O apoio dos Institutos Nacionais de Saúde (concede GM092804, DE019938) e Shriners Hospital Queimaduras.

1. Fabricação de dispositivos microfluídicos <ol><li> Usando técnicas de fotolitografia padrão, fabricar o wafer molde mestre em uma classe de 1000 sala limpa. Padrão da primeira camada de material fotosensitivo 3 mícrons-fina à base de epóxi negativo para definir os canais de migração de acordo com as instruções do fabricante. Padrão a segunda camada de 50 mícrons de espessura para definir as câmaras de célula de carga e quimiocinas. </li><li> Use o wafer padronizada para lançar dispositivos polidimetilsiloxano (PDMS). Misture vigorosamente PDMS (20 g) com o iniciador (2 g) durante 5 min usando garfo de plástico, em grande bandeja de pesagem de plástico. </li><li> Com cuidado, despeje PDMS para molde. </li><li> Desgasificar o PDMS pela colocação do molde com PDMS vertida num exsicador de vácuo durante 1 hora. </li><li> Asse e curar PDMS dispositivos de microfluidos para, pelo menos, 3 horas num forno regulado para 65 ° C. </li><li> Perfurar os WBLCs centrais usando um perfurador com um diâmetro da ponta de 1,5 mm. </li><li> Retire o dispositivos em forma de anel inteiras usando uma punçãoela com um diâmetro da ponta de 5,0 mm. </li><li> Remova as partículas a partir de dispositivos de rosca com fita adesiva. </li><li> Lavar a 12 poços placa com água deionizada e, em seguida, seco com nitrogênio. Colocar em placa a 60 ° C forno durante 5 min. </li><li> Plasma de oxigénio tratar a placa de 12 poços por duas vezes; uma vez sozinho por 35 segundos e, em seguida, novamente com os dispositivos de rosca para outros 35 seg. </li><li> Com cuidado, coloque os dispositivos nos poços da placa, usando uma pinça. </li><li> Asse placa com dispositivos ligados em uma placa quente definida como 80 ° C durante 10 min. </li></ol> 2. Microfluidic Ensaio Preparação <ol><li> Dispositivos de microfluidos Prime imediatamente após o tratamento por plasma de oxigénio, quando o dispositivo é hidrófilo e os efeitos capilares pode promover a iniciação dos pequenos canais no dispositivo. </li><li> Criar solução quimioatractiva pela mistura de 5 mL de fMLP [solução stock 10 uM] com 5 mL de fibronectina [solução de estoque de 1 mg / mL] e 490 uL de HBSS. </li><li> Lentamente pipeta &amp; #160; solução quimioatractiva em WBLC, utilizando uma ponta de carga do gel (Figura 1A). Pipeta um adicional de 20 ul de quimioatractor em torno do exterior do dispositivo. </li><li> Coloque a placa num secador durante 15 minutos. Através da aplicação de um vácuo para o dispositivo, a solução é instilada nos canais laterais do dispositivo e o ar deslocado difundido para fora através das PDMS. </li><li> Retire a placa de exsicador e confirmar umedecimento do canal dispositivo sob microscópio. Veja como bolha torna-se menor, como solução chemoattractant entra na câmara. Nenhuma bolha deve estar presente no interior do dispositivo após o tratamento a vácuo. </li><li> Lava-se a WBLC e fora do dispositivo completamente para remover o excesso de solução quimioatractor. Este passo produz um gradiente de quimioatractor de cada uma das câmaras quimiotáticos focais (FCC) para o centro do dispositivo. <ol><li> Encha uma seringa de 1 ml com PBS, adicionar uma 30 G contundente ponta da agulha de seringa. Gentilmente, insira a ponta da agulha da seringa nopara o centro do orifício de rosca. Suavemente empurrar 100 ul de PBS no orifício de modo a que uma gotícula de formas de PBS na parte superior do dispositivo. </li><li> Placa de inclinação e de pipeta de 1 ml de PBS em torno do dispositivo de modo a que o líquido se acumula na parte inferior do poço. Aspirar o processo líquido e repita para o total de 3x usando solução tampão fresco (media de uso em vez de buffer se os neutrófilos separados serão carregados em media) 9. </li></ol></li><li> Encher cada poço com a mídia até que os topos dos dispositivos estão submersos sob líquido. Deixe os dispositivos descansar por 15 min para permitir inclinação a se estabilizar. Os resultados experimentais e os dados teóricos obtidos em simulações de elementos finitos mostram que os gradientes em estes dispositivos são estáveis ​​até cerca de 24 horas, para as moléculas pequenas (por exemplo, fMLP) e até um de vários dias para o maior peso molecular (por exemplo, IL8). </li><li> Usando Dicas para colocar o gel, lentamente pipeta 2 mL de sangue (neutrófilos ou isoladas) em cada loa sangue totalding câmara (WBLC) (Figura 1A). </li></ol> 3. Preparação de Amostras Os neutrófilos humanas A partir de sangue capilar <ol><li> Coletar sangue capilar de punção digital de voluntários saudáveis, que está em nenhum imunossupressores. Todas as amostras dos pacientes foram obtidas com consentimento informado por escrito, e através de procedimentos aprovados pelo MGH e Shriners Conselhos de Revisão Institucional. <ol><li> Para a coleta de sangue de punção digital, lavar as mãos com água e sabão e seque a pele. Preparar a solução estoque anti-coagulant/stain adicionando 1 ml HBSS + 0,2% de HSA e 10 mL Hoechst mancha (32,4 mM) à heparina tubo de coleta de sangue (1,65 USP/50 mL de sangue). Picar o dedo usando uma lanceta segurança SurgiLance (profundidade de 2,2 mm, 22 G), limpe primeira gota de sangue e recolher 50 mL de sangue em tubo Eppendorf contendo o estoque de anti-coagulante e solução fluorescente mancha Hoechst (10 mL) e gentilmente misturar. </li></ol></li&gt; <li> Incubar o sangue e Hoechst mancha durante 10 min para permitir a coloração fluorescente núcleo. Execute amostra dentro de 1 hora da coleta de sangue. </li></ol> Neutrófilos humanos do sangue venoso <ol start="3"><li> Desenhar 10 ml de sangue periférico, venoso em tubos contendo 33 USP de heparina. </li><li> Adicionar 50 mL de sangue venoso para a mídia e Hoechst mancha como descrito anteriormente. Incubar o sangue e Hoechst mancha durante 10 min para permitir a coloração fluorescente núcleo. Execute amostra dentro de 1 hora da coleta de sangue. </li></ol> Neutrófilos Separação De Sangue - Controle Positivo <ol start="5"><li> Para separar os neutrófilos, utilizar técnicas estéreis para isolar os neutrófilos a partir de 10 ml da amostra de sangue total venoso usando Hetastarch (6% w / v) de gradiente de densidade, seguido por uma selecção kit de isolamento de esfera magnética negativa seguindo o protocolo do fabricante. </li><li> Ressuspensas a alíquota final, de neutrófilos a uma concentrção de 4.000 células / ul em 1X HBSS + 0,2% de albumina do soro humano. Manter as células a 37 ° C até que esteja pronto para executar o experimento. </li></ol> 4. Microscopia e Análise de Imagem para medições de quimiotaxia de neutrófilos <ol><li> Defina-se a temperatura biochamber a 37 ° C, humidade entre 80% e CO 2 a 5%. </li><li> Comece imaginando imediatamente o uso de imagens de lapso de tempo em um microscópio (10X ou superior ampliação). </li></ol> 5. Statistical Analysis <ol><li> Rastrear manualmente, pelo menos, 50 neutrófilos em cada amostra. </li><li> Contar as células que entram na FCC ao longo do tempo (Figura 2). </li><li> Calcular as velocidades de neutrófilos no canal entre WBLC e CCI, utilizando o ImageJ (NIH) (Figura 2). </li><li> Quantificar direccionalidade de neutrófilos através da contagem do número de células que passam a bifurcação e calcular a razão entre o número de células que voltam-se para o FCC e que as células de saída the dispositivo. As células que não são direcionais não seguem o gradiente quimiotático e, portanto, migrar em números iguais em direção à FCC e do canal de saída (Figura 2). </li></ol>

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Neste trabalho, foi desenvolvida uma plataforma microfluídica para medir quimiotaxia de neutrófilos a partir de uma gota de sangue (2 mL). A filtragem mecânica on-chip de hemácias de migração de neutrófilos ativamente contorna a necessidade de métodos complicados de separação de células, como a densidade gradientes 10, a seleção positiva 11, ou seleção negativa de 12, que são propensas a introduzir artefatos ativando neutrófilos. A filtragem mecânica de hemácias distin...

<html> Tráfico de neutrófilos desempenha um papel fundamental na determinação do progresso e resolução de muitas doenças inflamatórias, incluindo aterosclerose 1, infecção bacteriana ou sepsia 2, lesão por queimadura e 3. Por sua grande contribuição para as condições de saúde e doença, contagem de neutrófilos é parte da análise de sangue padrão muitas vezes considerado em laboratórios clínicos e de pesquisa. No entanto, apesar de ser um dos testes mais ubíquos, o valor de contagem de neutrófilos para o diagnóstico da infecção e sépsis tem sido frequentemente questionada 4. Por exemplo, um estudo de neutrófilos em pacientes com queimaduras revelou que a contagem de neutrófilos e função de migração de neutrófilos não se correlacionam; significando contar que neutrófilos por si só não é um indicador preciso do estado imunológico 3. Embora mais difícil de medir, competência funcional de neutrófilos tem sido proposta como mais valioso em uma ampla gama de condições. t &quot;&gt; É importante, muitos dos defeitos de neutrófilos são transientes e não são provocados por defeitos genéticos permanentes, uma distinção que tem sido largamente ignorada na clínica até recentemente. No contexto de queimaduras, a migração de neutrófilos pode ser monitorizada durante o curso de o tratamento de um paciente como um indicador do estado inflamatório ou infecção 3. ensaios de migração tradicionais actualmente utilizados no laboratório (câmara de Boyden, Dunn câmara, ensaio micropipeta) não pode ser traduzido em um ambiente clínico porque requerem grandes volumes de sangue e pesado demorado técnicas de isolamento de neutrófilos (Tabela 1). Estes ensaios também não pode ser usado para monitorar as mudanças transitórias na quimiotaxia de neutrófilos em animais de laboratório pequenos, tais como ratos, porque o volume de sangue necessário para o isolamento de neutrófilos permite apenas um exemplo e muitas vezes até mesmo exige conjugação de sangue de vários animais para um único ensaio. Por exemplo, um estudo envolvendo mcondições ultiple e tratamentos ao longo de vários pontos temporais poderiam exigir milhares de ratos usando ensaios de quimiotaxia atuais. Isso restringe a pesquisa biológica básica que pode ser feito para compreender a complexa dinâmica da função imune no contexto de lesão, infecção ou queimar freqüentemente estudados em modelos murinos 5. Para satisfazer a necessidade de um ensaio funcional de neutrófilos que é rápida e robusta, enquanto o volume de sangue que exige um mínimo, temos desenvolvido um dispositivo de microfluidos, que mede a quimiotaxia de neutrófilos directamente a partir de uma pequena gota de sangue completo. Sabe-se que muitos fatores em sangue total, incluindo soro 6 e 7 plaquetas, afetar a função dos neutrófilos. Por conseguinte, é benéfico que o doseamento de microfluidos sangue total minimiza o processamento da amostra para manter o microambiente in vivo dos neutrófilos ao medir variações de quimiotaxia com um ensaio in vitro 8. Esta abordagemreduz o tempo de coleta de sangue para testes de migração de neutrófilos de horas usando técnicas tradicionais, a poucos minutos (Tabela 1). A plataforma de microfluidos sangue total produz um gradiente linear quimioatractiva estável durante a duração da experiência, não tem partes móveis, e não necessita de uma fonte de pressão exterior (isto é, uma bomba de seringa). A característica-chave no desenho de conjunto do dispositivo de microfluidos de sangue é a incorporação de uma célula de sangue vermelho (RBC) pente filtração que filtra mecanicamente GVs de entrar no canal de migração do dispositivo. As voltas à direita deste pente filtração evitar a necessidade de filtração exclusão de tamanho, o que provavelmente seriam obstruídos por hemácias e, portanto, bloquear o gradiente chemoattractant de atingir os neutrófilos migram ativamente na WB. A incorporação de todo o dispositivo de microfluidos de sangue em uma placa de 12 ou 24 poços facilita o rastreio de vários mediadores de si quimiotaxia de neutrófilos humana ou murinataneamente.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Name of Material/ Equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:179px;">Catalog Number</td>
			<td style="width:191px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Device Fabrication</td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">SU8&#160;</td>
			<td style="width:191px;">Microchem</td>
			<td style="width:179px;">Y131273</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Polydimethylsiloxane (PDMS)</td>
			<td style="width:191px;">Ellsworth Adhesives</td>
			<td style="width:179px;">Sylgard 184 1.1 lb. Kit</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Standard glass slides</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:179px;">125495</td>
			<td style="width:191px;">1 X 3 inches</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Glass-bottom plate</td>
			<td style="width:191px;">MatTek</td>
			<td style="width:179px;">P12G-1.5-14-F</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Harris Uni-Core, Tip Diameter 5.0mm</td>
			<td style="width:191px;">Ted Pella, Inc.</td>
			<td style="width:179px;">15081</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Harris Uni-Core, Tip Diameter 1.5 mm</td>
			<td style="width:191px;">Ted Pella, Inc.</td>
			<td style="width:179px;">15072</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Microfluidic Assay Preparation and Analysis</td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Gel-loading pipet tip</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:179px;">02-707-139</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Syringe</td>
			<td style="width:191px;">Fisher Scientfic</td>
			<td style="width:179px;">309602</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Blunt tip needle, 30g &#189; in.</td>
			<td style="width:191px;">Brico Medical Supply</td>
			<td style="width:179px;">BN3005</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Vacutainer, Heparin</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;"></td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">HBSS&#160;</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Human serum albumin</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:179px;">A5843-5G</td>
			<td style="width:191px;">0.2% final concentration in HBSS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Fibronectin</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:179px;">F0895-1MG</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">fMLP</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:179px;">F3506-10MG</td>
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		</tr>
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			<td height="42" style="height:42px;width:211px;">SurgiLance safety lancet, 2.2mm depth, 22 gauge</td>
			<td style="width:191px;"></td>
			<td style="width:179px;">SLN240</td>
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			<td height="21" style="height:21px;width:211px;">Hoescht stain</td>
			<td style="width:191px;">Life Technologies</td>
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			<td height="21" style="height:21px;width:211px;">Positive Control&#160;</td>
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			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
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			<td height="42" style="height:42px;width:211px;">HetaSep</td>
			<td style="width:191px;">STEMCELL Technologies Inc.</td>
			<td style="width:179px;">7906</td>
			<td style="width:191px;"></td>
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			<td height="42" style="height:42px;width:211px;">EasySep Human Neutrophil Enrichment Kit</td>
			<td style="width:191px;">STEMCELL Technologies Inc.</td>
			<td style="width:179px;">19257</td>
			<td></td>
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			<td height="21" style="height:21px;width:211px;"></td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Equipment</td>
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			<td></td>
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			<td height="21" style="height:21px;width:211px;">Plasma Asher</td>
			<td style="width:191px;">March Instruments</td>
			<td style="width:179px;">P-250</td>
			<td></td>
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			<td height="21" style="height:21px;width:211px;">Lindberg/Blue M Oven</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:179px;">13-258-30C</td>
			<td></td>
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			<td height="42" style="height:42px;width:211px;">Stainless Steel Precision Tweezers</td>
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			<td style="width:179px;">758TW458</td>
			<td></td>
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			<td height="42" style="height:42px;width:211px;">Bel-Art Scienceware Chemical-Resistant Vacuum Desiccator</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:179px;">08-594-15A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Dataplate Digital Hot Plate</td>
			<td style="width:191px;">Alpha Multiservices</td>
			<td style="width:179px;">PMC 720&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Nikon TiE inverted microscope</td>
			<td style="width:191px;">Nikon - Micro Video Instruments Inc.</td>
			<td style="width:179px;">MEA53100</td>
			<td></td>
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			<td height="42" style="height:42px;width:211px;">CFI Plan Fluor DL 10x na 15.2wd Objective</td>
			<td style="width:191px;">Nikon - Micro Video Instruments Inc.</td>
			<td style="width:179px;">MRH20101</td>
			<td></td>
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			<td height="42" style="height:42px;width:211px;">Lumen 200 with 2 Meter Light Guide for Nikon</td>
			<td style="width:191px;">Nikon - Micro Video Instruments Inc.</td>
			<td style="width:179px;">500-L200NI2</td>
			<td></td>
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			<td height="63" style="height:63px;width:211px;">DAPI/Hoechst/AMCA Narrow Band 32mm Exciters - 25mm Emitters</td>
			<td style="width:191px;">Chroma - Micro Video Instruments Inc.</td>
			<td style="width:179px;">31013v2</td>
			<td></td>
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			<td height="42" style="height:42px;width:211px;">Retiga R 2000 cooled CCD Camera 1600x1200 pixels</td>
			<td style="width:191px;">Qimaging - Micro Video Instruments Inc.</td>
			<td>RET-2000R-F-M-12-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidic platform for measuring neutrophil chemotaxis from unprocessed whole blood

Os neutrófilos desempenham um papel essencial na proteção contra infecções e seus números no sangue são freqüentemente medido na clínica. Maiores número de neutrófilos no sangue são geralmente um indicador de infecções em curso, enquanto a baixa contagem de neutrófilos são um sinal de alerta para os riscos mais elevados para infecções. Para realizar as suas funções, os neutrófilos, também tem que ser capaz de se mover de forma eficaz a partir do sangue, onde eles passam a maior parte de sua vida, para os tecidos, onde ocorrem infecções. Consequentemente, quaisquer defeitos na capacidade dos neutrófilos para migrar pode aumentar os riscos de infecções, mesmo quando os neutrófilos estão presentes em números adequados no sangue. No entanto, a medição de neutrófilos capacidade de migração na clínica é uma tarefa desafiadora, que é demorado, exige grande volume de sangue, e conhecimento especializado. Para lidar com essas limitações, nós projetamos um robusto ensaios microfluídicos para a migração de neutrófilos, o que requer uma única gota de sangue não transformados, circumvents a necessidade de separação dos neutrófilos, e é fácil de quantificar num microscópio simples. Neste ensaio, os neutrófilos migram directamente a partir da gota de sangue, através de pequenos canais, no sentido da fonte do quimioatractor. Para evitar que o fluxo granular de células vermelhas do sangue através dos mesmos canais, implementamos filtros mecânicos com ângulo direito voltas que bloquear seletivamente o avanço das células vermelhas do sangue. Nós validamos o ensaio comparando a migração de neutrófilos de gotas de sangue coletadas de punção digital e sangue venoso. Também comparamos todo estes sangue (BM) fontes com migração de neutrófilos a partir de amostras de neutrófilos purificados e encontrou velocidade consistente e direcionalidade entre as três fontes. Esta plataforma microfluídica permitirá o estudo da migração de neutrófilos humano na clínica e no ambiente de pesquisa para ajudar a avançar a nossa compreensão das funções de neutrófilos na saúde e na doença.

O sangue (BM) ensaio quimiotaxia de neutrófilos todo foi validada medindo o acúmulo de neutrófilos para um gradiente fMLP (S1 Filme). Os resultados confirmam que os glóbulos vermelhos estão presos pelo pente filtração enquanto neutrófilos (azuis) são capazes de migrar ativamente de todo o sangue (Figura 3A e S1 Filme). O gradiente estável linear chemoattractant (verde), formado por todo o dispositivo micro sangue foi confirmado usando dextrano marcadas com FIT...

Watch this Scientific Journal Video about Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood at JoVE.com

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Este protocolo detalha um ensaio concebido para medir a quimiotaxia de neutrófilos humanos a partir de uma gota de sangue total com reprodutibilidade robusta. Esta abordagem evita a necessidade de separação de neutrófilos e requer apenas alguns minutos de ensaio tempo de preparação. O chip de microfluídica permite a medida repetida de quimiotaxia de neutrófilos ao longo do tempo em crianças ou pequenos mamíferos, onde o volume da amostra é limitado.

Plataforma microfluídica para medição de quimiotaxia de neutrófilos não transformados Sangue Total

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

microfluidic-platform-for-measuring-neutrophil-chemotaxis-from

Research

JoVE Journal

Bioengineering

11.2K visualizações.  Massachusetts General Hospital.  Este protocolo detalha um ensaio concebido para medir a quimiotaxia de neutrófilos humanos a partir de uma gota de sangue total com reprodutibilidade robusta. Esta abordagem evita a necessidade de separação de neutrófilos e requer apenas alguns minutos de ensaio tempo de preparação. O chip de microfluídica permite a medida repetida de quimiotaxia de neutrófilos ao longo do tempo em crianças ou pequenos mamíferos, onde o volume da amostra é limitado. 

Vídeo: Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Alto rendimento Gerador Protein Expression Usando uma plataforma microfluídica

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Bioengenharia

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 ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

O Chip Multi-organ - Uma plataforma microfluídica para longo prazo Coculture Multi-tecidos

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white blood cells and platelets that are suspended in blood plasma. Due to the abundance of the RBCs (40% to 45% of the blood volume), their behavior dictates the rheological behavior of blood especially in the microcirculation. At very low shear rates, RBCs are seen to assemble and form entities called aggregates, which causes the non-Newtonian behavior of blood. It is important to understand the conditions of the aggregates formation to comprehend the blood rheology in microcirculation. The protocol described here details the experimental procedure to determine quantitatively the RBC aggregates in microcirculation under constant shear rate, based on image processing. For this purpose, RBC-suspensions are tested and analyzed in 120 x 60 &#181;m poly-dimethyl-siloxane (PDMS) microchannels. The RBC-suspensions are entrained using a second fluid in order to obtain a linear velocity profile within the blood layer and thus achieve a wide range of constant shear rates. The shear rate is determined using a micro Particle Image Velocimetry (&#181;PIV) system, while RBC aggregates are visualized using a high speed camera. The videos captured of the RBC aggregates are analyzed using image processing techniques in order to determine the aggregate sizes based on the images intensities.

The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

Ambiente Microfluidic controlada de Investigação dinâmico de Agregação Glóbulo Vermelho

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

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 ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

Totalmente dispositivo automático centrífugo microfluídicos para a detecção de proteínas Ultrasensitive de Sangue Total

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Plataforma microfluídico com multiplexada detecção electrónica do Ordenamento do Rastreamento de Partículas

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by their natural environment, root hair morphology and function are difficult to study and often excluded from plant research. In recent years, microfluidic platforms have offered a way to visualize root systems at high resolution without disturbing the roots during transfer to an imaging system. The microfluidic platform presented here builds on previous plant-on-a-chip research by incorporating a two-layer device to confine the Arabidopsis thaliana main root to the same optical plane as the root hairs. This design enables the quantification of root hairs on a cellular and organelle level and also prevents z-axis drifting during the addition of experimental treatments. We describe how to store the devices in a contained and hydrated environment, without the need for fluidic pumps, while maintaining a gnotobiotic environment for the seedling. After the optical imaging experiment, the device may be disassembled and used as a substrate for atomic force or scanning electron microscopy while keeping fine root structures intact.

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

A raiz do cabelo morfologia de plântulas de Arabidopsis em uma plataforma Microfluidic de duas camadas de imagem

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

Uma plataforma Microfluidic para Imaging Longitudinal em Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Uma plataforma Microfluidic de ultra alta taxa de transferência para o sequenciamento do genoma de célula única

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...