This work is financially supported by the Ministry of Science and Technology, Taiwan (Contract no. MOST 103-2113-M-001 -003 -MY2) and the Research Program on Nanoscience and Nanotechnology, Academia Sinica, Taiwan.

1. Projeto e Fabricação do Chip MDF <ol><li> Desenhe um padrão de camada de acrílico individual utilizando software comercial, como AutoCAD, e salvar o padrão. <ol><li> Rever o desenho do padrão de folha de acrílico de quatro camadas na Figura 1A e confirmar as conexões inter-camada. </li></ol></li><li> Fabricar todas as chapas acrílicas ea fita dupla face por ablação a laser utilizando um CO 2 riscador laser (Figuras 1B, 2A, e 2B). 16 <ol><li> Ligue o riscador de laser e ligar o aparelho ao computador portátil de controle. </li><li> Execute o software comercial e abrir o padrão desenhado na etapa 1.1, clicando em &quot;arquivo padrão desenvolvido.&quot; </li><li> Coloque um pedaço de folha de acrílico branco ou fita dupla face no palco XYZ do riscador laser. </li><li> Definir o foco do feixe de laser na superfície da folha de acrílico ou fita adesiva de dupla face com uma auto-alignment vara fornecida pelo fabricante do riscador laser. </li><li> Envie o padrão projetado para o riscador laser para usinagem direta da chapa acrílica ou fita dupla face. </li></ol></li><li> Retire o papel protetor das placas de acrílico usando uma pinça e soprar a superfície limpa com azoto gasoso. </li><li> Empilhar as folhas de acrílico e Bond-los juntos sob uma pressão de 2 kg / cm 2, num Bonder térmico durante 45 min a 110 ° C para formar o conjunto do canal de fluxo de estimulação / eléctrica. </li><li> Prepara-se o vidro de cobertura de microscópio como o substrato de cultura de células no chip. <ol><li> Coloque a tampa de vidro em uma tina de coloração e encher o frasco com uma diluição de dez vezes do detergente listado na lista de material. </li><li> Coloque a tampa de vidro e tina de coloração em uma steri-limpador ultra-sônico e limpar o vidro da tampa para 15 min. </li><li> Despeje o detergente diluído para fora da tina de coloração, encher o frasco com água destilada e repita o passo 1.5.2 3 vezes. </li><li> Seque o vidro da tampa limpos por sopro com azoto gasoso antes de aderir-lo para a fita dupla face. </li></ol></li><li> Aderem a tampa de vidro limpos para o conjunto do canal de estimulação do fluxo / elétrico com a fita dupla face modelado no passo 1.2. </li><li> Aderem 13 peças de acrílico adaptadores para as aberturas individuais em uma camada do conjunto de chips MDF com super cola. O conjunto de chip de MDF é então completo (Figura 2D). </li><li> Esteriliza-se o conjunto completo de chip MDF com 30 min de irradiação UV antes de ser utilizado. </li></ol> 2. Configuração da Ponte de Rede do Chip MDF Sal <ol><li> Antes da utilização, esterilizar todos os tubos de plástico, porcas com os dedos, e tubos de microcentrífuga mostrados na Figura 3B, definindo um tempo de retenção de 15 min a 121 ° C na autoclave. </li><li> Ligue os tubos Fluoroplastic (Figura 3B-i) ao conjunto de chips MDF através do meio de entradae adaptadores de saída mostrado na Figura 1A. </li><li> Conecte o cone Luer do meio de entrada e saída de tubos de plástico na Figura 3B-i para o 3-way torneiras. </li><li> Retirar 2,5 ml de CO 2 salina tamponada com fosfato -equilibrated (PBS) utilizando uma seringa de 3 ml e ligar-se a seringa para a torneira de 3 vias do tubo de entrada de plástico. Ligar uma seringa de 3 ml vazio à torneira de 3 vias do tubo de saída de plástico. As duas seringas são assim interligados por meio do chip de MDF. NOTA: Incubar a PBS no 37 ° C 5% de CO 2 incubadora de cultura de células O / N para se obter CO 2 -equilibrated PBS. </li><li> Selar as aberturas do azul e os adaptadores verdes (Figura 1A) na folha de PMMA Camada 1 com nozes dedo-tight sólidos brancos (Figura 3B-iii). </li><li> Preencha os canais de ponte de sal e câmaras de cultura com o CO 2 -equilibrated PBS na seringa descrito na configuração do passo 2.4. Evitara formação de bolhas. </li><li> Em seguida, coloque o CO 2 -equilibrated PBS contendo chip de MDF no 37 ° C 5% CO 2 celular cultura incubadora de O / N incubação. Isso permite que o ar dissolvido na fita de dupla face para formar bolhas nas câmaras. </li><li> Lave as bolhas para longe nos canais por um fluxo rápido de PBS no canal utilizando as duas seringas. Bombear o PBS e para trás, se necessário. </li><li> Drenar o PBS nos canais através da torneira de 3 vias ligado ao tubo de saída média. </li><li> Utilizando uma nova seringa de 3 ml, retirar 2,5 ml de CO 2 -equilibrated de Eagle Modificado por Dulbecco (DMEM) (ver passo 3.5), e substituir a seringa ligada à entrada com o novo. Reabastecer os canais com o meio. </li><li> Preparação da rede de ponte de sal. <ol><li> Temporalmente remover os sólidos porcas (Figura 3B-III) nos adaptadores verde (Figura 1A) e injectar 3% quente (&gt; 70 ° C) de agarose para o sal BRIdge canal através da abertura na adaptadores verde (Figura 1A). NOTA: Preparação de agarose quente: Dissolve-se 1,5 g de pó de agarose em 50 ml de PBS. Esterilizar por fixação de um tempo de retenção de 20 min a 121 ° C na autoclave. </li><li> Pare injectando a agarose, quando o líquido enche três quartos do comprimento do canal de ponte salina. </li><li> Selar os poros nos adaptadores verde (Figura 1A) apertando as porcas de sólidos (Figura 3B-III) após a injecção de agarose. </li><li> Substitua as porcas sólidas sobre as placas azuis (Figura 1A) com as porcas de dedo-apertado tubulares translúcidos (Figura 3B). </li><li> Carregar a 3% de agarose pré-aquecida para as porcas tubulares (Figura 3B-IV). </li><li> Incorporar os eléctrodos Ag / AgCl (Figura 3B-v) nas porcas tubulares (Figura 3B-iv) antes dosolidificação da agarose. </li></ol></li><li> Após concluir a configuração da rede de ponte de sal, injetar as células de câncer de pulmão CL1-5 para as câmaras de cultura, uma câmara de cada vez. </li></ol> 3. Preparação das células cancerosas e configurar para Electrotactic Experiment <ol><li> Cultura regular da linha de células de câncer de pulmão CL1-5. <ol><li> Cultura das células de cancro do pulmão, CL1-5 obtidos a partir de Prof. Pan-Chyr Yang, 17 em meio completo num frasco de cultura de células a 37 75T ° C numa atmosfera de CO2 a 5%. O meio completo é composto de DMEM e 10% de soro fetal de bovino (FBS). Sub-cultura das células a cada 3 a 4 dias. As células utilizadas para a realização de experimentos electrotaxis têm menos de 25 passagens da fonte original. </li></ol></li><li> Adicionar 2 ml de tampão de tripsina a 0,25% para as células em crescimento exponencial CL1-5 e incubar durante 2 minutos em 37 o ° C 5% de CO 2 incubadora para cultura de células de células detachment. </li><li> Terminar o processo de desprendimento com 6 ml de meio DMEM FBS a 10% e centrifuga-se as células a 300 g durante 5 min à temperatura ambiente. Depois descarte a forma e suspender o sedimento de células com 5 ml de PBS. </li><li> Contar o número de células em PBS. Então, 1 x 10 6 células e centrifugar a 300 g durante 5 min à temperatura ambiente. </li><li> Descarte o PBS. Suspender as células pré-aquecido com CO 2 -equilibrated DMEM e ajustar a densidade celular para ser de 1 x 10 6 células / ml. NOTA: Incubar o meio completo no 37 ° C 5% de CO 2 incubadora de cultura de células O / N para se obter o CO 2 -equilibrated DMEM. </li></ol> 4. Configurar para Electrotactic Experiment <ol><li> A partir da saída do chip MDF, injectar 0,3 ml, 1 x 10 6 / ml, as células no chip. </li><li> Incubar o chip MDF célula-semeado na incubadora de cultura celular (fixada em 37 ° C e 5% CO2) durante 2-4 horas. </li><li> Configuração de MDF microsistema fluídico. <ol><li> Instale o sistema microfluídico MDF em um aquecedor de vidro transparente de óxido de índio-estanho (ITO). Medir a temperatura do chip de MDF com um termopar tipo K grampeado entre o chip de MDF e o vidro ITO. Controlar o aquecedor ITO com um controlador proporcional-integral-derivativo (PID). Defina o MDF temperatura de incubação sistema microfluídico para 37 ° C com o controlador PID. NOTA: O fabrico do aquecedor de ITO e a configuração do sistema de aquecimento de cultura celular são descritos em relatórios anteriores 18,19. </li><li> Montar o conjunto de chips MDF com temperatura controlada no XYZ estágio motorizado controlado por computador em um microscópio invertido. O uso da platina motorizada é descrito na etapa 5.1. Bombear o meio completo para dentro das câmaras através das entradas de cultura com uma bomba de seringa de quatro canais. </li></ol></li><li> Bombear o meio completo para o chip de MDF com um caudal de 20 l / h. Os resíduos da cultura para foravamos é coletada nos tubos de microcentrífuga (indicados como &quot;resíduos&quot; na Figura 3A). </li><li> Incubar as células para outra 16-18 h a 37 ° C no chip de MDF. </li><li> Para substituir o meio, bombear o meio contendo 50, 25, 5 e 0 uM de inibidor de cinase, respectivamente, em cada câmara de cultura a uma taxa de fluxo de 20 mL / min durante 10 min. NOTA: Prepara-se uma solução estoque de 10 mM do inibidor de proteína-quinase associada a Rho-Y27632 por adição de 3 ml de água estéril para o inibidor de cinase de 10,6 mg. Dilui-se a solução stock de 10 mM para 50, 25, e 5 uM, respectivamente, usando 10% de FBS DMEM. </li><li> Incubar as células em que o inibidor de cinase durante mais 60 minutos a 37 ° C. Definir a taxa de fluxo médio de 20 l / h. Use a mesma temperatura da cultura e taxa acima descrito em etapas posteriores fluir. </li><li> Use fios eléctricos para ligar uma fonte de alimentação DC para o chip de MDF através dos eléctrodos de Ag / AgCl sobre o chip. </li><li> Serially conectar um amperímetro in o circuito elétrico. Use o amperímetro para monitorar a corrente elétrica no chip MDF. </li><li> Ligue a fonte de alimentação DC e definir o amperímetro atual para 86,94 mA, ajustando a tensão de alimentação entre 15 e 19 V. NOTA: Tendo em conta a lei de Ohm, E = I / (σA FEP), onde I é a corrente eléctrica que flui através do material a granel, ou seja, o meio de cultura na câmara electrotactic, σ (= 1,38 Ω -1 M - 1) é o condutividade do meio de cultura, um FEP (= 0,21 mm2 para uma largura de 3 mm x 0,07 mm de altura) é a área em secção transversal efectiva da câmara de electrotactic, e a corrente eléctrica (I) necessária para gerar uma 300 mV / mm EF no chip MDF é 86,94 mA. </li></ol> 5. Aquisição e Análise de Imagens de celular <ol><li> Controlar o estágio motorizado usando um programa MATLAB GUI. Com o sistema de microfluidos de MDF montado no microscope, mover o chip para as regiões de observação usando o estágio motorizado. </li><li> Imagens de células gravar usando uma câmera de single-lens reflex digitais (SLR) montado no microscópio. </li><li> Tome imagens microscópicas, utilizando uma lente de objectiva de 4X em intervalos de 15 minutos durante 2 horas. </li><li> A análise de migração celular. <ol><li> Execute NIH ImageJ 1,47 V. </li><li> Analisar a distância a partir do inicial para as posições finais do centróide da célula. </li><li> Vá em &quot;Arquivo&quot; → &quot;Importar&quot; → &quot;Sequência de Imagens&quot;, e importar nove imagens para análise de migração celular. A primeira imagem é o resultado do tempo zero, e a última imagem é o resultado de 120 min. </li><li> Vá em &quot;Analisar&quot; → &quot;Definir Medidas&quot; e marque a caixa ao lado de &quot;Centroid&quot; </li><li> Clique no ícone &quot;Freehand Seleções&quot;. </li><li> Descrevem a borda das células seleccionadas na primeira imagem. </li><li> Vá em &quot;Editar&quot; → </em&gt; &quot;Seleção&quot; → &quot;Adicionar ao Manager&quot; </li><li> Ir para o nono imagem e descrever o bordo das mesmas células seleccionadas na primeira imagem. As posições de células pode ser rastreada usando a segunda e a oitava imagens. </li><li> Vá em &quot;Editar&quot; → &quot;Seleção&quot; → &quot;Adicionar ao Manager&quot; </li><li> Repita os passos 5.4.5 a 5.4.9 para coletar dados de 90 a 100 células. </li><li> Vá em &quot;Analisar&quot; → &quot;Measure&quot; para obter o resultado das posições iniciais e finais das células selecionadas. NOTA: a velocidade de migração é definida como o comprimento médio do movimento das células por hora. O direcionamento é definido como co-seno, onde é o ângulo entre o vector da dcEF (do ânodo para o cátodo) e o vector a partir do ponto de partida de uma célula para a sua posição final. O direcionamento é -1 para as células migram para o ânodo e um para as células que migram em direção ao cátodo. Para um grupo de aleatoriamente migradeamento de células, o direcionamento é 0. 2 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Encontramos o processo de adesão adaptadores de acrílico para Camada 1 do chip MDF a ser complicado. A aplicação de apenas 1 a 2 ul de super-cola é suficiente para aderir firmemente o adaptador no chip MDF. Grandes quantidades de cola resultou em uma polimerização incompleta do super-cola e falta em aderir. Uma vez que os adaptadores de acrílico foram firmemente aderido no chip MDF, fuga de líquido no sistema microfluídico raramente ocorria. Além disso, O / N incubação dentro da câmara de vácuo ajudou a r...

O comportamento de células aderentes que se deslocam em relação um ânodo ou cátodo sob uma corrente eléctrica directa de campo (dcEF) é referido como electrotaxis. O comportamento electrotactic de células desempenha um papel importante na embriogénese, a regeneração do nervo, e a cicatrização de feridas. 1 As células de tumor, tais como células de cancro da próstata de rato, células de cancro da mama, 2 e 3 de adenocarcinoma do pulmão de células 4-8 mostraram movimento electrotactic sob uma dcEF aplicada . O EF fisiológica foi medido em tecidos glandulares. 9,10 Electrotaxis também tem sido relatada em células de tumor da glândula associada a 2,3. Tomados em conjunto, os electrotaxis de células cancerosas é considerado um factor de metástase. 11 Controlando a orientação eléctrica de células cancerosas sob dcEF pode ser uma abordagem potencial para o tratamento futuro de cancro. No entanto, hoje, o mecanismo molecular detalhada de electrotaxis permanece controverso. Portanto, uma investigação do influence da estimulação elétrica sobre a migração de células de câncer pode facilitar o desenvolvimento de estratégias para o tratamento do câncer. Recentemente, dispositivos bio-microfluídicos foram fabricadas para estudar as respostas celulares a fluir força de cisalhamento, 12 gradientes químicos, 13 e estímulos elétricos 4 in vitro. O fabrico de dispositivos de microfluidos que utilizam bio-polidimetilsiloxano (PDMS) ou polimetilmetacrilato (PMMA, também conhecido como acrílico) conseguiu reduzir a taxa de falha de tais experiências. Além disso, a utilização de dispositivos de microfluidos de base acrílica como um protótipo para investigar temas biológicos é mais simples do que utilizando PDMS fichas. Várias funções em dispositivos à base de acrílicos têm sido desenvolvidos para electrotaxis estudo. No entanto, nenhum dos modelos anteriores são capazes de testar simultaneamente os efeitos de várias condições químicas e o campo eléctrico sobre as células para electrotaxis estudo. Assim, foi desenvolvido um dispositivo microfluídico-o multichannel dual-elétrico-campo (MDF) de quatro canais independentes de cultura e oito condições experimentais diferentes em um único chip contendo chips. O chip de MDF à base de acrílico, relatada pela primeira vez por Hou et ai., 8 integra estimulação eléctrica e vários canais quimicamente isolado. Estes canais quimicamente isoladas podem ser usadas para a cultura de células de diferentes tipos em um experimento. O dcEF nos canais é produzido por uma fonte de alimentação elétrica. Dois campos elétricos independentes, um com a força aplicada de campo elétrico (EFS) e outro com 0 EFS, são realizados em cada canal quimicamente isolado. Deste modo, o chip fornece EF-coexistente controlado melhor e estimulação química. Além disso, os resultados da simulação numérica da difusão química dentro do chip MDF indicar que não ocorreu contaminação cruzada entre os canais, após um período experimental de 24 horas. 8 Em comparação com o device relatado por Li et al., 14 o chip MDF fornece uma área de cultura em geral, o que permite, para posterior análise bioquímica das células estimuladas electricamente. Além disso, com a área de observação maior do chip MDF, mais células podem ser observados no teste, de modo que a análise de velocidade de migração ou direcionamento das células estimuladas electricamente é mais preciso. Os desenhos de chips de um único canal de estudos anteriores relatados por Huang et al. 4 e Tsai et al. 15 permitir que apenas um tipo de célula ou de produto químico a ser testado. No entanto, o chip de MDF podem ser utilizados para investigar os efeitos de vários produtos químicos na electrotaxis, bem como os efeitos da estimulação eléctrica sobre diferentes tipos de células. Em outras palavras, o chip MDF permite o estudo eficiente de dependências químicas de dose.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Reagent</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >DMEM medium</td>
			<td >Gibco,Invitrogen, USA</td>
			<td >12800-017</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum</td>
			<td >Gibco,Invitrogen, USA</td>
			<td >16000-044</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin</td>
			<td >Gibco,Invitrogen, USA</td>
			<td >25200-072</td>
			<td></td>
		</tr>
		<tr >
			<td >PBS</td>
			<td >Basic Life</td>
			<td >BL2651</td>
			<td></td>
		</tr>
		<tr >
			<td >Y-27632 (hydrochloride)</td>
			<td >Cayman Chemical Co</td>
			<td >10005583</td>
			<td></td>
		</tr>
		<tr >
			<td >agarose</td>
			<td >LONZO, USA</td>
			<td >SeaKem LE AGAROSE</td>
			<td></td>
		</tr>
		<tr >
			<td >syringe</td>
			<td>Terumo</td>
			<td>3 ml with Luer taper</td>
			<td></td>
		</tr>
		<tr >
			<td >3-way stopcock</td>
			<td>Nipro</td>
			<td >with Luer taper</td>
			<td></td>
		</tr>
		<tr >
			<td >PMMA (acrylic)</td>
			<td >HiShiRon Industries CO., Ltd, Taiwan</td>
			<td ></td>
			<td >thickness 1mm, 2mm</td>
		</tr>
		<tr >
			<td >acrylic adaptor</td>
			<td >KuanMin Technology Co., Ltd, Taichung, Taiwan</td>
			<td >1/4-28 port, 10x10x6 mm</td>
			<td>customized</td>
		</tr>
		<tr >
			<td >nut</td>
			<td >Thermo Fisher Scientific Inc.</td>
			<td >UPCHURCH:P-206x, P-200x, F120x, P-659, P-315x</td>
			<td></td>
		</tr>
		<tr >
			<td >Microscope cover glass</td>
			<td >Deckgl&#228;ser, Germany</td>
			<td >24x60 mm</td>
			<td></td>
		</tr>
		<tr >
			<td >double-sided tape</td>
			<td >3M</td>
			<td >PET 8018</td>
			<td></td>
		</tr>
		<tr >
			<td >super glue</td>
			<td >3M</td>
			<td >Scotch Liquid Plus Super Glue</td>
			<td></td>
		</tr>
		<tr >
			<td >Teflon tube</td>
			<td >HENG YI ENTERPRISE CO., LTD., Taiwan</td>
			<td >UPTB_06, DUPONT TEFLON BRAND RESIN FEP TUBING</td>
			<td>outer diameter 1/16 in., inner diameter 0.03 in.; Upchurch Scienti&#64257;c</td>
		</tr>
		<tr >
			<td >TFD4 detergent</td>
			<td >Franklab, France</td>
			<td >TFD4</td>
			<td></td>
		</tr>
		<tr >
			<td >ultrasonic steri cleaner</td>
			<td >LEO ULTRASONIC CO., LTD., Taiwan</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Thermo bonder</td>
			<td >KuanMin Technology Co., Ltd, Taichung, Taiwan</td>
			<td>customized</td>
			<td></td>
		</tr>
		<tr >
			<td >CO2 laser scriber</td>
			<td >LTT group, Taiwan</td>
			<td >ISL-II</td>
			<td></td>
		</tr>
		<tr >
			<td >indium tin oxide glass (ITO glass)</td>
			<td >AimCore Technology Co., Ltd</td>
			<td >TN/STN, &#8806;10&#937;</td>
			<td></td>
		</tr>
		<tr >
			<td >proportional-integral-derivative (PID) controller</td>
			<td>JETEC Electronics Co., Japen</td>
			<td >TTM-J40-R-AB,</td>
			<td></td>
		</tr>
		<tr >
			<td >K-type thermocouple</td>
			<td >TECPEL</td>
			<td >TPK-02A</td>
			<td></td>
		</tr>
		<tr >
			<td >4-channel syringe pump</td>
			<td >KdScientific, USA</td>
			<td >250P</td>
			<td></td>
		</tr>
		<tr >
			<td >DC power supply</td>
			<td >GWInstek, Taiwan</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >X-Y-Z motor stage</td>
			<td >TanLian, E-O Co. Ltd., Taiwan</td>
			<td>customized</td>
			<td></td>
		</tr>
		<tr >
			<td >inverted microscope</td>
			<td >Olympus, Japan</td>
			<td >CKX41</td>
			<td></td>
		</tr>
		<tr >
			<td >digital SLR camera</td>
			<td >Canon, Japan</td>
			<td >60D</td>
			<td></td>
		</tr>
	</tbody>
</table>

electrotaxis studies of lung cancer cells using a multichannel dual-electric-field microfluidic chip

O comportamento de migração direccional de células sob uma corrente eléctrica directa de campo (dcEF) é referido como electrotaxis. O papel fisiológico significativo de dcEF em guiar o movimento de células durante o desenvolvimento embrionário, a diferenciação celular, a cicatrização de feridas e tem sido demonstrada em vários estudos. Através da aplicação de chips microfluídicos para um ensaio electrotaxis, o processo de investigação é encurtado e erros experimentais são minimizados. Nos últimos anos, os dispositivos microfluidicos feitos de substâncias poliméricas (por exemplo, polimetilmetacrilato, PMMA, ou acrílico) ou polidimetilsiloxano (PDMS) têm sido amplamente utilizados no estudo das respostas das células à estimulação eléctrica. No entanto, ao contrário de numerosos passos necessários para fabricar um dispositivo de PDMS, a construção simples e rápido dos micro chip de acrílico fl uidic o torna adequado tanto para dispositivo de prototipagem e produção. No entanto, nenhum dos dispositivos relatados facilitar o estudo da química eficiente e simultânea DCEF efeitos sobre as células. Neste relatório, nós descrevemos nosso projeto e fabricação de um chip de múltiplos canais de base acrílica dual-elétrico-campo (MDF) para investigar o efeito simultâneo de química e estimulação elétrica em células de câncer de pulmão. O chip MDF fornece oito combinações de estímulos elétricos / químicos em um único teste. O chip não só reduz drasticamente o tempo necessário experimental, mas também aumenta a precisão na electrotaxis estudos.

Fabricação e montagem do dispositivo de MDF Um diagrama esquemático do chip MDF à base de acrílicos é mostrado na Figura 1A. Quatro placas de acrílico, uma tampa de vidro, 13 adaptadores de acrílico, e um pedaço de fita dupla face foram utilizados na montagem do chip MDF concluída (Figura 2D). Há apenas quatro canais independentes de cultura no disp...

Watch this Scientific Journal Video about Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip at JoVE.com

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

Electrotaxis Estudos de pulmão células de câncer usando um Multichannel Dual-elétrico-campo Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

electrotaxis-studies-lung-cancer-cells-using-multichannel-dual

Research

JoVE Journal

Bioengineering

8.6K visualizações.  Academia Sinica. Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

Vídeo: Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

Métodos de detecção de fluorescência para as plataformas de gotículas microfluídicos

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Bioengenharia

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

Electrotaxis baseados em microfluídica para análise quantitativa On-demand de<em&gt; Caenorhabditis elegans</em&gt; &#39;Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

Fabricação de chips microfluídicos e método para detectar a gripe

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

Um chip microfluídico para a Versátil Análise Química de células individuais

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

Um chip microfluídico para ICPMS Amostra Introdução

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

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 ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Co-cultura de Vida Microbiome com Intestinal Microengineered Humano Villi num Gut-on-a-Chip Microfluidic Dispositivo

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

Película seca baseada em fotorresiste eletroquímica Microfluidic Biosensor plataforma: Fabricação de dispositivo, ensaio em-microplaqueta preparação e operação de sistema

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Imobilização de indivíduos ao vivo Caenorhabditis elegans , usando um Chip de Microfluidic polidimetilsiloxano ultrafino com retenção de água

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

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 ...