Damos las gracias a T. Hunt, M. Rosenbluth, y el Laboratorio de Lam por sus consejos y discusiones útiles. Queremos agradecer el apoyo de G. Spinner y el Instituto de Electrónica y Nanotecnología en el Instituto de Tecnología de Georgia. El apoyo financiero para este trabajo fue proporcionado por una subvención del NIH K08-HL093360, premio UCSF REAC, un Centro de Desarrollo de Nanomedicina NIH Premio PN2EY018244, y la financiación del Centro para la Biología de las células endoteliales de la Salud de los Niños de Atlanta.

1. La fabricación de la Microdevice endotelial <ol><li> Crear una fotomáscara mediante la presentación de un equipo de diseño asistido (CAD) de dibujo del dispositivo de microfluidos a un vendedor de la máscara exterior. La máscara utilizada estaba compuesta de una capa de cromo sobre vidrio de sosa y cal. En este caso la anchura del canal de microfluidos fue de 30 micras. </li><li> Limpiar una oblea de silicio al descubierto con piraña (10:1 relación de ácido sulfúrico y peróxido de hidrógeno) durante 15 minutos y sumergir en ácido fluorhídrico durante 30 segundos. Enjuague con agua desionizada (DI) de agua durante aproximadamente 10 segundos. </li><li> Usando una revestidora de giro, giro Microchem SU-8 2025 fotorresistente sobre la oblea a una altura de 30 micras. Por SU-8 2025, una velocidad de centrifugado de 3000 rpm se recomienda. Las otras viscosidades de SU-8 están disponibles, que alcanzará esta altura. Las instrucciones completas para el uso de SU-8 están disponibles en <a href="http://www.microchem.com" target="_blank">www.microchem.com</a> </li><li> Colocar la oblea con SU-8 sobre una placa caliente a 95 ° C durante5 minutos para eliminar el exceso de solvente. Nota: Un horno se seque la oblea diferente a una zona de cocción y no es recomendable. </li><li> Coloque una máscara con la forma característica deseada sobre la oblea, y exponer a la luz UV (160 mJ / cm 2 medido a 365 nm) en un alineador de máscara (Karl Suss, MA-6). Esta cruz une el fotoresist. </li><li> Colocar la oblea de nuevo en una placa caliente a 95 ° C durante 5 minutos adicionales para acelerar aún más la polimerización de SU-8. </li><li> Sumergir la oblea en SU-8 Desarrollador, compuesto principalmente de PGMEA (propileno glicol metil éter acetato) durante 4 minutos para eliminar la no reticulado SU-8. </li><li> Enjuague la oblea de nuevo desarrollo con 100% de alcohol isopropílico (IPA) durante 10 s. La oblea se puede secar con nitrógeno a presión o al permitir que el disolvente se evapore de la oblea en una campana de humos limpia durante varios minutos. </li><li> Cinta de los bordes de la oblea en seco con estampado SU-8 en una placa de Petri para preventilar el movimiento. </li><li> Aplicar 1 ml de Sigmacote a la oblea usando una pipeta, cubrir con la parte superior de la placa de Petri, y la oblea remolino para asegurar el revestimiento completo de la oblea, quitar la tapa, y permitir que la oblea que se seque durante varios minutos hasta que todo el disolvente se ha evaporado. </li></ol> 2. PDMS (polidimetilsiloxano) Preparación <ol><li> Mezcla PDMS polímero y el agente de curado en una proporción 10:1 (w / w) y eliminar las burbujas de aire utilizando un desecador de vacío. La longitud de tiempo necesario para desgasificar el PDMS varía en la fuerza del sistema de vacío disponible, pero normalmente oscila entre varios minutos a una hora. Para un plato de petri seis pulgadas sin previamente vertió PDMS, un volumen total de 60 ml de polímero se recomienda. </li><li> Verter la mezcla sobre la oblea, aproximadamente 5 mm de espesor. También se vierte en un plato de fondo plano para crear una lámina delgada de PDMS, aproximadamente 1 mm de espesor. Curación a 60 ° C en un horno durante la noche. </li><li> Con un cuchillo o un bisturí, cortar alrededor de la cured dispositivo de PDMS y sacarlo de la oblea. Además, cortar una hoja delgada de PDMS ligeramente mayor que el dispositivo. </li><li> Crear orificios de entrada y salida en el dispositivo de PDMS utilizando un perforador 1,0 mm. Esto puede lograrse utilizando un tornillo de banco pasador, dispositivo de Harris Uni-Core, o similar. </li><li> Limpie las superficies del dispositivo y la lámina con cinta adhesiva. </li><li> El uso de un limpiador de plasma, exponer las superficies del dispositivo PDMS y PDMS hoja de plasma de oxígeno durante 30 s. El plasma de oxígeno crea especies reactivas en la superficie expuesta del PDMS que enlace cuando se pone en contacto físico. </li><li> Conectar más pequeño tubo a una pequeña longitud (varios centímetros) de tubo grande, que a su vez está conectado a una jeringa con una aguja roma de punto, lleno de 50 mg / ml de fibronectina de plasma humano en PBS. El tubo y la aguja están dimensionados de tal manera que un ajuste de fricción se crea entre la tubería. Insertar más pequeño tubo en la entrada de PDMS y una microdispositivoplique presión a la jeringa para llenar los canales completamente con la solución de fibronectina y para crear una pequeña gota 100 l en el puerto de salida, para asegurar que los canales de permanecer húmedo. Incubar el dispositivo a 37 ° C durante 40-60 minutos. </li><li> Conecte una jeringa llena con PBS fresco a la aguja de punta roma. Aplicar presión a la jeringa para enjuagar el dispositivo con PBS. </li></ol> 3. Sembrando el dispositivo de microfluidos con las células endoteliales <ol><li> Preparar 1.000.000 células / ml de células humanas endoteliales de vena umbilical (HUVEC) en medios de crecimiento endoteliales con 8% de dextrano. Una gama de 500.000 a 2.000.000 células por mL ha sido utilizado con éxito. La adición de dextrano al medio de carga de células aumenta la viscosidad del fluido, lo que disminuye la velocidad de las células endoteliales al entrar en el sistema de microfluidos fibronectina recubierto. Esto, a su vez, aumenta la probabilidad de que las células se adhiere y la cultura con éxito dentro de la microdispositivo. </li><li> Conecte la nueva jeringa y el tubo como en el paso 2.7. pero con un tubo más largo (aproximadamente 1 metro de longitud). </li><li> Para optimizar el rendimiento de este sistema, es crítico en este punto para evitar cualquier fuga o burbujas en el sistema de perfusión entero; cualquier fuga de la solución o la presencia de burbujas en los medios va a cambiar el flujo y evitar la siembra con éxito de células endoteliales. Por lo tanto, ajustada de la tubería es esencial para eliminar las fugas entre las conexiones. Las burbujas en la jeringa y / o el tubo debe ser eliminada antes de células se introducen en el microdispositivo y el sistema de perfusión completa debe ser cebado con la solución de células antes de conectar el tubo a la microfluidos para evitar la introducción de burbujas de aire. </li><li> Utilizando una bomba de jeringa, infundir suspensión de células en el dispositivo de PDMS en velocidad de flujo volumétrico de 1,23 l / min durante 2 horas a 37 ° C y 5% de CO 2. Los resultados óptimos se logran cuando la bomba de jeringa estaba en el<html> misma altura que el dispositivo de PDMS. Para la entrada, el tubo más largo, que puede ser enrollada dentro de la incubadora, asegura que las células y los medios están adecuadamente calentado antes de entrar en contacto con el dispositivo. En nuestro sistema, la velocidad de flujo volumétrico de 1,23 l / min se aproxima a una velocidad medio de la corriente de ~ 1 mm / s en los más pequeños microcanales, correspondientes a una tensión de cizallamiento de ~ 1 dinas / cm 2 en aquellos canales que utilizan la sangre entera. </li><li> Usando la bomba misma jeringa y el tubo largo, perfunden medio de crecimiento fresco durante 2-8 días a razón de 1,23 l / min. Un dispositivo exitoso tendrá una monocapa de células endoteliales que crecen en el interior del dispositivo dentro de 24-48 horas. Experimentos previos han demostrado que monocapas confluentes adecuadamente expresar VE-cadherina en las uniones célula-célula a través del dispositivo 14. </li><li> Inyectar sangre o suspensión de células en el sistema para la experimentación. </li></ol> 4. Los resultados representativos _content &quot;&gt; El uso de este protocolo, las técnicas estándar de microfabricación litográficas se utilizan para crear el molde necesario para producir los canales de microfluidos que fisiológicamente imitan la sizescale de la microvasculatura (Figura 1A). Utilizando una técnica de perfusión optimizado, las células endoteliales a continuación, las semillas y la cultura confluentemente toda la superficie interna del sistema de microfluidos el plazo de 24-48 horas de la siembra de células (fig. 1B). A medida que el sistema de microfluidos es transparente, el microdispositivo se puede colocar todo en un campo claro / platina del microscopio de fluorescencia para la imagen y la recopilación de datos. Nuestro sistema puede ser aplicado al estudio de enfermedades hematológicas que impliquen alteración de las propiedades biofísicas, como la enfermedad de células falciformes, en la que el aumento de la rigidez de los glóbulos rojos falciformes y la adhesión de leucocitos y células endoteliales aberrante contribuyen a la obstrucción microvascular. Un medicamento clínicamente aprobada, la hidroxiurea, alivia los síntomas, pero su direct efecto sobre el flujo microvascular es desconocido. Nuestro ensayo tiene en cuenta tanto la rigidez y la adhesión celular, y demuestra que la hidroxiurea aminora significativamente el flujo en la enfermedad de células falciformes (Figura 2). Enfermedad de células falciformes es sólo un ejemplo de una aplicación para la microvasculatura-en-un-chip, ya que este sistema es ideal para estudiar cualquier proceso hematológico en el que las células sanguíneas interactuar con cada uno y otras células endoteliales en la microvasculatura. Otras aplicaciones clínicamente relevantes incluyen los trastornos inflamatorios, lesiones sepsis / pulmón, microangiopatías trombóticas, la malaria y la metástasis del cáncer, mientras que las aplicaciones más básicas incluyen la biología de leucocitos y la biología de células madre hematopoyéticas, entre muchos otros. <img src="/files/ftp_upload/3958/3958fig1.jpg" alt="Figura 1"/> Figura 1. A) el primer dispositivo de microfluidos PDMS antes de endotelización. Aquí, el microdispositivo se inyecta ingenioh colorante de alimentos para ilustrar el diseño sizescale y en general del sistema. B) la microscopía de campo claro se muestra el sistema de microfluidos es completamente endotelizado plazo de 48 horas de la siembra de células utilizando el protocolo descrito aquí. <img src="/files/ftp_upload/3958/3958fig2.jpg" alt="Figura 2"/> Figura 2. A) La microvasculatura-en-un-chip con microcanales se aproxima al tamaño de post-capilares vénulas (30 micras), el sitio de la mayoría de los eventos de células falciformes obstructivas microvasculares. La sangre entera de pacientes con anemia drepanocítica que recibieron hidroxiurea y de pacientes que no recibieron hidroxiurea se hace fluir a través de dos diferentes microvasculatura-en-un-chip de los dispositivos. B) Toda la sangre de pacientes con anemia de células falciformes que recibieron los flujos de hidroxiurea con relativa facilidad dentro de los microcanales endotelizado. C) En las condiciones hemodinámicas mismos, toda la sangre de pacientes con anemia drepanocítica que no recibieron hidroxiurea, sin embargo, muestra un flujo mucho más lento con microchannel obstrucción. El canal inferior está completamente obstruido sin flujo y las velocidades de flujo en los microcanales endotelizado otros son significativamente menores que en la condición hidroxiurea. La barra de escala en las tres imágenes es de 30 micras.

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No hay conflictos de interés declarado.

Nuestro sistema microdispositivo endotelizado es el más adecuado cuando se utiliza en conjunción con los experimentos in vivo, y su enfoque reduccionista puede ayudar a aclarar los mecanismos biofísicos de los procesos hematológicos que se observan en los seres humanos y modelos animales. Además, nuestro sistema no está exento de limitaciones. Por ejemplo, nuestros canales de microfluidos son cuadrados en sección transversal. Aunque técnicamente microcanales circulares se pueden fabricar 1...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> aguja de punta roma </td><td> OK International </td><td> 920050-TE </td><td> Precisión TE aguja de calibre 20 x 1/2 &quot;, de color rosa </td></tr><tr><td> dextrano </td><td> Sigma-Aldrich </td><td> 31392 </td><td></td></tr><tr><td> La fibronectina </td><td> Sigma-Aldrich </td><td> F0895 </td><td></td></tr><tr><td> Perforador (tornillo de banco pines) </td><td> Las innovaciones técnicas </td><td></td><td></td></tr><tr><td> Las células endoteliales del cordón umbilical (HUVEC) </td><td> Lonza </td><td> CC-2519 </td><td></td></tr><tr><td> Plasma cleaner </td><td> Plasma </td><td> PDC-326 </td><td></td></tr><tr><td>Polidimetilsiloxano (PDMS) </td><td> Fisher Scientific </td><td> NC9285739 </td><td> Sylgard 184 Elastómero de silicona KIT </td></tr><tr><td> Sigmacote </td><td> Sigma-Aldrich </td><td> SL2 </td><td></td></tr><tr><td> SU-8 2025 </td><td> Microchem </td><td> Y111069 </td><td></td></tr><tr><td> SU-8 para desarrolladores </td><td> Microchem </td><td> Y020100 </td><td></td></tr><tr><td> Jeringa bomba </td><td> Aparato de Harvard </td><td> 70-3008 </td><td> PHD-ULTRA </td></tr><tr><td> tubo (más grande) </td><td> Cole-Parmer Instrument Company </td><td> 06418-02 </td><td> Tygonreg microbore tubo, 0.020 &quot;x 0.060&quot; OD </td></tr><tr><td> tubería (más pequeño) </td><td> Cole-Parmer Instrument Company </td><td> 06417-11 </td><td> PTFE tubo microperforado, 0,012 &quot;x 0,030&quot; de diámetro exterior </td></tr></tbody></table>

endothelialized microfluidics for studying microvascular interactions in hematologic diseases

Los avances en técnicas de microfabricación han permitido la producción de sistemas de microfluidos de bajo costo y reproducible para la realización de experimentos biológicos y bioquímicos en el 1,2 micro y nanoscales. Además, también se han microfluídica se utilizan específicamente para analizar cuantitativamente los procesos hematológicos y microvascular, debido a su capacidad de controlar fácilmente el entorno dinámico fluídico y condiciones biológicas 3-6. Como tal, los investigadores han utilizado más recientemente sistemas de microfluidos para estudiar la deformabilidad de las células sanguíneas, la agregación de células de sangre, el flujo sanguíneo microvascular, y la sangre endoteliales interacciones célula-célula 6-13. Sin embargo, estos sistemas de microfluidos o bien no incluyen células endoteliales cultivadas o eran más grandes que la correspondiente a la sizescale microvasculares procesos patológicos. Una plataforma de microfluidos con cultivos de células endoteliales, que recapitula la precisión celular, físico, y hemodynel medio ambiente amic de la microcirculación que se necesita para avanzar en nuestra comprensión de la fisiopatología subyacente biofísica de enfermedades hematológicas que involucran la microvasculatura. Aquí se presenta un método para crear una &quot;endotelizado&quot; modelo in vitro de la microvasculatura, utilizando un proceso sencillo, solo la máscara de microfabricación en conjunción con las técnicas de cultivo de células endoteliales, para estudiar las interacciones patológicas microvasculares biofísicos que se producen en la enfermedad hematológica. Este &quot;microvasculatura-en-un-chip&quot; proporciona al investigador un sólido ensayo que se controla estrictamente biológica, así como las condiciones biofísicas y se maneja mediante una bomba de jeringa estándar y claro / microscopía de fluorescencia. Parámetros tales como las condiciones hemodinámicas microcirculación, el tipo de células endoteliales, el tipo de células sanguíneas (s) y la concentración (s), droga / concentración inhibitoria etc, todos pueden ser fácilmente controlado. Como tal, nuestro microsistema ofreceun método para investigar cuantitativamente los procesos de enfermedad en la que se altera el flujo microvascular, debido a alteraciones en la adhesión celular, la agregación y la deformabilidad, la capacidad disponible con los ensayos existentes.

Watch this Scientific Journal Video about Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases at JoVE.com

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Un método para la cultura de una monocapa de células endoteliales en toda la superficie interior en 3D de un dispositivo de microfluidos con microvascular del tamaño de los canales (&lt;30 micras) se describe. Este<em&gt; In vitro</em&gt; Modelo de microvasculatura permite el estudio de las interacciones biofísicas entre las células sanguíneas, células endoteliales, y factores solubles en enfermedades hematológicas.

Microfluídica endotelizado para estudiar las interacciones microvasculares en Enfermedades Hematológicas

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

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15.8K vistas.  Emory University School of Medicine. Un método para la cultura de una monocapa de células endoteliales en toda la superficie interior en 3D de un dispositivo de microfluidos con microvascular del tamaño de los canales (

Video: Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

Mezcladores de microfluidos para el estudio plegamiento de proteínas

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Investigación

Bioingeniería

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Determinación de la velocidad de transporte de xenobióticos y Nanomateriales través de la placenta mediante el<em&gt; Ex vivo</em&gt; Modelo perfusión placentaria humana

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

Procedimiento para el Desarrollo de la Multi-profundidad Circular transversal endotelizado microcanales-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

El uso de chips de microfluídica para imágenes en vivo y de estudio de las respuestas de lesiones en<em&gt; Drosophila</em&gt; Las larvas

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

La impresión Sumergido de células sobre una superficie modificada utilizando un flujo continuo Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microentornos-Sandwich como para aprovechar Celulares / Materiales Interacciones

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Multicolor detección por fluorescencia de la gotita de microfluídica el uso de fibras ópticas

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

Aislamiento de murino tejido adiposo derivado de microvascular Fragmentos como Unidades Vascularización para ingeniería de tejidos

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

Microfluídica de entretela con arreglos de microelectrodos para el estudio de la comunicación Neuronal y Axonal señal propagación

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

Organoides neuronales humanos para el estudio del cáncer cerebral y las enfermedades neurodegenerativas

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...