Damos las gracias a T. Shatova útil para el debate sobre el diseño experimental y análisis de datos. La ayuda y la experiencia de G. Paradis, el personal de la citometría de flujo básico en el Instituto Koch, y el personal del Laboratorio de Tecnología de Microsistemas en el Instituto de Tecnología de Massachusetts Se agradecen. Este trabajo fue financiado por los Institutos Nacionales de Salud Subvenciones RC1 EB011187-02, DE013023, DE016516, EB000351, y en parte por el Instituto Nacional del Cáncer Centro del Cáncer de Apoyo (Core) Subvenciones P30-CA14051 y MPP-09Call-Langer-60.

1. Almacenamiento <ol><li> Guarde los depósitos, soportes, juntas tóricas y los dispositivos de microfluidos en etanol al 70%. Use un recipiente (por ejemplo, frasco o vaso de precipitados) que tiene una tapa para evitar la evaporación y la contaminación por partículas de polvo o fuera. Coloque los dispositivos en un contenedor (1), embalses y juntas tóricas en un segundo recipiente (2), y los titulares en el tercer (3). </li></ol> Nota: El uso de etanol al 70% para el almacenamiento es para mantener la esterilidad. Si los únicos componentes de la solución son el etanol y el agua (es decir, sin agentes desnaturalizantes), todos los componentes del sistema deben ser totalmente compatibles y no se degradarán con el tiempo. <ol start="2"><li> Cambie solución de etanol en recipientes (2) y (3) antes de cada uso para evitar la contaminación cruzada a través de experimentos y reducir al mínimo la presencia de partículas no deseadas que pueden causar la obstrucción. </li></ol> 2. Preparación experimental <ol><li> Ponga sus recipientes (2) y (3) en un baño de ultrasonido for 5-10 min antes de cada uso. Esto ayuda a eliminar las partículas contaminantes de los experimentos anteriores. </li><li> Espacio de trabajo limpio en cabinas de seguridad con una solución de etanol al 70%. </li><li> Rocíe todos los materiales (3 contenedores y pinzas) con una solución de etanol al 70% antes de colocarlos en el interior de la cabina de bioseguridad. </li></ol> 3. Montaje <ol><li> Deposite 2-3 toallitas bajo pelusa en el área de trabajo. </li><li> Eliminar depósitos de plástico de su contenedor con unas pinzas y ponerlos en las toallitas para facilitar la evaporación de la solución de etanol a partir de superficies interiores. Golpear suavemente los depósitos en la superficie o soplar el aire a través de ellos para facilitar la eliminación de la solución de etanol. </li><li> Inserte las juntas tóricas en su ranura correspondiente de los depósitos. </li><li> Retire el soporte y las fichas de los contenedores respectivos y permitir que el etanol se evapore (~ 1-2 min). </li><li> Use pinzas para colocar el chip boca arriba deseada (es decir, los agujeros de acceso de seguridad) en el soporte. Levante el soportecon el chip a nivel de los ojos para asegurarse de que el chip está plana en el soporte y ajuste si es necesario con las pinzas. IMPORTANTE: Si el dispositivo no encaja correctamente en su soporte, existe el riesgo de que se rompa durante las etapas posteriores. </li><li> Luego, coloque suavemente los embalses en el soporte y alinearlos con los clips. Tenga cuidado de que las juntas tóricas no se salgan de sus ranuras durante este proceso. </li><li> Presione suavemente hacia abajo en los embalses hasta que encajen en su lugar. Asegúrese de que ambos lados de los embalses sean seguros y que el chip parece estar en la posición correcta. </li></ol> 4. Preparación de células <ol><li> Para células adherentes (líneas primarias o establecidas: células de la placa) 1-2 días antes del experimento de manera que sean no más de 80% de confluencia en el día del experimento. </li><li> Coloque las células en suspensión (en PBS o medio relevante) y aspirar a una concentración de funcionamiento de 1,0 x 10 6 células / ml a 1,0 x 10 7 cells / ml. NOTA: No se han observado cambios significativos en el rendimiento de la entrega debido a la concentración de células. </li><li> Mezclar las células y el material de entrega deseado en un tubo separado para obtener la concentración de material deseado para el experimento. IMPORTANTE: Debido a que el método de entrega descrito se basa en la difusión para facilitar la entrega, una concentración de material de más alto producirá la entrega superior. Si es posible, se recomienda utilizar una solución 1 M de material deseado para los ensayos iniciales. Esta concentración puede entonces ser titulada en futuros experimentos, según sea necesario. La concentración más baja reportada utilizado con este dispositivo es 10 nM 21. </li></ol> 5. Operación <ol><li> Mezcla de pipetas de células y el material de entrega en un depósito (diseño actual tiene una capacidad máxima de 150 l). NOTA: La mayoría de los diseños de dispositivos son totalmente reversibles, por tanto, la dirección del flujo a través de los canales, no importa. Las muestras pueden ser cargados en cualquiera de los dos depósitos. </li&gt; <li> Conecte el tubo de presión para el depósito lleno y apriete la tuerca para garantizar el cierre (apretado el dedo suele ser suficiente). </li><li> Ajustar la presión al nivel deseado en el regulador. Esto controla la velocidad a la que las células viajan a través del dispositivo. Tenga en cuenta que el flujo de células no se inicia hasta que se pulse el botón. </li></ol> NOTA: En el trabajo anterior, nitrógeno o aire comprimido han trabajado igualmente bien como gas portador. <ol start="4"><li> Elevar dispositivo a nivel de los ojos y orientarla de modo que el líquido en el depósito es fácilmente visible. NOTA: Seguimiento de la columna de líquido para apagar el sistema antes de vaciar el depósito. </li><li> Pulse el botón para presurizar el depósito y comenzar el flujo de células. </li><li> Cuando el nivel de líquido es de aproximadamente 2 mm de la parte inferior del depósito, gire rápidamente el regulador a 0 psi para detener el flujo. IMPORTANTE: Si uno no logra detener el flujo antes de vaciar el depósito, se corre el riesgo de expulsar la muestra de la CollectioN depósito. También tenga en cuenta que si la columna de fluido no se está moviendo a un ritmo apreciable, o ha disminuido sustancialmente en relación a su caudal inicial (por ejemplo, 3 veces más lento), el dispositivo montado probablemente obstruido y necesita ser intercambiado. La operación de un dispositivo obstruido puede conducir a una mayor muerte celular. </li><li> Recoger las células tratadas desde el depósito correspondiente y colocarlos en el tubo de recogida / plato deseado. IMPORTANTE: No diluir las células recogidas en cualquier búfer en esta etapa ya que las células porated continuarán con la captación de material para un máximo de 10 min 20. Después de esta ventana ha pasado, diluir las células en los medios de comunicación / tampón deseada. </li><li> Para recoger las células tratadas más o tratar condiciones experimentales alternativos, repita los pasos 5.1 a 5.7, según sea necesario. Recordemos que las fichas son reversibles, por lo tanto, las muestras se pueden montar en cualquiera de los depósitos. Asegúrese de cambiar fichas según sea necesario si se obstruyen. Deseche dispositivos tapados. </li></ol> 6. Desmontaje <ol><li&gt; Desconecte con cuidado los depósitos del titular principal pulsando un lado las armas de clip. </li><li> Coloca cada pieza en el recipiente de almacenamiento adecuado (que se detallan en la sección 1). </li><li> Coloque los chips usados ​​en un contenedor separado para su eliminación. </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>

Los autores Armon Sharei, Robert Langer, y Klavs F. Jensen son accionistas de SQZ Biotecnologías Empresa que produce los dispositivos de microfluidos utilizados en este artículo.

Pueden necesitar ser optimizado en función del tipo de células y material de suministro del sistema se aplica a ciertos aspectos del procedimiento experimental descrito (es decir, factores distintos de diseño de chips y la velocidad de funcionamiento). La discusión que sigue trata sobre algunos de los factores más comunes a tener en cuenta en el diseño de experimentos. Para mejorar la señal de entrega de compuestos marcados con fluorescencia, se necesita para hacer frente a l...

La entrega de macromoléculas a la citoplasma de la célula es un paso crítico en aplicaciones terapéuticas y de investigación. Nanopartículas mediada por la entrega, por ejemplo, ha demostrado su potencial en la terapia génica de 1,2, mientras que la entrega de proteínas es un medio prometedor para afectar la función celular en tanto clínicos y de laboratorio 3 4 ajustes. Otros materiales, tales como fármacos de moléculas pequeñas, puntos cuánticos, o nanopartículas de oro, son de interés en aplicaciones que van desde la terapéutica del cáncer 5,6 a 7,8 etiquetado intracelular, y una sola molécula de seguimiento 9. La membrana celular es en gran medida impermeable a macromoléculas. Muchas de las técnicas existentes utilizan nanopartículas poliméricas, liposomas 10,11 12, o modificaciones químicas 13 para facilitar la ruptura de la membrana o la entrega de endocitosis. En estos métodos, la eficiencia en la entrega y la viabilidad celular son a menudo depende de la estructura de the molécula diana y el tipo de célula. Estos métodos pueden ser eficaces en la entrega de los materiales estructuralmente uniformes, tales como ácidos nucleicos, pero a menudo son poco adecuado para la entrega de más estructuralmente diversos materiales, tales como las proteínas y los nanomateriales 14,15 7. Por otra parte, el mecanismo de interrupción endosoma que la mayoría de estos métodos se basan en es a menudo ineficaz, por lo tanto, dejando mucho material atrapado en estructuras vesiculares 16. Por último, los métodos que a menudo se desarrollan para su uso con las líneas celulares establecidas no se traducen bien a las células primarias. Métodos poración de membrana, tales como la electroporación 17,18 y sonoporación 19, son una alternativa atractiva en algunas aplicaciones, sin embargo, se sabe que causan la viabilidad celular baja y puede ser limitado por la carga del material de entrega esperados. Deformación mecánica rápida de las células, un enfoque de microfluidos para la entrega, tiene recientemente demonstrATED sus ventajas sobre las técnicas actuales en el contexto de la reprogramación celular 20 y la entrega nanomaterial 21. Este método se basa en la rotura mecánica o la membrana celular para facilitar la entrega citosólica de los materiales presentes en el tampón circundante. El sistema ha demostrado que permite el potencial para desafiar previamente tipos de células (por ejemplo, células inmunes primarias y las células madre) y materiales (por ejemplo, anticuerpos y nanotubos de carbono). En este documento, se describe el procedimiento general para utilizar estos dispositivos para la entrega intracelular de macromoléculas diana. El procedimiento es generalizable a la mayoría de tipos de células y materiales de entrega, sin embargo, se recomienda que uno Realizar una pequeña optimización de las condiciones, como se detalla en nuestras pautas de diseño de informes anteriores 20, para cualquier aplicación que no reportados previamente. Hasta la fecha, el sistema ha sido utilizado con éxito para la entrega de ARN, ADN, las nanopartículas de oro, puntos cuánticos, nanotubos de carbono, proteínass, y 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

Deformación mecánica rápida de las células se ha convertido en un método prometedor, libre de vectores para el suministro intracelular de macromoléculas y los nanomateriales. Esta tecnología ha demostrado su potencial en el tratamiento de las solicitudes previamente difíciles, como son, la entrega de las células primarias inmunes, la reprogramación celular, nanotubos de carbono, y la entrega de puntos cuánticos. Esta plataforma de microfluidos libre de vectores se basa en la rotura mecánica de la membrana celular para facilitar la entrega citosólica del material objetivo. En este documento, se describe el método detallado de uso de estos dispositivos de microfluidos, incluyendo, conjunto de dispositivo, preparación de células, y la operación del sistema. Este enfoque requiere la entrega de un breve optimización del tipo de dispositivo y condiciones de funcionamiento de las aplicaciones antes publicados. Las instrucciones que se proporcionan son generalizables a la mayoría de tipos de células y materiales de entrega ya que este sistema no requiere tampones especializados o pasos modificación química / de conjugación. Este trabajo también proporcionans de recomendaciones sobre cómo mejorar el rendimiento del dispositivo y localizar averías problemas potenciales relacionados con la obstrucción, la eficiencia de entrega de baja, y la viabilidad celular.

La figura 1 contiene un esquema descriptivo del sistema de suministro de microfluidos. Figuras 2a-b ilustran resultados típicos de tratamiento de las células HeLa con diferentes diseños de dispositivos en la presencia de fluorescencia conjugado 3 kDa de dextrano 20. Si el procedimiento se sigue correctamente, el rendimiento del sistema será sensible al tipo de dispositivo y la velocidad de operación. Por lo tanto, se debe optimizar estas condiciones para una aplicación ...

Watch this Scientific Journal Video about Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform at JoVE.com

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Deformación mecánica rápida de las células se ha convertido en un método prometedor, libre de vectores para el suministro intracelular de macromoléculas y los nanomateriales. Este protocolo proporciona instrucciones detalladas sobre cómo utilizar el sistema para una amplia gama de aplicaciones.

Exprimir la célula como un robusto, Microfluidic intracelular Delivery Platform

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 vistas.  Massachusetts Institute of Technology.  Deformación mecánica rápida de las células se ha convertido en un método prometedor, libre de vectores para el suministro intracelular de macromoléculas y los nanomateriales. Este protocolo proporciona instrucciones detalladas sobre cómo utilizar el sistema para una amplia gama de aplicaciones. 

Video: 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 una plataforma 3D para la propagación y diferenciación de las células estaminales embrionarias humanas (CMEH) para los diferentes linajes

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

Investigación

Bioingeniería

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.

Proteínas de alto rendimiento del generador de expresiones utilizando una plataforma de microfluidos

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.

El chip de varios órganos - Una plataforma de microfluidos para el largo plazo co-cultivo de múltiples tejidos

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.

Una plataforma microfluídica para el aislamiento de gran procesamiento de celda única y 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 de microfluidos con multiplexado de detección electrónica para el seguimiento espacial de las partículas

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

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

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

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

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

La morfología del pelo de la raíz de plántulas de Arabidopsis en una plataforma de microfluídica de dos capas de imagen

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

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

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

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

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

Una plataforma de microfluidos para la proyección de imagen Longitudinal en 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.

Una plataforma de ultra rendimiento microfluidos para la secuenciación del genoma de unicelular

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.

Una plataforma microfluídica multicapa para la conducción de la expresión génica sin 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.

Una plataforma microfluídica para estimular los condrocitos con compresión dinámica

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