This work was supported by a grant from the National Health Research Institutes (03-A1 BNMP11-014).

Nota: Os desenhos fotomáscara para a fabricação de dispositivos microfluídicos foram desenhadas usando um software de desenho assistido por computador (CAD). Os desenhos foram então utilizados para fabricar máscaras de crómio usando um serviço comercial. Os dispositivos PDMS foram feitas usando técnicas de litografia suave 11. 1. Fabricação de Mestre Moldes por litografia <ol><li> Antes do processo de fotolitografia 12, utilizar as bolachas de silício de 4 polegadas como um substrato e desidratar as bolachas em um forno convencional a 120 ° C durante 10 min. </li><li> Limpar as bolachas de silício desidratados usando oxigénio tratamento com plasma a 100 watts durante 30 segundos em um aspirador de plasma. </li><li> Pré-aqueça duas placas a 65 ° C e 95 ° C, respectivamente, para o seguinte processo de cozimento. </li><li> Brasão de 5 g de fotorresiste negativo (PR) sobre as pastilhas de silício limpo por um revestidor de rotação; de centrifugação a 1.200 rpm (SU-8 de 50) durante 30 segundos para produzir a camada de microcanais. </li><li> Coloque o PRrevestidos bolacha numa placa de aquecimento pré-aquecido a 65 ° C durante 12 min e transferi-lo para uma outra placa de aquecimento pré-aquecido a 95 ° C durante 33 min (para os padrões de 100 um de espessura) para executar um processo de cozer macio. </li><li> Após o cozimento, coloque a pastilha de silício PR revestido sobre o titular de um alinhador semi-automatizado máscara e alinhá-lo a um photomask transparência 25.400 dpi de resolução. </li><li> Expor a bolacha de silício revestido de RP à luz UV (365 nm) a uma dose de 500 mJ / cm 2 para criar o padrão de PR na bolacha de silício. </li><li> Remova o wafer do alinhador e colocá-lo numa placa de aquecimento para pós-cozimento a 95 ° C por 12 min. </li><li> Mergulhe as bolachas em SU-8 desenvolvedor de soluções (acetato de éter propilenoglicol monometílico, PGMEA) para lavar PR não reticulada de 12 min e secar delicadamente com azoto para expor as marcas de alinhamento. </li><li> Mais uma vez, revestimento 5 g de fotorresiste negativo sobre as bolachas por um revestidor de rotação; rotação de 700 rpm (SU-8 100) durante 30 segundos e 1.200 rpm (SU-8 10) por 30 s para 300 &amp;# 181; m padrão de espessura e 27 mm padrão de espessura, respectivamente, para fazer a camada de micropoços. </li><li> Coloque a bolacha PR revestido numa placa de aquecimento a 65 ° C durante 4 minutos e a 95 ° C durante 8 minutos (para 27 uM camada de captura de poços profundos); e a 65 ° C durante 40 min e a 95 ° C durante 110 minutos (para 300 uM camada cultura poços profundos). </li><li> Após arrefecimento, colocar a pastilha de silício revestido PR no alinhador máscara equipado com luz UV. </li><li> Expor a bolacha de silício revestido de PR para a luz UV (365 nm) a uma dose de 250 mJ / cm 2 (27 uM para padrão de espessura) e 700 mJ / cm 2 (por 300 um de espessura padrão). </li><li> Coza as bolachas a 95 ° C durante 5 min (27 uM padrão de espessura) e 30 min (300 um de espessura padrão), respectivamente. </li><li> Lavar o PR não reticulado pela PGMEA durante 6 minutos (27 uM padrão de espessura) e 25 min (300 um de espessura padrão), respectivamente, e em seguida secá-los com azoto gasoso. </li><li> Medir a altura do padrão apresenta emo wafer com uma profilometer de varredura a laser, colocando a bolacha sobre o xy-estágio de um profilometer de varredura a laser. <ol><li> Ajuste o plano focal para mostrar claramente o padrão apresenta na bolacha usando o modo de observação &quot;visão da câmera&quot; sob uma lente objetiva de 20X. </li><li> Mude o modo de observação de &quot;visão da câmera&quot; para &quot;visão laser&quot;, e definir as posições superior e inferior dos recursos. </li><li> Defina o modo de medição, área, qualidade, e z-pitch até transparente (em cima), 1 linha (1.024 x 1), de alta precisão e 0,5 mm, respectivamente, e, em seguida, pressione a parte inferior start para iniciar a medição. </li></ol></li></ol> 2. Preparação de dispositivos PDMS para isolamento de células Individual <ol><li> Antes de fundição PDMS, silanizar os moldes mestres com trichlorosilane para criar uma superfície hidrofóbica, o que torna mais fácil descolar as réplicas PDMS dos moldes mestre. <ol><li> Coloque os moldes de mestre e um barco ponderação contendo200 ul de triclorossilano num secador, e aplicar vácuo (-85 kPa) durante 15 min. </li><li> Pare o vácuo e, em seguida, deixar os moldes mestres no exsicador para silanizar os moldes mestres à temperatura ambiente durante pelo menos 1 h. </li><li> Retire os moldes mestres do exsicador e coloque cada molde mestre em um 10 centímetros placa de Petri. </li></ol></li><li> Misturar uma quantidade total de polímero de PDMS kit de 17,6 g contendo uma base e um agente de cura a uma razão de 10: 1, e em seguida, despejar o PDMS no molde mestre na placa de Petri. </li><li> Colocar a placa de Petri num exsicador e aplicar vácuo (-85 kPa) durante 1 h para remover as bolhas de ar nas PDMS. </li><li> Remova a placa de Petri do exsicador e colocá-lo num forno convencional a 65 ° C durante 3-6 horas para curar o PDMS. </li><li> Retirar as réplicas PDMS curado a partir dos moldes mestres e dois furos como uma entrada e uma saída nas duas extremidades do microcanal no array PDMS réplica captura poços por um perfurador com diâmetro interior de 0.75 mm para o canal de fluidos (Figura 2A). </li><li> Use fita para limpar a superfície das réplicas de PDMS, e, em seguida, colocar o PDMS réplicas em um produto de limpeza de plasma por tratamento com plasma de oxigénio breve (100 watts durante 14 seg). </li><li> Remover o PDMS réplicas da máquina de plasma de oxigénio. </li><li> Alinhar uma réplica superior (contendo os micropoços de captura) e um PDMS inferior (contendo os micropoços de cultura) com a mão sob um microscópio estereoscópico e trazê-los em contato. </li><li> Coloque as réplicas PDMS alinhados num forno a 65 ° C durante 24 horas para conseguir a ligação permanente entre as réplicas PDMS para formar o dispositivo final. </li><li> Embeber o dispositivo PDMS em uma desionizada (DI) -água recipiente cheio e colocar o recipiente em um exsicador sob vácuo (-85 kPa) durante 15 min para remover o ar a partir do microcanal do dispositivo de PDMS. </li><li> Coloque o dispositivo PDMS cheio de água desionizada em um capuz de cultura de tecidos e utilizam a luz UV (comprimento de onda da luz: 254 nm) para esterilizar o dispositivo para 30 min. </li><li> Substituir a água Dl no dispositivo de PDMS com uma albumina de soro bovino a 5% (BSA) em solução de PBS 1x e incubar a 37 ° C durante 30 min para evitar que as células se colem à superfície do PDMS. Nota: O revestimento de BSA é crítica para melhorar a eficiência da transferência de células isoladas a partir de captura de poços para cultura poços. </li><li> Substituir a solução de BSA a 5% no dispositivo de PDMS com solução 1x PBS esterilizado. </li></ol> 3. Preparação da suspensão de uma única célula <ol><li> Cultura Neural stem KT98 célula, e A549 de células de carcinoma humano e MDA-MB-435 que, em uma placa de Petri em incubadora de cultura de célula convencional (37 ° C, 5% de CO 2, e 95% de humidade) para preparar as células para a experiência celular dispositivo PDMS . </li><li> Remover e descartar o meio de cultura gasto (meio basal DMEM fornecido com antibióticos 10% de soro fetal bovino e 1%) a partir do prato de cultura com células crescidas a 70-80% de confluência. </li><li> Lavar cuidadosamente as células com esterilizados PBS três vezes. &lt;/ Li&gt; <li> Retirar e descartar o PBS e adicionar 2 ml de uma mistura de enzima recombinante com proteolíticas, e atividades DNase colagenolíticas (por favor consulte a lista de materiais para informações detalhadas). </li><li> Incubar a placa de cultura à temperatura ambiente durante 5 min, e depois toque no prato de cultura para facilitar a separação celular. </li><li> Adicione 4 ml de PBS esterilizado para dispersar as células, e em seguida, transferir a suspensão celular para um tubo de 15 ml. </li><li> Centrifuga-se o tubo a 300 xg durante 3 min e remover o sobrenadante. </li><li> Suavemente ressuspender o sedimento celular em 1 ml de PBS esterilizado e contar o número de células vivas utilizando o método de exclusão com Trypan Blue padrão 13. Nota: Esta etapa de ressuspensão é crítica para a preparação de suspensão de uma única célula bem dissociado para melhorar a eficiência de isolamento de uma única célula. </li></ol> 4. Isolamento de célula única e Cultura Clonal <ol><li> Carga de 50 ul de suspensão celular a uma concentração de 2,2-2,5 x 10 <sup&gt; 6 células / ml no microcanal do dispositivo PDMS via orifício de saída do dispositivo com uma pipeta de mão. Nota: O carregamento de pipeta suspensão de células precisa ser parado e realizada na primeira posição de paragem para evitar a introdução de bolhas de ar dentro do microcanal. </li><li> Carregar mais 50 ul de suspensão de células através do orifício de entrada para preencher uniformemente-se todo o microcanal com células. </li><li> Fecha-se o orifício de saída com uma ficha (A, 1 mm de diâmetro cortado em nylon linha de pesca 3 milímetros de comprimento) para evitar o escoamento induzido pela pressão hidrostática das gotas em orifícios de entrada e de saída. Nota: Os tampões foram esterilizados UV dentro de um capuz de cultura de tecidos antes da utilização. </li><li> Encha uma seringa estéril 1 ml com meio de cultura, retire as bolhas de ar, e configurá-lo em uma bomba de seringa. </li><li> Ligar a forma carregada seringa para o orifício de entrada do dispositivo de PDMS através de uma agulha G 23 sem corte e uma tubagem de poli-tetrafluoroethene (PTFE). </li><li> Retire a ficha da furo de saída e todosow um intervalo de tempo de 2 min para permitir que as células a instalar-se nos unicelulares captura de poços por força gravitacional. </li><li> Lavar as células não confinadas com 300 ul de meio de cultura a uma taxa de fluxo de 600 mL / min impulsionado pela bomba de seringa. </li><li> Espera durante 2 min para estabilizar o aparelho, e vedar os orifícios de entrada e de saída com fichas de modo a formar um sistema de cultura fechada. </li><li> Virar o dispositivo com a mão para transferir as células individuais capturados para os micropoços de cultura. </li><li> Coloque o dispositivo PDMS em uma placa de cultura de tecido de 100-mm, e adicionar 10 ml de PBS esterilizado em torno do dispositivo de cultura para evitar a evaporação do meio do microcanal. </li><li> Mover o prato de cultura para uma incubadora convencional de cultura de células (37 ° C, 5% de CO 2, e 95% de humidade) para a cultura clonal das células individuais. </li></ol> 5. Meio de Cultura Reposição <ol><li> Depois de 1 d de cultura, substituir o meio de cultura no dispositivo de PDMS com meio fresco para melhorar PR célulaoliferation. </li><li> Colocar duas gotas de meio de cultura na parte superior do dispositivo de PDMS perto das áreas de entrada e saída de buracos para evitar a introdução de bolhas de ar para dentro do microcanal, enquanto executa o passo seguinte. </li><li> Perfurar dois buracos da parte superior do dispositivo de PDMS perto das duas extremidades do microcanal como uma entrada e uma saída para o microcanal. Nota: Não perfurar toda a duas camadas do dispositivo PDMS; em vez disso, apenas perfurar através da camada superior do PDMS. </li><li> Conecte uma seringa de plástico de 1 ml contendo meio fresco para a entrada através de um G agulha romba 23 e tubos de PTFE. </li><li> Fluxo médio de 120 ul fresco para dentro do dispositivo durante 5 minutos para substituir o meio velho. </li><li> Inserir dois tampões para selar a entrada e a saída de furos, e devolver o dispositivo para a incubadora de cultura de células. </li><li> Refrescar o meio de cultura a cada 2 d durante o período de cultura, removendo os tampões de vedação, seguido pela repetição dos passos 5,4-5,5. </li></ol>

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The authors declare that they have no competing financial interests.

Sistemas de dispositivo baseado em micropoços 6,14 têm sido utilizados para a manipulação de uma única célula e análise, tal como uma única célula em larga escala prendendo 6 e proliferação de células-tronco hematopoiéticas único 15. Embora o tamanho bem, número e forma podem ser ajustados a aplicações específicas, a eficiência de isolamento de uma única célula está sempre comprometida quando o tamanho do poço é aumentada. 9,15 <p class="jove_conten...

Actualmente colocando células isoladas individualmente num espaço de cultura é geralmente conseguido através de diluição limitante ou de células activadas por fluorescência (FACS). Para muitos laboratórios, diluição limitante é um método conveniente, uma vez que requer apenas uma pipeta e placas de cultura de tecidos, as quais estão prontamente disponíveis. Neste caso, uma suspensão de células é diluída em série até uma densidade celular apropriada, e, em seguida, colocados em poços de cultura, utilizando uma pipeta manual. Estas células individuais compartimentados são então utilizados para análise de células, tais como heterogeneidade genética e rastreio de uma colónia de formação 2. No entanto, este método é de baixo rendimento e de trabalho intensivo, sem a utilização de um braço robótico para assistência, devido à natureza distribuição de Poisson do método de diluição limitante restringe eventos de uma única célula a uma probabilidade máxima de 37% 3. máquinas de FACS com um braço robótico integrado pode superar a limitação da distribuição de Poisson pela precisão placção de uma única célula em um poço de cultura de cada vez 4. No entanto, a alta tensão de cisalhamento mecânico (assim, reduzido a viabilidade celular) 5 e compra da máquina e os custos operacionais têm limitado o seu uso em muitos laboratórios. Para superar as limitações acima, em microescala dispositivos têm sido desenvolvidos para carregar altamente eficiente de células individuais em micropoços 6. No entanto, os micropoços não proporcionar um espaço adequado para as células carregadas com a proliferar, devido à necessidade de tornar o tamanho de cada micropoço próximo do de uma única célula de carga para maximizar a probabilidade de uma única célula. Como são necessários ensaios de cultura em muitas aplicações baseadas em células (por exemplo, ensaio clonogénico 7), micropoços maiores (90-650 um de diâmetro ou de comprimento lateral) também têm sido utilizados para permitir a culturas celulares prolongadas. No entanto, como o método da diluição limitante, eles também possuem baixas eficiências de carga de células individuais, que variam de 10 -. 30% 8, 9 Anteriormente, nós desenvolvemos uma plataforma de microfluidos de alto rendimento para isolar células individuais em micropoços individuais e demonstrar a sua aplicação no ensaio clonogénico das células isoladas. 10 O dispositivo foi feito com poli-dimetilsiloxano (PDMS), e compreende dois conjuntos de matrizes de micropoços com diferentes tamanhos de microcavidades, que podem melhorar em grande medida a eficiência no carregamento de uma única célula em um micropoço cujo tamanho é significativamente maior do que a célula. Notavelmente, este conceito &quot;dupla bem&quot; permite que o tamanho da área de cultura a ser ajustada de forma flexível, sem afectar a eficiência da captação de uma única célula, tornando-a simples de ajustar o desenho do dispositivo para se adequar a diferentes tipos de células e aplicações. Este método de elevada eficiência deveria ser útil para experiências de cultura de células de longo prazo para os estudos de heterogeneidade da célula e estabelecimento linha celular monoclonal.

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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 100 negative photoresist</td>
			<td>MicroChem</td>
			<td>Y131273</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spin coater</td>
			<td>Synrex Co., Ltd.</td>
			<td>SC-HMI 2&#34; ~ 6&#34;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hotplate</td>
			<td>YOTEC company</td>
			<td>YS-300S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Msak aligner</td>
			<td>Deya Optronic CO.</td>
			<td>A1K-5-MDA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 developer</td>
			<td>Grand Chemical Companies</td>
			<td>GP5002-000000-72GC</td>
			<td>Propylene glycol monomethyl ether acetate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scanning laser profilometer</td>
			<td>KEYENCE</td>
			<td>VK-X 100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trichlorosilane</td>
			<td>Gelest, Inc</td>
			<td>SIT8174.0</td>
			<td style="width:463px;">TRIDECAFLUORO-1,1,2,2-TETRAHYDROOCTYL.&#160; Hazardous. Corrosive to the respiratory tract., reacts violently with water.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Desiccator</td>
			<td>Bel-Art Products&#160;</td>
			<td>F42020-0000</td>
			<td>SPACE SAVER VACUUM DESICCATOR 190MM WHITE BASE</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polydimethylsiloxane (PDMS) kit</td>
			<td>Dow corning</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Harris Uni-Core puncher</td>
			<td>Ted Pella Inc.</td>
			<td>15072</td>
			<td>with 0.75 mm inner-diameter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Removable tape</td>
			<td>3M Company</td>
			<td>Scotch Removable Tape 811</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereomicroscope</td>
			<td>Leica Microsystems</td>
			<td>Leica E24</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin (BSA)</td>
			<td>Bersing Technology</td>
			<td>ALB001.500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM basal medium</td>
			<td>Gibco</td>
			<td style="width:235px;">12800-017</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>Thermo Hyclone</td>
			<td>SH30071.03HI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotics</td>
			<td>Biowest</td>
			<td>L0014-100</td>
			<td>Glutamine-Penicillin-Streptomycin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recombinant enzyme mixture</td>
			<td>Innovative cell technology</td>
			<td style="width:235px;">AM-105</td>
			<td>Accumax</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DiIC12(3) cell membrane dye</td>
			<td>BD Biosciences</td>
			<td style="width:235px;">354218</td>
			<td>Used as a cell tracker</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td style="width:235px;">703007</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic syringe (1 mL)</td>
			<td>BD Biosciences</td>
			<td style="width:235px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">23 gauge blunt needles</td>
			<td>Ever Sharp Technology, Inc.</td>
			<td style="width:235px;">TD21</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Poly-tetrafluoroethene (PTFE) tubing</td>
			<td>Ever Sharp Technology, Inc.</td>
			<td style="width:235px;">TFT-23T</td>
			<td>&#160;inner diameter, 0.51 mm; outer diameter, 0.82 mm</td>
		</tr>
	</tbody>
</table>

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.

A plataforma de microfluidos para o isolamento de uma única célula e de cultura compreende um micro-canal (200 um de altura) com dois conjuntos de matrizes de micropoços (Figura 2A). Os dois conjuntos de matrizes de micropoços são denominados como captura de poços (25 um de diâmetro e 27 uM em profundidade) e cultura poços (285 um de diâmetro e 300 mm de profundidade) para o isolamento de uma única célula e a cultura, respectivamente, e cada captura-se bem pos...

Watch this Scientific Journal Video about A Microfluidic Platform for High-throughput Single-cell Isolation and Culture at JoVE.com

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

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 ...

a-microfluidic-platform-for-high-throughput-single-cell-isolation

Research

JoVE Journal

Bioengineering

11.2K visualizações.  National Health Research Institutes, Taiwan. 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. 

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

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

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

Alta taxa de transferência de célula única e múltipla de células Micro-encapsulamento

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

CometChip: A alta taxa de transferência de plataforma 96-Bem, para avaliação dos danos causados ​​no DNA em células Microarrayed Humanos

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging techniques.
The one-time use packaging technique employs UV-based and temperature curing epoxies to connect microtubes to access holes, wire-bonding for integrated circuit connections, and silver epoxy for electrical connections. This method is based on a robust assembly technique that can support relatively high pressure close to 1 psi and does not need any support to strengthen the microfluidic architecture.
Reusable packaging techniques consist of PDMS-based microtube interconnectors and anisotropic adhesive films for electrical connections. These devices are more sensitive and fragile. Consequently, Plexiglas support is added to the microfluidic structure to improve the electrical contact when anisotropic adhesive films are used, and also to strengthen the microfluidic architecture. In addition, a micromanipulator is needed to maintain tubes while using a thin PDMS layer to connect them to the access holes. Different PDMS layer thicknesses, ranging from 0.45-3 mm, are tested to compare the best adherence versus injection rates. Applied injection rates are varied from 50-300 &#956;l/hr for 0.45-3 mm PDMS layers, respectively. These techniques are mainly applicable for low-pressure applications. However, they can be extended for high-pressure ones through plasma-oxygen process to permanently seal the PDMS to glass substrates. The main advantage of this technique, besides the fact that it is reusable, consists of keeping the device observable when the microchannel length is very short (in the range of 3 mm or lower).

In this article we describe different techniques for microfluidic rapid prototyping platforms. The proposed techniques are based on ultraviolet (UV) sensitive and temperature curing epoxies, polydimethylsiloxane (PDMS) based tubing, wire-bonding, and anisotropic adhesive films. The assembling procedures presented are developed for both one-time use devices as well as reusable microfluidic systems.

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging ...

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 ...

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 ...

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

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

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

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

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

A 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 ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

Uma plataforma Microfluídico para estimular condrócitos com compressão dinâmica

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...