestida com uma monocamada de células / substratos relevantes e a interacção entre as células e a superfície perfundidos sob fluxo de cisalhamento é então explorado 7. No entanto, PPFC é uma baixa, reagente-consuming, e método bastante tedioso, com formação de bolhas, vazamento e refluxo mal controlada apresentando grandes inconvenientes. Uma técnica alternativa para o PPFC tradicional é um sistema de microfluídica placa multi-bem, o que permite maior desempenho de transferência de ensaios celulares (até 10 vezes maior do que PPFCs) sob, o fluxo de cisalhamento controlado por computador precisa, com baixo consumo de reagente 1,10. Experiências de rolamento celular são realizados no interior dos canais de microfluidos, que pode ser revestido com as monocamadas de células ou substratos modificados e usi fotografadang de um microscópio, com propriedades de rolamento facilmente analisados ​​utilizando um software adequado. Neste estudo, demonstramos a capacidade deste sistema de microfluídica placa multi-bem, estudando as propriedades de rolamento de leucemia promielocítica humana (HL-60) células em diferentes superfícies. HL-60 de rolamento em substratos como a P-e E-sel, bem como sobre monocamadas de células que expressam diferentes receptores de rolamento, foi analisado. Além disso, o anticorpo (Ab) bloqueio foi usado para demonstrar o envolvimento directo de selectinas específicas na mediação do movimento de rolamento de células HL-60 sobre essas superfícies. Experimentos rolamento foram realizados com maior produção, sob fluxo de cisalhamento estável, com o mínimo consumo de reagente / celular, permitindo a análise eficiente dos parâmetros-chave do rolamento tais como a velocidade de rolamento, o número de células de rolamento, e rolando propriedades do caminho.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cells</td>
		</tr>
		<tr>
			<td>Human Lung Microvascular Endothelial Cells</td>
			<td>Lonza</td>
			<td>CC-2527</td>
			<td></td>
		</tr>
		<tr>
			<td>P-selectin-expressing Chinese Hamster Ovary Cells (CHO-P)</td>
			<td>Kind gift by Dr. Barbara Furie11,12</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HL-60 Cells</td>
			<td>ATCC</td>
			<td>CCL-240</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cell Culture Reagents</td>
		</tr>
		<tr>
			<td>Endothelial Basal Medium</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr>
			<td>EBM-2 Media</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Basal Medium Supplements</td>
			<td>Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr>
			<td>EGM-2 MV SingleQuots</td>
			<td>Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM - Iscove&#39;s Modified Dulbecco&#39;s Medium 1x</td>
			<td>Gibco</td>
			<td>12440</td>
			<td></td>
		</tr>
		<tr>
			<td>F-12 (1x) Nutrient Mixture (Ham)</td>
			<td>Gibco</td>
			<td>11765-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin (P/S)</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (L/G) 200 mM</td>
			<td>Gibco</td>
			<td>25030</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>Sa550</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>BD Falcon</td>
			<td>BD-353003</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Cell Culture Dish, Tissue-Culture Treated Polystyrene</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes (15 ml polypropylene conical tubes)</td>
			<td>MedSupply Partners</td>
			<td>TC1500</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 Flasks</td>
			<td>BD Falcon</td>
			<td>353136</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin Solution (2%)</td>
			<td>Sigma</td>
			<td>G1393</td>
			<td></td>
		</tr>
		<tr>
			<td>dPBS (without calcium chloride and magnesium chloride)</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA Solution (10x)</td>
			<td>Sigma</td>
			<td>T4174</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Antibodies</td>
		</tr>
		<tr>
			<td>Anti-hE-Selectin/CD62E</td>
			<td>R&#38;D Systems</td>
			<td>BBA21</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Conjugated Mouse IgG1</td>
			<td>R&#38;D Systems</td>
			<td>BBA21</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-hP-Selectin</td>
			<td>R&#38;D Systems</td>
			<td>BBA34</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Conjugated Mouse IgG1</td>
			<td>R&#38;D Systems</td>
			<td>BBA34</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Mouse IgG&#173;1 &#954; Isotype Control</td>
			<td>BD Bioscience</td>
			<td>555748</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-SLeX /CD15s Ab, Clone: 5F18</td>
			<td>Santa Cruz</td>
			<td>SC70545</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Conjugated</td>
			<td>Santa Cruz</td>
			<td>SC70545</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Mouse IgM-FITC Isotype Control</td>
			<td>Santa Cruz</td>
			<td>SC2859</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse Anti-Human CD162, Clone: KPL-1</td>
			<td>BD Pharmingen</td>
			<td>556055</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse IgG1 k Isotype Control</td>
			<td>BD Pharmingen</td>
			<td>550617</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-P-Selectin Ab (AK4)</td>
			<td>Santa Cruz</td>
			<td>SC19996</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-E-Selectin Ab, Clone P2H3</td>
			<td>Millipore</td>
			<td>MAB2150</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG1 Isotype Control</td>
			<td>Santa Cruz</td>
			<td>SC3877</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Other Reagents</td>
		</tr>
		<tr>
			<td>Recombinant Human TNF-alpha</td>
			<td>PeproTech</td>
			<td>300-01A</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Trace CFSE Cell Proliferation Kit - For Flow Cytometry</td>
			<td>Invitrogen</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr>
			<td>Human P-selectin-FC recombinant protein</td>
			<td>R&#38;D Systems</td>
			<td>137-PS-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Human E-selectin-FC recombinant protein</td>
			<td>R&#38;D Systems</td>
			<td>724-ES-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin Human, Plasma</td>
			<td>Invitrogen</td>
			<td>33016-015</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Bioflux 1000</td>
			<td>Fluxion Biosciences</td>
			<td>Bioflux Montage was the software used to run the experiments and analyze the data</td>
			<td></td>
		</tr>
		<tr>
			<td>BioFlux 48-well plates</td>
			<td>Fluxion Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Accuri C6 Flow Cytometer</td>
			<td>BD Bioscience</td>
			<td>CFlow Plus was the software used to run the experiments and analyze the data</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti-S</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CoolSnap HQ2 CCD camera</td>
			<td>Photometrics</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

systematic analysis of in vitro cell rolling using a multi-well plate microfluidic system

Um grande desafio para a terapia de base celular é a incapacidade para atingir sistemicamente uma grande quantidade de células viáveis ​​com eficiência elevada para os tecidos de interesse após a infusão intravenosa ou intra-arterial. Consequentemente, o aumento homing celular é atualmente estudada como uma estratégia para melhorar a terapia celular. Célula de rolamento sobre o endotélio vascular é um passo importante no processo de homing célula e podem ser sondados in vitro utilizando uma câmara de fluxo de placas paralelas (PPFC). No entanto, este é um ensaio de transferência extremamente fastidioso, de baixo, com as condições de fluxo mal controlada. Em vez disso, foi utilizado um sistema de microfluídica multi-bem placa que permite estudo das propriedades de rolamento celular em um maior rendimento sob precisamente controlado, fluxo de cisalhamento fisiologicamente relevante 1,2. Neste artigo, vamos mostrar como as propriedades de rolamento do HL-60 (leucemia promielocítica humana) células em superfícies E-revestido-selectina P-e, assim como em superfícies revestidas de monocamada de células pode ser readily examinada. Para melhor simular as condições inflamatórias, a superfície do canal microfluídico foi revestida com células endoteliais (ECs), que foram, em seguida, activados com fator de necrose tumoral-α (TNF-α), aumentando significativamente as interacções com células HL-60 em condições dinâmicas. A taxa de transferência melhorada e multi-parâmetro plataforma de análise de software integrado, que permite a rápida análise de parâmetros como rolando velocidades e caminho de rolamento, são vantagens importantes para avaliar as propriedades da célula rolando in-vitro. Permitindo a análise rápida e precisa de abordagens de engenharia destinados a impactar rolamento celular e homing, esta plataforma pode ajudar a terapia baseada em células exógena antecedência.

Células HL-60 rolam em superfícies P-e E-selectin, mas não sobre f ibronectina Células HL-60 são considerados &quot;rolos&quot; padrão de ouro em que expressam uma variedade de ligandos homing, incluindo os ligantes de rolamento P-glicoproteína ligando sel-1 (PSGL-1) e sialil-Lewis X (sLex) 5,14 (Figura 1A ). A proteína de superfície PSGL-1 age como um andaime para o tetra-sacarídeos Slex, mediando a interação específica com P-e E-sel, que são sobre-reg...

Watch this Scientific Journal Video about Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System at JoVE.com

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

Este estudo utilizou um sistema de microfluídica multi-bem placa, aumentando significativamente o rendimento dos estudos rolantes célula sob fluxo de cisalhamento fisiologicamente relevante. Dada a importância da laminagem celular na célula de multi-passo homing cascata e a importância de homing celular após entrega sistémica de populações exógenas de células em pacientes, este sistema oferece a possibilidade de uma plataforma de pesquisa para melhorar a terapia de base celular.

Análise sistemática de<em&gt; In Vitro</em&gt; Célula de rolamento usando um sistema microfluídicos Multi-bem placa

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

systematic-analysis-vitro-cell-rolling-using-multi-well-plate

Research

JoVE Journal

Bioengineering

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

11.9K visualizações.  Brigham and Women's Hospital.  Este estudo utilizou um sistema de microfluídica multi-bem placa, aumentando significativamente o rendimento dos estudos rolantes célula sob fluxo de cisalhamento fisiologicamente relevante. Dada a importância da laminagem celular na célula de multi-passo homing cascata e a importância de homing celular após entrega sistémica de populações exógenas de células em pacientes, este sistema oferece a possibilidade de uma plataforma de pesquisa para melhorar a terap...

Vídeo: Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

Dispositivo microfluídicos para Recriando um microambiente do tumor<em&gt; In Vitro</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Bioengenharia

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

Estudando Trajetórias celular Rolamento no Patterns Receptor Asymmetric

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

A utilização de uma alta taxa de transferência<em&gt; In Vitro</em&gt; Microfluidic Sistema de Desenvolvimento Oral Biofilms Multi-espécies

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Irritação dos olhos Test (EIT) para identificação de perigos de olho irritantes químicos usando reconstruída Humano Córnea-like epitelial (RHCE) Modelo Tissue

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

A Method for Determination and Simulation of Permeability and Diffusion in a 3D Tissue Model in a Membrane Insert System for Multi-well Plates

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as well as substance testing. These models are mostly cultivated in membrane-insert systems, their permeability toward different substances being an essential factor. Typically, applied methods for determination of these parameters usually require large sample sizes (e.g., Franz diffusion cell) or laborious equipment (e.g., fluorescence recovery after photobleaching (FRAP)). This study presents a method for determining permeability coefficients directly in membrane-insert systems with diameter sizes of 4.26 mm and 12.2 mm (cultivation area). The method was validated with agarose and collagen gels as well as a collagen cell model representing skin models. The permeation processes of substances with different molecular sizes and permeation through different cell models (consisting of collagen gel, fibroblast, and HaCaT) were accurately described.
Moreover, to support the above experimental method, a simulation was established. The simulation fits the experimental data well for substances with small molecular size, up to 14 x 10-10 m Stokes radius (4,000 MW), and is therefore a promising tool to describe the system. Furthermore, the simulation can considerably reduce experimental efforts and is robust enough to be extended or adapted to more complex setups.

A method for determination of permeability in a membrane insert system for multi-well plates and in silico parameter optimization for the calculation of diffusion coefficients using simulation are presented.

Um método para a determinação e simulação da permeabilidade e difusão em um modelo 3D do tecido em uma membrana inserir sistema para placas multi bem

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as ...

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. Unfortunately, many researchers spend large amounts of time and resources developing and optimizing workflows that are only relevant to their own experiments. In this article, we describe a protocol that can be used by those with access to a widefield high-content analysis system (WHCAS) to image any slide-mounted tissue, with options for customization within pre-built modules found in the associated software. Not originally intended for slide scanning, the steps detailed in this article make it possible to acquire slide scanning images in the WHCAS which can be imported into the associated software. In this example, the automated segmentation of brain tumor slides is demonstrated, but the automated segmentation of any fluorescently-labeled nuclear or cytoplasmic marker is possible. Furthermore, there are a variety of other quantitative software modules including assays for protein localization/translocation, cellular proliferation/viability/apoptosis, and angiogenesis that can be run. This technique will save researchers time and effort and create an automated protocol for slide analysis.

Here we describe a protocol for the automated segmentation of fluorescently labeled tissues on slides using a widefield high-content analysis system (WHCAS). This protocol has wide-ranging applications in any field which involves the quantitation of fluorescent markers in biological tissues including the biological sciences, medical engineering, and health sciences.

Digitalização de slides automatizado e segmentação em tecidos fluorescente-rotulado, usando um sistema de análise de Widefield alto teor

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

Análise de expressão genética das células endoteliais expostos para Shear Stress usando múltiplas câmaras de fluxo paralelo-placa

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, including Pseudomonas aeruginosa (P. aeruginosa). Here, we will describe how the system can be used to study the growth kinetics and the morphological properties such as the surface roughness and textural entropy of P. aeruginosa strain PA01 that expresses an enhanced green fluorescent protein (PA01-EGFP). A detailed protocol will describe how to grow and seed PA01-EGFP cultures, how to set up the microscope and autorun, and conduct the image analysis to determine growth rate and morphological properties using a variety of shear forces that are controlled by the microfluidic device. This article will provide a detailed description of a technique to improve the study of PA01-EGFP biofilms which eventually can be applied towards other strains of bacteria, fungi, or algae biofilms using the microfluidic platform.

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

Here, we describe the use of a higher-throughput microfluidic bioreactor coupled with a fluorescent microscope for the analysis of shear stress effects on Pseudomonas aeruginosa biofilms expressing green fluorescent proteins, including instrument set up, the determination of biofilm coverage, growth rate, and morphological properties.

Ao vivo celular análise de tensão de cisalhamento na Pseudomonas aeruginosa , usando um sistema automatizado de Microfluidic superior-taxa de transferência

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

Estabelecendo co-culturas baseadas em uma única célula de forma determinística com um chip Microfluídico

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...