Esta investigación fue apoyada por el programa de investigación de ciencia básica a través de la nacional investigación Fundación de Corea (NRF) financiado por el Ministerio de ciencia, TIC y planeación de futuro (NRF-2014R1A1A2057527 y NRF-2016R1D1A1B03934418).

1. elaboración de matriz de imán y placa de aluminio por-agujero matriz
<ol><li>Preparar dos de 50 x 50 mm (o mayor) placas de aluminio. El espesor de cada placa fue 300 μm que es la mitad del diámetro del grano.</li>
	<li>Forman una matriz de 30 x 30 a través del agujero en una de las placas de aluminio utilizando un CNC grabador rotatorio con una Φ550-μm micro broca de 30 mm/s de tasa de penetración y 8000 RPM de velocidad del huso. La distancia entre cada agujero (de centro a centro) fue de 1 mm (Figura 1a y Figura 2a, ).</li>
	<li>Formar una matriz de 30 x 30 de Φ750 μm a través de agujeros en la otra placa de aluminio, utilizando el mismo procedimiento que se describe en 1.2 (Figura 1a y Figura 2a, ii).</li>
	<li>Coloque las dos placas entre sí utilizando una cinta adhesiva y formar los agujeros de alineación de Φ3 mm en cada una de las cuatro esquinas de dos placas de aluminio.</li>
	<li>Remojar las placas de aluminio en ácido sulfúrico de 15% durante 12 h para limpiar sus superficies. Ya que la fina capa de óxido de aluminio sobre la superficie del aluminio que sea resistente a la corrosión, el diámetro de agujero y el grosor de la placa no cambia este tratamiento con ácido.</li>
	<li>Forman una matriz de 30 × 30 de 1 x 1 x 1 mm neodimio imanes (con una fuerza magnética de 0.363 N). Asegúrese de que cada imán es la polaridad opuesta a su vecino. Para evitar la ruptura o la dispersión de la matriz de imán, fije una placa de aluminio de 30 x 30 mm en la parte inferior de la matriz de imán usando cinta de doble cara (Figura 2a, la iii y la inserción en figura 2).</li>
</ol>2. proceso de captura de grano
<ol><li>Alinear y apilar las dos placas de aluminio (top placa: placa de orificio de 750 μm, placa inferior: placa de orificio de 550 μm) usando los orificios de alineación dispuestos en las cuatro esquinas de cada placa (Figura 1b).</li>
	<li>Traben las dos placas mediante la inserción de los tornillos M3 en los agujeros de alineación y luego fije los tornillos con tuercas (Figura 1b).</li>
	<li>Coloque el ensamblaje de placa de aluminio en el arreglo de imán preparado (Figura 1b, 2by 2C). Alinee el conjunto de imanes y la matriz de agujeros en la placa de aluminio durante el proceso de apilado. Utilizar una cinta adhesiva para fijar la posición de la matriz de imán.</li>
	<li>Lugar un número suficiente de acero de Φ600 mm. SUJ2 granos en el conjunto de placa y manipular los granos con un acrílico (o no metálico) de la placa tal que un grano queda atrapado en cada orificio (figura 1C, 1Dy 1e) mientras que al mismo tiempo quitar el exceso granos que no han presentado en los agujeros.</li>
	<li>Retire con cuidado la placa superior para evitar la dispersión no deseada y la dislocación de los granos atrapados (figura 1f).</li>
</ol>3. cóncavo micropocillos fabricación
<ol><li>Mover el molde cóncavo micropocillos, producido en pasos 2.1 a 2.5, arriba, a una placa Petri.</li>
	<li>La mezcla de monómero de polidimetilsiloxano (PDMS) y el agente endurecedor según las instrucciones de los fabricantes19 con un monómero PDMS: cociente de 10:1 del agente de curado.</li>
	<li>Gas de la mezcla PDMS utilizando un desecador y una bomba de vacío para eliminar las posibles burbujas en la mezcla PDMS.</li>
	<li>Vierta la mezcla PDMS en el molde cóncavo micropocillos y gas de nuevo utilizando el mismo procedimiento que se describe en 3.3 (figura 1f).</li>
	<li>Cueza al horno la mezcla PDMS en la placa caliente a 80 ° C por 2 h para formar un sustrato PDMS integrado de grano (figura 1 g).</li>
	<li>Quite el sustrato PDMS curado del molde (figura 1 g). En el proceso de extracción, pulverización metanol con botella de lavado para separar el sustrato PDMS del molde.</li>
	<li>Con un Φ15 x imán de 2 mm, retire los granos de acero atrapados el sustrato PDMS (figura 1 h). Para este proceso, puede utilizarse cualquier imán que es suficientemente fuerte para extraer los granos del substrato de PDMS.</li>
</ol>4. cultura de esferoide
<ol><li>Corte cóncavo sustrato PDMS micropocillos-patrón utilizando un punzón de biopsia Φ14 mm para montarse en placa de 24 pozos en este estudio.</li>
	<li>Esterilizar el sustrato resultante de PDMS de mm Φ14 en un esterilizador de la autoclave a 121 ° C y 15 psi.</li>
	<li>Coloque el sustrato esterilizado de PDMS en una placa bien 24.</li>
	<li>La capa del sustrato PDMS todo con pluronic F-127 solución al 4% (w/v) durante la noche para evitar la fijación de células a la superficie de los micropocillos. Durante el proceso de capa, eliminar posibles burbujas de aire atrapadas en los micropocillos cóncavos mediante pipeteo o utilizando un limpiador ultrasónico.</li>
	<li>Descargue la solución de F-127 tres veces mediante el uso de solución salina tamponada con fosfato (PBS).</li>
	<li>Sembrar 1 mL de solución de medio celular (medio de Eagle modificado de Dulbecco) (que contiene 2 x 106 células) en el sustrato PDMS. Tenga en cuenta que la densidad de siembra puede cambiar según el tamaño del esferoide de destino o tipo de celda de destino. Aquí, se utilizaron células madre procedentes de adiposo (ASC).</li>
	<li>Aspirar 1 mL del medio usando una pipeta de 1000 μL para quitar cualquier exceso células que no fueron atrapadas en los pocillos (figura 3).</li>
	<li>Incubar las células en 36,5 ° C, humedad de > 95% y 5% CO2 condición. En el caso de la ASC en nuestro estudio, las células se agregan a un esferoide en 48 h.</li>
</ol>

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Los autores no tienen conflictos de interés divulgar.

El reto principal de este método de fabricación era la segura fijación de los granos de la matriz a través del agujero en la placa de aluminio. Para resolver este desafío, la fuerza magnética en la forma de una matriz de 30 x 30 imán fue utilizada para fijar los granos con seguridad, como se muestra en las figuras 6 y 7. La densidad de flujo magnético de la matriz de imán, que tiene la polaridad opuesta, es más fuerte en el centro de cada superficie del imán. Porque la fuerza ...

Las células cultivadas en forma de esferoide son más similares a real tejido en el cuerpo de un plano bidimensional de la cultura1. Teniendo en cuenta esta ventaja, el uso de esferoides se ha adoptado para mejorar el estudio de la interacción de célula a célula2,3, activación inmune4,5y6de la diferenciación de la detección de drogas. Además, esferoides con múltiples tipos de células se han aplicado recientemente a organoides (cerca de-fisiológico tridimensional (3D) el tejido), que son muy útiles para estudiar el desarrollo y la enfermedad humana7. Varios métodos se han utilizado para producir esferoides. El método más simple consiste en la utilización de una superficie no adhesiva, que las células se agregan entre sí y esferoides de forma. Un plato de Petri pueden tratarse con albúmina de suero bovino, pluronic F-127 o un polímero hidrofóbico (por ejemplo metacrilato de 2-hydroxyethl de poly) para hacer su superficie no adhesiva89. El método de frasco de spinner es otra forma bien conocida de producir grandes cantidades de esferoides10,11. En este método, las células se llevan a cabo en suspensión agitando para evitar de ser unido al sustrato. Por el contrario, la flotación células agregado esferoides de forma. El método superficie no adhesiva y el método de frasco de spinner pueden producir grandes cantidades de esferoides. Sin embargo, están sujetas a limitaciones incluyendo dificultades en controlar el tamaño del esferoide, así como el seguimiento y monitoreo de cada esferoide. Como un remedio para este tipo de problemas, otro método de producción del esferoide, a saber, el colgante gota método pueden ser empleados12. Se trata de depositar las gotas de la suspensión de células en la parte inferior de la tapa de una placa de cultivo. Estas gotas son generalmente 15 a 30 μL en tamaño y contienen aproximadamente de 300 a 3000 células13. Cuando se invierte la tapa, las gotas se llevan a cabo en lugar por la tensión superficial. El ambiente de microgravedad en cada gota concentra en las células, que entonces forman esferoides solo en la interfase líquido aire libre. Los beneficios de colgar el método de caída son que ofrece una distribución de tamaño bien controlados, si bien es fácil de rastrear y monitorear cada esferoide, en relación con los métodos de matraz superficie y spinner no adhesiva. Sin embargo, este método tiene una desventaja en que la producción masiva de esferoides y el propio proceso de producción es excesivamente trabajo intensivo.
Un conjunto de micropocillo es un plano de la placa con muchos pozos de tamaño micro, cada uno con un diámetro que van desde 100 hasta 1000 μm. El principio de producción del esferoide cuando utilice micropocillos es similar a la del método de superficie no adhesiva. Los beneficios incluyen el hecho de que los micropocillos proporcionan espacios entre los micropocillos para separar las células o esferoides, que es fácil controlar el tamaño del esferoide, mientras que también hace fácil supervisar cada esferoide sola. Con un gran número de los micropocillos, producción de esferoide alto rendimiento también es posible. Otra ventaja de los micropocillos es la opción a los pozos de la forma de diferentes formas (hexaedros, cilíndricos, trigonal prismático) dependiendo de propósitos experimentales únicas de los usuarios. En general, sin embargo, una forma tridimensional (3D) de cóncavo (o hemisférica) se considera el más adecuado para la producción de esferoides solo tamaño uniforme. Por lo tanto, la utilidad de los micropocillos cóncavos se ha divulgado para muchos estudios de Biología de la célula tales como examinar la diferenciación de cardiomiocitos de células madre embrionarias de14,15, racimos de la secreción de insulina de la célula del islote la actividad enzimática de los hepatocitos16y la resistencia a los medicamentos de tumor esferoides17.
Desafortunadamente, la fabricación de los micropocillos a menudo requiere instalaciones especializadas micropatterning; métodos de fotolitografía convencionales requieren exposición e instalaciones en vías de desarrollo mientras que los reactivos ion-aguafuerte-métodos necesitan equipo de plasma y viga de ion. Ese equipo es costoso que, junto con el proceso de fabricación complicado, presenta una alta barrera de entrada para los biólogos que no tienen acceso a la microtecnología. Para superar estos problemas, otros métodos rentables como hielo litografía18 (usando las gotas de agua congeladas) y el método de membrana flexible14 (usando una membrana, sustrato por-agujero y un vacío) se han sugerido. Sin embargo, estos métodos también conllevan graves inconvenientes como que es difícil controlar el tamaño del patrón, el logro de altos cocientes de aspecto y la producción de micropocillos de área más grande.
Para superar estas cuestiones, proponemos un método de fabricación de novela micropocillos cóncavo utilizando un substrato a través del agujero, bolas de acero y una variedad de imán. Usando este método, se pueden fabricar cientos de micropocillos esféricos cóncavos aprovechando el mecanismo de cuentas metálicas autobloqueantes asistida de fuerza magnética (figura 1). El proceso de fabricación implica el uso de muy pocas instalaciones costosas y complicadas y no requiere de muchas habilidades avanzadas. Como tal, personas aún pueden realizar fácilmente este método de fabricación. Para demostrar el método propuesto, humanos-adiposo-derivados de las células madre fueron cultivadas en los micropocillos cóncavos para producir esferoides.

<table><tbody><tr><td>CNC rotary engraver</td><td>Roland DGA</td><td>EGX-350</td><td></td></tr><tr><td>Micro drill bit</td><td>HAM Pr&amp;auml;zision</td><td>30-1301 TA</td><td>&amp;Phi; 0.55 and 0.75 mm</td></tr><tr><td>Sulfuric acid 98%</td><td>Daejung</td><td>7683-4100</td><td>For cleaning aluminum plate.&lt;br/&gt; Dilute with distilled water with 15% solution</td></tr><tr><td>Neodymium magnet</td><td>Supermagnete</td><td>W-01-N</td><td>1 x 1 x 1 mm</td></tr><tr><td>Bearing ball</td><td>Agami Modeling</td><td>SUJ2</td><td>&amp;Phi; 600 &amp;mu;m steel bead</td></tr><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dowcorning</td><td>Sylgard 184</td><td></td></tr><tr><td>Pluronic F-127</td><td>Sigma Aldrich</td><td>p2443</td><td>Dilute with phosphate buffered saline to 4% (w/v) solution</td></tr><tr><td>Dulbecco&#039;s modified eagle&#039;s medium (DMEM)</td><td>ATCC</td><td>30-2002</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline (D-PBS)</td><td>ATCC</td><td>30-2200</td><td></td></tr><tr><td>Fetal bovine serum</td><td>ATCC</td><td>30-2020</td><td></td></tr><tr><td>Adipose-derived mesenchymal stem cells</td><td>ATCC</td><td>ATCC PCS-500-011</td><td></td></tr></tbody></table>

a paired bead and magnet array for molding microwells with variable concave geometries

Un esferoide es una herramienta útil para comprender el comportamiento celular en que proporcione un en vivo-como el entorno tridimensional. Métodos de producción de varios esferoide como superficies no-adhesiva, frascos de spinner, gotas colgantes y micropocillos se han utilizado en estudios de interacción de célula a célula, activación inmune, de detección de drogas tallo diferenciación celular y la generación de organoide. Entre estos métodos, los micropocillos con una geometría cóncava tridimensional han ganado la atención de científicos e ingenieros, dados sus ventajas de generación esferoide de tamaño uniforme y la facilidad con que pueden ser las respuestas de los esferoides seguimiento. Aunque se han propuesto métodos rentables como el uso de membranas flexibles y litografía de hielo, estas técnicas incurren en graves inconvenientes como la dificultad para controlar el tamaño del patrón, logro de proporciones de alto y la producción de áreas más grandes de los micropocillos. Para superar estos problemas, proponemos un método robusto para la fabricación de los micropocillos cóncavos sin necesidad de instalaciones complejas de alto costo. Este método utiliza una matriz de 30 x 30 por-agujero, varios acero cien micrómetros-orden granos y la fuerza magnética para fabricar 900 micropocillos en un sustrato de polidimetilsiloxano (PDMS) de 3 cm x 3 cm. Para demostrar la aplicabilidad de nuestro método para aplicaciones biológicas de la célula, se cultivan células madre adiposas para 3 días y producido con éxito utilizando nuestra plataforma de micropocillos de esferoides. Además, se realizó una simulación de magnetostática para investigar el mecanismo, por el que se utilizó fuerza magnética para atrapar los granos de acero en los agujeros. Creemos que el método de fabricación de micropocillos propuesto puede aplicarse a muchos estudios celulares basado en el esferoide como screening de drogas, regeneración de tejidos, diferenciación de la célula de vástago y metástasis del cáncer.

Un molde convexo y un patrón de micropocillos fueron fabricados con éxito siguiendo los pasos 2.1 a 3.7. (Figura 4). Los granos de acero comerciales quedaron atrapados en la matriz por-agujero de 30 x 30. Los granos se mantuvieron firmemente sin espacios entre los granos y los correspondientes agujeros (figura 4a). La forma de micropocillos cóncava fabricado es cóncavo semiesférico, con un diámetro de 600 μm, que es lo mis...

Watch this Scientific Journal Video about A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries at JoVE.com

A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

Este manuscrito presenta un método robusto de la fabricación de los micropocillos cóncavos sin necesidad de instalaciones complejas de alto costo. Usando la fuerza magnética, granos de acero y una matriz a través del agujero, varios cientos los micropocillos fueron formados en un sustrato de polidimetilsiloxano (PDMS) de 3 cm x 3 cm.

Un grano emparejado y arreglo de imán para moldear los micropocillos con geometrías variables cóncavos

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various ...

a-paired-bead-magnet-array-for-molding-microwells-with-variable

Universidad de Cartagena UDEC

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JoVE Core

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Physics

Bioengineering

Unit 1

Magnetic Forces and Fields

SCIENTISTS IN ACTION

8.6K vistas.  Chung-Ang University. Este manuscrito presenta un método robusto de la fabricación de los micropocillos cóncavos sin necesidad de instalaciones complejas de alto costo. Usando la fuerza magnética, granos de acero y una matriz a través del agujero, varios cientos los micropocillos fueron formados en un sustrato de polidimetilsiloxano (PDMS) de 3 cm x 3 cm.

Video: A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

Microbubble Fabrication of Concave-porosity PDMS Beads

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is particularly useful for separations applications. Porous polydimethylsiloxane (PDMS) beads can be produced by heat-curing such an emulsion, allowing the interface between the aqueous and aliphatic phases to mold the morphology of the polymer. In the procedures described here, both polymer and crosslinker (triethoxysilane) are sonicated together in a cold-bath sonicator. Following a period of cross-linking, emulsions are added dropwise to a hot surfactant solution, allowing the aqueous phase of the emulsion to separate, and forming porous polymer beads. We demonstrate that this method can be tuned, and the SAV ratio optimized, by adjusting the electrolyte content of the aqueous phase in the emulsion. Beads produced in this way are imaged with scanning electron microscopy, and representative SAV ratios are determined using Brunauer&#8211;Emmett&#8211;Teller (BET) analysis. Considerable variability with the electrolyte identity is observed, but the general trend is consistent: there is a maximum in SAV obtained at a specific concentration, after which porosity decreases markedly.

Procedures used to generate microstructured concave-porosity polydimethylsiloxane beads are presented. Effects of electrolyte concentration and identity within the aqueous phase are particularly emphasized.

Fabricación de microburbujas de Cuentas PDMS Cóncavo porosidad

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is ...

Investigación

Química

Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies

Embryoid bodies (EB) are aggregates of embryonic stem cells. The most common way of creating these aggregates is the hanging drop method, a laborious approach of pipetting an arbitrary number of cells into well plates. The interactions between the stem cells forced into close proximity of one another promotes the generation of the EBs. Because the media in each of the wells has to be manually exchanged every day, this approach is manually intensive.
Moreover, because environmental parameters including cell-cell, cell-soluble factor interactions, pH, and oxygen availability can be functions of EB size, cell populations obtained from traditional hanging drops can vary dramatically even when cultured under identical conditions. Recent studies have indeed shown that the initial number of cells forming the aggregate can have significant effects on stem cell differentiation. We have developed a simple, rapid, and scalable culture method to load pre-defined numbers of cells into microfabricated wells and maintain them for embryoid body development. Finally, these cells are easily accessible for further analysis and experimentation. This method is amenable to any lab and requires no dedicated equipment. We demonstrate this method by creating embryoid bodies using a red fluorescent mouse cell line (129S6B6-F1).

We show a simple and rapid method to load pre-defined numbers of cells into microfabricated wells and maintain them for embryoid body development.

Shrinky-Dink colgante Gotas: Una manera sencilla de Forma y Cultura cuerpos embrioides

Embryoid bodies (EB) are aggregates of embryonic stem cells. The most common way of creating these aggregates is the hanging drop method, a laborious ...

Biología

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its transparency. Due to the softness of the PDMS material, however, it is challenging to use PDMS for building nanochannels. The channels tend to collapse easily during plasma bonding. In this paper, we present an evaporation-driven self-assembly method of silica colloidal nanoparticles to create nanofluidic junctions with sub-50 nm pores between two microchannels. The pore size as well as the surface charge of the nanofluidic junction is tunable simply by changing the colloidal silica bead size and surface functionalization outside of the assembled microfluidic device in a vial before the self-assembly process. Using the self-assembly of nanoparticles with a bead size of 300 nm, 500 nm, and 900 nm, it was possible to fabricate a porous membrane with a pore size of ~45 nm, ~75 nm and ~135 nm, respectively. Under electrical potential, this nanoporous membrane initiated ion concentration polarization (ICP) acting as a cation-selective membrane to concentrate DNA by ~1,700 times within 15 min. This non-lithographic nanofabrication process opens up a new opportunity to build a tunable nanofluidic junction for the study of nanoscale transport processes of ions and molecules inside a PDMS microfluidic chip.

We propose a simple self-assembly technique of silica colloidal nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane (PDMS). Using this technique, a nanoporous bead membrane with a pore size down to ~45 nm was built inside a microchannel and applied to electrokinetic preconcentration of DNA samples.

Creación de Sub-50 Nm nanofluídico uniones en PDMS microfluidos de chips VIA proceso de autoensamblaje de partículas coloidales

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its ...

Ingeniería

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

Formación de biomembrana Microarrays con un método basado en la Asamblea Rasero

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

Bioingeniería

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

Fabrication polimérico matriz de microagujas por fotolitografía

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

Fácil y Precisa mecanoterapia perfiles en micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

Micropatterning y Montaje de Microvasos 3D

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 &#181;m in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

Microfluidics is a powerful tool for the development of diagnostic tests. However, expensive equipment and materials, as well as laborious fabrication and handling techniques, are often required. Here, we detail the fabrication protocol of an acrylic microfluidic device for magnetic micro- and nanoparticle-based immunoassays in a low-cost and simple-to-use setting.

Microfresado de control numérico computarizado de un dispositivo acrílico microfluídico con una restricción escalonada para inmunoensayos magnéticos basados en nanopartículas

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or ...

High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are therefore poised to be useful in the field of mechanobiology. Since the method commonly relies on image-based tracking of magnetic beads, the speed limit in recording and analyzing images, as well as the thermal fluctuations of the beads, has long hampered its application in observing small and fast structural changes in target molecules. This article describes detailed methods for the construction and operation of a high-resolution MT setup that can resolve nanoscale, millisecond dynamics of biomolecules and their complexes. As application examples, experiments with DNA hairpins and SNARE complexes (membrane-fusion machinery) are demonstrated, focusing on how their transient states and transitions can be detected in the presence of piconewton-scale forces. We expect that high-speed MTs will continue to enable high-precision nanomechanical measurements on molecules that sense, transmit, and generate forces in cells, and thereby deepen our molecular-level understanding of mechanobiology.

Here, we describe a high-speed magnetic tweezer setup that performs nanomechanical measurements on force-sensitive biomolecules at the maximum rate of 1.2 kHz. We introduce its application to DNA hairpins and SNARE complexes as model systems, but it will be also applicable to other molecules involved in mechanobiological events.

Pinzas magnéticas de alta velocidad para mediciones nanomecánicas en elementos sensibles a la fuerza

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are ...

Bioquímica

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Fabricación de una Matriz microambiente celular Artificial multiplexados

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...