Agradecemos T. Shatova para discussão útil em delineamento experimental e análise de dados. A assistência e competências de G. Paradis, o pessoal da citometria de fluxo do núcleo do Instituto Koch, e os funcionários do Laboratório de Tecnologia Microsystems no Instituto de Tecnologia de Massachusetts estão reconhecido agradecimento. Este trabalho foi financiado pelo National Institutes of Grants Saúde RC1 EB011187-02, DE013023, DE016516, EB000351, e parcialmente pelo Instituto Nacional do Câncer Cancer Center Support (Core) Concede-P30 CA14051 e MPP-09Call-Langer-60.

1. Armazenamento <ol><li> Armazene os reservatórios, suportes, anéis de vedação e dispositivos microfluídicos em etanol 70%. Use um recipiente (por exemplo, jarra ou copo) que tem uma tampa para evitar a evaporação e contaminação por poeira ou partículas de fora. Colocar os dispositivos em um recipiente (1), reservatórios e os O-rings em um segundo recipiente (2), e os detentores na terceira (3). </li></ol> Nota: O uso do etanol de 70% para o armazenamento é manter a esterilidade. Se os únicos componentes da solução são o etanol e água (ou seja, sem agentes de desnaturação), todos os componentes do sistema deve ser totalmente compatível e não se degrada com o tempo. <ol start="2"><li> Alterar solução de etanol em recipientes (2) e (3) antes de cada utilização, para evitar a contaminação cruzada através de experiências e minimizar a presença de partículas indesejáveis ​​que podem causar o entupimento. </li></ol> 2. Experiência Preparação <ol><li> Coloque os recipientes (2) e (3) em um banho de ultra-som for 5-10 min, antes de cada utilização. Isso ajuda a remover as partículas contaminantes a partir de experiências anteriores. </li><li> Limpe espaço de trabalho na cabine de segurança biológica com solução de etanol a 70%. </li><li> Spray de todos os materiais (3 recipientes, e pinças) com uma solução de álcool 70% antes de colocá-los dentro da cabine de segurança biológica. </li></ol> 3. Montagem <ol><li> Pouse 2-3 toalhetes baixo fiapos na área de trabalho. </li><li> Remover reservatórios de plástico a partir de seu recipiente com uma pinça e colocá-las sobre os lenços para facilitar a evaporação da solução de etanol a partir de superfícies internas. Bater levemente os reservatórios sobre a superfície ou sopro de ar através delas a fim de facilitar a remoção da solução de etanol. </li><li> Insira o-rings em seu slot apropriado dos reservatórios. </li><li> Retire o suporte e fichas respectivos recipientes e permitir que o etanol evapore (~ 1-2 min). </li><li> Use uma pinça para colocar o chip virado para cima desejado (ou seja, furos de acesso para cima) no suporte. Levante a titularcom o chip para eyelevel para garantir que o chip está deitado no suporte e ajustar, se necessário, com a pinça. IMPORTANTE: Se o dispositivo não encaixar corretamente em seu suporte, há um risco de que ele vai quebrar durante as etapas subseqüentes. </li><li> Em seguida, coloque delicadamente os reservatórios no suporte e alinhá-las com os clipes. Tenha cuidado para que o-rings não caem de seus slots durante este processo. </li><li> Pressione gentilmente os reservatórios até que eles se encaixem no lugar. Assegure-se que ambos os lados dos reservatórios são seguras e que o chip parece estar na posição correcta. </li></ol> 4. Preparação celular <ol><li> Para as células aderentes (linhas primárias ou estabelecidas): células da placa de 1-2 dias antes da experiência de modo a que eles não são confluentes mais do que 80% no dia da experiência. </li><li> Coloque as células em suspensão (em PBS ou mídia relevante) e apontar para uma concentração operacional de 1,0 x 10 6 células / ml a 1,0 x 10 7 cells / ml. NOTA: Nós não observamos mudanças significativas no desempenho de entrega, devido à concentração de células. </li><li> Misturar as células e o material de entrega desejado num tubo separado para obter a concentração de material desejado para a experiência. IMPORTANTE: Como o método de entrega descrito depende de difusão para facilitar a entrega, uma concentração maior de material trará entrega superior. Se possível, é recomendado o uso de uma solução 1 mM de o material desejado para ensaios iniciais. Esta concentração pode ser, em seguida, titulada para baixo em futuras experiências, conforme necessário. A concentração mais baixa relatado utilizado com este dispositivo é de 10 nM 21. </li></ol> 5. Operação <ol><li> Pipeta mistura de células e material de entrega em um reservatório (projeto atual tem um máximo de capacidade de 150 mL). NOTA: A maioria dos projetos de dispositivos são totalmente reversíveis, portanto, sentido do fluxo através dos canais não importa. As amostras podem ser carregados em qualquer um dos dois reservatórios. </li&gt; <li> Prenda a tubulação de pressão para o reservatório cheio e apertar a porca para garantir vedação adequada (dedo apertado é muitas vezes suficiente). </li><li> Regule a pressão para o nível desejado no regulador. Este controla a velocidade à qual as células de viajar através do dispositivo. Note-se que o fluxo de células não começar até que o botão é pressionado. </li></ol> NOTA: Num trabalho prévio, o azoto ou o ar comprimido pode trabalhar igualmente bem como o gás de transporte. <ol start="4"><li> Elevar dispositivo ao nível dos olhos e orientá-la de modo a que o líquido no reservatório é facilmente visível. NOTA: Acompanhe a coluna de líquido para desligar o sistema antes de o reservatório é esvaziado. </li><li> Pressione o botão para pressurizar o reservatório e iniciar o fluxo de celular. </li><li> Quando o nível de líquido é de cerca de 2 mm a partir do fundo do reservatório, desligar rapidamente o regulador de 0 psi até parar o fluxo. IMPORTANTE: Se um falhar para interromper o fluxo antes que o reservatório é esvaziado, se corre o risco de ejetar a amostra do collection reservatório. Observe também que, se a coluna de fluido não está se movendo em um ritmo apreciável, ou diminuiu substancialmente em relação ao seu caudal inicial (por exemplo, 3x mais lento), o dispositivo montado é provavelmente entupida e precisa ser trocado. A operação de um dispositivo obstruído pode levar à morte da célula superior. </li><li> Recolha as células tratadas a partir do reservatório apropriado e coloque-os no tubo de coleta / placa desejada. IMPORTANTE: Não diluir as células coletadas em todos os buffers, nesta fase, as células rado continuará a captação de material para até 10 min 20. Após esta janela já passou, diluir as células na mídia / tampão desejado. </li><li> Para coletar mais células tratadas ou tente condições experimentais alternativos, repita os passos de 5,1-5,7, conforme necessário. Recorde-se que as batatas fritas são reversíveis, por conseguinte, as amostras podem ser montados em qualquer reservatório. Certifique-se de trocar chips como necessário se entupir. Descarte dispositivos entupidos. </li></ol> 6. Desmontagem <ol><li&gt; Desligar suavemente os reservatórios do titular principal, empurrando de lado as armas de clipe. </li><li> Coloque cada parte no recipiente apropriado (detalhado na seção 1). </li><li> Coloque chips usados ​​em um recipiente separado para a eliminação. </li></ol>

<ol>
	<li>Schaffert, D., Wagner, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+progress+and+prospects:+synthetic+polymer-based+systems.">Gene therapy progress and prospects: synthetic polymer-based systems.</a> Gene. Ther. 15, 1131-1138 (2008).</li><li>Whitehead, K. A., Langer, R., Anderson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knocking+down+barriers:+advances+in+siRNA+delivery.">Knocking down barriers: advances in siRNA delivery.</a> Nat. Rev. Drug Discov. 8, 129-138 (2009).</li><li>Leader, B., Baca, Q. J., Golan, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+therapeutics:+a+summary+and+pharmacological+classification.">Protein therapeutics: a summary and pharmacological classification.</a> Nat. Rev. Drug Discov. 7, 21-39 (2008).</li><li>Kim, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Human+Induced+Pluripotent+Stem+Cells+by+Direct+Delivery+of+Reprogramming+Proteins.">Generation of Human Induced Pluripotent Stem Cells by Direct Delivery of Reprogramming Proteins.</a> Cell Stem Cell. 4, 472-476 (2009).</li><li>Dhar, S., Daniel, W. L., Giljohann, D. A., Mirkin, C. A., Lippard, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyvalent+Oligonucleotide+Gold+Nanoparticle+Conjugates+as+Delivery+Vehicles+for+Platinum(IV)+Warheads.">Polyvalent Oligonucleotide Gold Nanoparticle Conjugates as Delivery Vehicles for Platinum(IV) Warheads.</a> J. Am. Chem. Soc. 131, 14652-14653 (2009).</li><li>Jiang, Z. -. X., Zhang, Z. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+PTPs+with+small+molecule+inhibitors+in+cancer+treatment.">Targeting PTPs with small molecule inhibitors in cancer treatment.</a> Cancer and Metastasis Reviews. 27, 263-272 (2008).</li><li>Derfus, A. M., Chan, W. C. W., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Delivery+of+Quantum+Dots+for+Live+Cell+Labeling+and+Organelle+Tracking.">Intracellular Delivery of Quantum Dots for Live Cell Labeling and Organelle Tracking.</a> Adv. Mater. 16, 961-966 (2004).</li><li>Michalet, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantum+Dots+for+Live+Cells+in+Vivo+Imaging,+and+Diagnostics.">Quantum Dots for Live Cells in Vivo Imaging, and Diagnostics.</a> Science. 307, 538-544 (2005).</li><li>Dahan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+Dynamics+of+Glycine+Receptors+Revealed+by+Single-Quantum+Dot+Tracking.">Diffusion Dynamics of Glycine Receptors Revealed by Single-Quantum Dot Tracking.</a> Science. 302, 442-445 (2003).</li><li>Slowing, I. I., Trewyn, B. G., Lin, V. S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesoporous+Silica+Nanoparticles+for+Intracellular+Delivery+of+Membrane-Impermeable+Proteins.">Mesoporous Silica Nanoparticles for Intracellular Delivery of Membrane-Impermeable Proteins.</a> J. Am. Chem. Soc. 129, 8845-8849 (2007).</li><li>Pack, D. W., Hoffman, A. S., Pun, S., Stayton, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+development+of+polymers+for+gene+delivery.">Design and development of polymers for gene delivery.</a> Nat. Rev. Drug Discov. 4, 581-593 (2005).</li><li>Joseph, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+lipids+used+in+gene+transfer.">Cationic lipids used in gene transfer.</a> Advanced Drug Delivery Reviews. 27 (97), 17-28 (1997).</li><li>Verma, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface-structure-regulated+cell-membrane+penetration+by+monolayer-protected+nanoparticles.">Surface-structure-regulated cell-membrane penetration by monolayer-protected nanoparticles.</a> Nat. Mater. 7, 588-595 (2008).</li><li>Yan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+intracellular+protein+delivery+platform+based+on+single-protein+nanocapsules.">A novel intracellular protein delivery platform based on single-protein nanocapsules.</a> Nat. Nano. 5, 48-53 (2010).</li><li>Shi Kam, N. W., Jessop, T. C., Wender, P. A., Dai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotube+Molecular+Transporters:+Internalization+of+Carbon+Nanotube-Protein+Conjugates+into+Mammalian+Cells.">Nanotube Molecular Transporters: Internalization of Carbon Nanotube-Protein Conjugates into Mammalian Cells.</a> J. Am. Chem. Soc. 126, 6850-6851 (2004).</li><li>Varkouhi, A. K., Scholte, M., Storm, G., Haisma, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endosomal+escape+pathways+for+delivery+of+biologicals.">Endosomal escape pathways for delivery of biologicals.</a> Journal of Controlled Release. 151, 220-228 (2011).</li><li>li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+Gene+Therapy:+New+Developments+In+Vivo+and+In+Vitro.">Electroporation Gene Therapy: New Developments In Vivo and In Vitro.</a> Current Gene Therapy. 4, 309-316 (2004).</li><li>Fox, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+cells+in+microfluidic+devices:+a+review.">Electroporation of cells in microfluidic devices: a review.</a> Anal. Bioanal. Chem. 385, 474-485 (2006).</li><li>Miller, D. L., Pislaru, S. V., Greenleaf, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonoporation:+Mechanical+DNA+Delivery+by+Ultrasonic+Cavitation.">Sonoporation: Mechanical DNA Delivery by Ultrasonic Cavitation.</a> Somatic Cell and Molecular Genetics. 27, 115-134 (2002).</li><li>Sharei, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+vector-free+microfluidic+platform+for+intracellular+delivery.">A vector-free microfluidic platform for intracellular delivery.</a> Proc. Natl. Acad. Sci. U.S.A. 110, 2082-2087 (2013).</li><li>Lee, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonendocytic+Delivery+of+Functional+Engineered+Nanoparticles+into+the+Cytoplasm+of+Live+Cells+Using+a+Novel,+High-Throughput+Microfluidic+Device.">Nonendocytic Delivery of Functional Engineered Nanoparticles into the Cytoplasm of Live Cells Using a Novel, High-Throughput Microfluidic Device.</a> Nano Lett. 12, 6322-6327 (2012).</li></ol>

Os autores Armon Sharei, Robert Langer e Klavs F. Jensen são acionistas da SQZ Biotechnologies empresa que produz os dispositivos microfluídicos usados ​​neste artigo.

Certos aspectos do procedimento experimental descrito (isto é, com excepção de design de chips e a velocidade de funcionamento factores) pode necessitar de ser optimizada, dependendo do tipo de célula e material de entrega que o sistema é aplicado. A discussão que se segue aborda alguns dos fatores mais comuns a considerar ao projetar experimentos. Para melhorar o sinal de entrega para compostos marcados com fluorescência, é preciso abordar as fontes de fluorescência de fun...

A entrega de macromoléculas para o citoplasma da célula é um passo crítico para aplicações terapêuticas e de pesquisa. Nanopartículas de entrega mediada, por exemplo, revelou potencial na terapia de genes de 1,2, enquanto que a entrega de proteína é uma forma promissora de afectar a função celular, tanto clínicas e laboratoriais 3 4 configurações. Outros materiais, tais como medicamentos de pequenas moléculas, pontos quânticos, ou nanopartículas de ouro, são de interesse em aplicações que vão desde o tratamento do câncer de 5,6 para 7,8 rotulagem intracelular, e única molécula de rastreamento 9. A membrana da célula é largamente impermeável a macromoléculas. Muitas técnicas existentes usar nanopartículas poliméricas 10,11, lipossomas 12 ou 13 modificações químicas para facilitar a ruptura da membrana ou a entrega endocítica. Nestes métodos, a eficiência de entrega e a viabilidade celular são muitas vezes dependentes da estrutura do the molécula-alvo e do tipo de célula. Estes métodos podem ser eficientes na entrega de materiais estruturalmente uniformes, tais como os ácidos nucleicos, mas são muitas vezes mal adaptados para a entrega de mais estruturalmente diversos materiais, tais como proteínas e os nanomateriais 14,15 7. Além disso, o mecanismo de interrupção do endossoma que a maioria destes métodos baseiam-se é muitas vezes ineficaz, deixando, portanto, muito material aprisionado em estruturas vesiculares 16. Finalmente, os métodos que são muitas vezes desenvolvidos para utilização com as linhas de células estabelecidas, não se traduz bem a células primárias. Métodos Membrane poração, como eletroporação 17,18 e sonoporação 19, são uma alternativa atraente em algumas aplicações, no entanto, eles são conhecidos por causar a viabilidade celular de baixo e pode ser limitada pela carga do material de entrega alvo. Deformação mecânica rápida de células, uma abordagem microfluídicos para entrega, tem recentemente demonstrated suas vantagens em relação às técnicas actuais no contexto de reprogramação das células 20 e 21 de fornecimento de nanomateriais. Este método baseia-se o desmembramento mecânico da membrana celular para facilitar a entrega citosólica de materiais presentes no tampão circundante. O sistema demonstrou permitindo potencial anteriormente desafiando tipos de células (por exemplo, células imunes primárias e as células-tronco) e materiais (por exemplo, anticorpos e os nanotubos de carbono). Aqui, o processo geral para utilizar estes dispositivos para a entrega intracelular de macromoléculas alvo é descrita. O procedimento é generalizável para a maioria dos tipos de células e materiais de entrega, no entanto, recomenda-se que uma realização de uma breve otimização das condições, conforme detalhado em nossas diretrizes previamente relatados projeto 20, para todas as aplicações anteriormente não declaradas. Até à data, o sistema tem sido utilizado com sucesso para a entrega de RNA, DNA, as nanopartículas de ouro, quantum dots, nanotubos de carbono, proteínas, e polímeros de dextrano 20,21.

<table border="0" cellpadding="0" cellspacing="0" width="777">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:375px;">Device Holder &#38; Plastic reservoir</td>
			<td style="width:136px;">SQZ Biotechnologies</td>
			<td style="width:149px;">Holder</td>
			<td style="width:117px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LSRFortessa Analyzer</td>
			<td>Becton Dickinson</td>
			<td>N/A</td>
			<td>Flow cytometry machine used at the Koch Institute Core Facilities</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microfluidic device</td>
			<td>SQZ Biotechnologies</td>
			<td>Cell Squeeze</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">O-Rings</td>
			<td>McMaster</td>
			<td>9452K311</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pressure system to operate device</td>
			<td>SQZ Biotechnologies</td>
			<td>Pressure System</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrasound bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

cell squeezing as a robust, microfluidic intracellular delivery platform

Deformação mecânica rápida de células surgiu como um método promissor, livre do vector para a entrega intracelular de macromoléculas e nanomateriais. Esta tecnologia tem mostrado potencial para enfrentar aplicações previamente desafiantes, incluindo, a entrega de pilhas imunes, reprogramação celular, nanotubos de carbono, e entrega ponto quântico. Esta plataforma de microfluidos livre do vector depende de ruptura mecânica da membrana celular para facilitar a entrega citosólica do material alvo. Relata-se o método detalhado de uso para esses dispositivos microfluídicos, incluindo, a montagem do dispositivo, a preparação celular, e operação do sistema. Esta abordagem requer a entrega de uma breve otimização do tipo de dispositivo e as condições de operação para aplicações previamente não-relatados. As instruções fornecidas são generalizáveis ​​para a maioria dos tipos de células e materiais de entrega, este sistema não requer buffers especializados ou etapas modificação química / conjugação. Este trabalho também fornecers recomendações sobre como melhorar o desempenho do dispositivo e de problemas disparar potenciais problemas relacionados com a obstrução, a eficiência de entrega baixos, ea viabilidade celular.

A Figura 1 apresenta um esquema descritivo de o sistema de entrega de microfluidos. Figuras 2a-b ilustram os resultados típicos de tratamento de células HeLa com diferentes modelos de dispositivos, na presença de fluorescência 3 conjugado dextrano 20 kDa. Se o procedimento for seguido corretamente, o desempenho do sistema será sensível ao tipo de dispositivo e velocidade de operação. Portanto, deve-se otimizar essas condições para uma determinada aplicação antes d...

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Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Deformação mecânica rápida de células surgiu como um método promissor, livre do vector para a entrega intracelular de macromoléculas e nanomateriais. Este protocolo fornece etapas detalhadas sobre como usar o sistema para uma ampla gama de aplicações.

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 ...

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12.7K visualizações.  Massachusetts Institute of Technology.  Deformação mecânica rápida de células surgiu como um método promissor, livre do vector para a entrega intracelular de macromoléculas e nanomateriais. Este protocolo fornece etapas detalhadas sobre como usar o sistema para uma ampla gama de aplicações. 

Vídeo: Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Microcápsulas de alginato como uma plataforma 3D para Propagação e Diferenciação de Células-Tronco Embrionárias (hESC) para diferentes linhagens

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Bioengenharia

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 ...

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 ...

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

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

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

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

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

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 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 ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Recombinante colágeno peptídeo Microcarriers para expansão da célula e seu potencial uso como sistema de entrega de células em um bioreator modelo

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

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

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

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

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

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

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 ...