Name: micropatterning and assembly of 3d microvessels
Uploaded: 2016-09-09T15:00:00.000+00:00
Duration: 13 min 5 s
Description: Watch this Scientific Journal Video about Micropatterning and Assembly of 3D Microvessels at JoVE.com

Los autores desean reconocer la Lynn y Mike Garvey Laboratorio de Imagen en el Instituto de Células Madre y Medicina Regenerativa, así como el Fondo de Washington nanofabricación de la Universidad de Washington. También reconocen el apoyo financiero del Instituto Nacional de Salud otorga DP2DK102258 (a YZ), y la formación otorga T32EB001650 (a SSK y MAR) y T32HL007312 (MAR).

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Los autores no tienen nada que revelar.

Microvasos de ingeniería son un modelo in vitro, donde están presentes y sintonizable características fisiológicas, tales como la geometría luminal, las fuerzas hidrodinámicas, y las interacciones multicelulares. Este tipo de plataforma es de gran alcance, ya que ofrece la posibilidad de modelar y estudiar el comportamiento del endotelio en una variedad de contextos en los que las condiciones de cultivo in vitro en puede ser igualada a la del microambiente en cuestión. Por ejemplo, los mecanismo...

La microvasculatura en cada órgano ayuda a definir el microambiente del tejido, mantener la homeostasis del tejido y regulan la inflamación, la permeabilidad, la trombosis, y la fibrinólisis 1,2. Endotelio microvascular, en particular, es la interfaz entre el flujo sanguíneo y el tejido circundante y por lo tanto juega un papel crítico en la modulación de la función del órgano vascular y en respuesta a estímulos tales como las fuerzas hidrodinámicas y citoquinas circulantes y hormonas 3 - 5. La comprensión de las interacciones detalladas entre el endotelio, la sangre y el microambiente tejido circundante es importante para el estudio de la biología vascular y progresión de la enfermedad. Sin embargo, los avances en el estudio de estas interacciones se ha visto obstaculizado por limitada en herramientas in vitro que no recapitulan en la estructura microvascular vivo y la función 6,7. Como resultado, el campo y el avance terapéutico se ha basado en gran medida en costoso y tiempo-el consumo de modelos animales que a menudo no traducir del éxito en seres humanos 8 - 10. Mientras que los modelos in vivo son de gran valor en el estudio de mecanismos de la enfermedad y funciones vasculares, que son complejas y a menudo carecen de un control preciso del individuo celular, bioquímica, y las señales biofísicas. Vasculatura en todo el cuerpo posee una estructura jerárquica madura en conjunción con lechos capilares expansivas, proporcionando la perfusión optimizado y el transporte de nutrientes al mismo tiempo 11. Inicialmente, las formas vasculatura como un plexo primitiva que reorganiza a una red jerárquica ramificada durante el desarrollo temprano 12,13. Aunque muchas de las señales implicadas en estos procesos se conocen bien 14 - 16, sigue siendo difícil de alcanzar, tales cómo el patrón vascular se determina 15. A su vez, la recapitulación de este proceso in vitro para diseñar redes vasculares organizados tiene abejan. Muchas plataformas difíciles existentes in vitro para modelar la vasculatura, por ejemplo, dos cultivos de células endoteliales dimensionales, carecen de características importantes tales como la proximidad multi-celular, tres geometría luminal dimensional, flujo, y de la matriz extracelular. Los ensayos de formación de tubos en 3D (hidrogeles de colágeno o fibrina) 17 - 19 o invasión ensayos de 20,21 se han utilizado para estudiar la función endotelial en 3D y sus interacciones con otras vasculares 17,22 celulares o tejidos tipos 23. Sin embargo, ensamblados lúmenes en estos ensayos carecen de interconectividad, el flujo hemodinámico, y la perfusión adecuada. Además, la propensión a la regresión vascular en estos ensayos de formación de tubo 24 impide que la cultura a largo plazo y la maduración que limita el grado de estudios funcionales que se pueden realizar. Por lo tanto, hay una necesidad creciente para diseñar plataformas in vitro de redes microvasculares que pueden modelar adecuadamente ESendotelial características y son capaces de cultivo a largo plazo. Una variedad de técnicas de ingeniería vasculares han surgido en los últimos años para aplicaciones médicas para reemplazar o vasos de derivación afectada en pacientes con enfermedad vascular. Vasos de gran diámetro fabricados con materiales sintéticos tales como tereftalato de polietileno (PET), y politetrafluoroetileno (PTFE) han tenido un considerable éxito terapéutico con la permeabilidad a largo plazo (promedio 95% la permeabilidad de más de 5 años) 25. Aunque de pequeño diámetro injertos sintéticos (&lt;6 mm) por lo general se enfrentan a complicaciones tales como hiperplasia de la íntima y trombopoyesis 26 - 28, ingeniería tisular injertos de pequeño diámetro hechos con material biológico han hecho progresos significativos 29,30. A pesar de los avances de este tipo, los vasos de ingeniería en la microescala han seguido siendo un reto. Para modelar adecuadamente la microvasculatura, es necesario para generar patrones de red complejas con sufciente resistencia mecánica para mantener la permeabilidad y con una composición de matriz que permite tanto la permeación de nutrientes para las células del parénquima y remodelación celular. Este protocolo presenta una novedosa red de vasos artificiales perfusable que imita un nativo in vivo con un ajuste ajustable y controlable microambiente el 31 de - de 34. El método descrito genera microvasos Diseñado con diámetros del orden de 100 micras. microvasos ingeniería se fabrican mediante la perfusión de las células endoteliales a través de un canal de microfluidos que está incrustado dentro del tipo I colágeno suave hidrogel. Este sistema tiene la capacidad de generar redes modelados con estructura luminal abierta, replicar las interacciones multicelulares, modular la composición de la matriz extracelular, y aplicar fuerzas hemodinámicas fisiológicamente relevantes.

<table><tbody><tr><td>&lt;strong&gt;Wafer Fabrication&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AutoGlow Plasma System</td><td>AutoGlow</td><td></td><td></td></tr><tr><td>Headway Spin Coater</td><td>Headway Research, Inc&amp;nbsp;</td><td>PWM32 Spin Coater&amp;nbsp;</td><td></td></tr><tr><td>ABM Contact Aligner</td><td>AB-M</td><td></td><td></td></tr><tr><td>Alpha Step Profilometer</td><td>Tencor</td><td>Alpha Step 200</td><td></td></tr><tr><td>SU-8 Developer</td><td>Microchem</td><td>Y020100</td><td></td></tr><tr><td>SU-8 Resist</td><td>Microchem</td><td>SU-8 2000</td><td></td></tr><tr><td>8&quot; silicon wafer</td><td>Wafer World Inc.</td><td></td><td></td></tr><tr><td>Tabletop Micro Pattern Generator</td><td>Heidelberg Instruments</td><td>&amp;mu;PG 101</td><td>For generation of photomask</td></tr><tr><td>Hot plate</td><td>VWR</td><td>97042-646</td><td></td></tr><tr><td>Ispropyl alcohol</td><td>Avantor Performance Materials</td><td>9088</td><td></td></tr><tr><td>Petri dishes (120 x 120 mm, square)</td><td>Sigma-Aldrich</td><td>Z617679</td><td></td></tr><tr><td>Trichloro(3,3,3-trifluoropropyl)silane</td><td>Sigma-Aldrich</td><td>MKBG3805V</td><td></td></tr><tr><td>Polydimethylsiloxane (PDMS) elastomer base and curing agent</td><td>Dow Corning</td><td>Sylgard 184</td><td>Mixed at 10:1 (w/w)</td></tr><tr><td>Vacuum desiccator</td><td>Sigma-Aldrich</td><td>Z119024-1EA</td><td></td></tr><tr><td>Oven</td><td>VWR</td><td>9120976</td><td></td></tr><tr><td>&lt;strong&gt;Device Fabrication and Culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>poly(methyl methacrylate) (PMMA)</td><td>Plexiglas</td><td></td><td></td></tr><tr><td>Corona Treater</td><td>Electro-Technic Products, Inc.</td><td>BD-20</td><td>Handheld device for plasma treatment of PMMA devices and PDMS molds</td></tr><tr><td>Soldering Iron</td><td>Weller&amp;nbsp;</td><td>WTCPS</td><td></td></tr><tr><td>Stainless Steel Truss Head Slotted Machine Screw</td><td>McMaster-Carr&amp;nbsp;</td><td>91785A096</td><td></td></tr><tr><td>Stainless steel dowel pins</td><td>McMaster-Carr&amp;nbsp;</td><td>93600A060</td><td></td></tr><tr><td>Tweezers&amp;nbsp;</td><td>Miltex</td><td>24-572</td><td>Any similar tweezers may be used</td></tr><tr><td>Spatula (Micro Spoon)</td><td>Electron Microscopy Services</td><td>62410-01</td><td></td></tr><tr><td>Screw driver</td><td></td><td></td><td>Any flat head screwdriver may be used, autoclaved</td></tr><tr><td>Glass coverslips (22 x 22 mm)</td><td>Fisher Scientific</td><td>12-542B</td><td></td></tr><tr><td>Bleach</td><td>Clorox</td><td>4460030966</td><td></td></tr><tr><td>Petri dishes (150 x 25 mm)</td><td>Corning</td><td>430599</td><td></td></tr><tr><td>Petri dishes (100 x 20 mm)</td><td>Corning</td><td>2909</td><td></td></tr><tr><td>Cotton, cut into 1 cm x 3 cm pieces</td><td></td><td></td><td>Autoclaved</td></tr><tr><td>Polyethyleneimine (PEI)</td><td>Sigma-Aldrich</td><td>P3143</td><td>Dilute to 1% in cell culture grade water</td></tr><tr><td>Glutaraldehyde</td><td>Sigma-Aldrich</td><td>G6257</td><td>Dilute to 0.1% in cell culture grade water</td></tr><tr><td>Sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>Autoclaved DI H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Type I collagen, dissolved in 0.1% acetic acid</td><td></td><td></td><td>Isolated from rat tails as described in Rajan &lt;i&gt;et. al.&lt;/i&gt; 2006 (ref #37)</td></tr><tr><td>1 ml syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>10 ml syringe</td><td>BD</td><td>309604</td><td></td></tr><tr><td>15 ml conical tubes</td><td>Corning</td><td>352097</td><td></td></tr><tr><td>30 ml conical tubes</td><td>Corning</td><td>352098</td><td></td></tr><tr><td>M199 10x Media&amp;nbsp;</td><td>Life Technologies</td><td>11825-015</td><td></td></tr><tr><td>1 N NaOH (sterile)</td><td>Sigma-Aldrich</td><td>415413</td><td>Dilute to 1 N in cell culture grade water</td></tr><tr><td>HUVECs</td><td>Lonza</td><td></td><td></td></tr><tr><td>Endothelial growth media</td><td>Lonza</td><td>CC-3124</td><td></td></tr><tr><td>Trypsin</td><td>Corning</td><td>25-052-CI</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermofisher Scientific</td><td>10082147</td><td></td></tr><tr><td>Dextran from Leuconostoc spp. (70&amp;nbsp;kDa)</td><td>Sigma-Aldrich</td><td>31390</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Hemocytometer</td><td>Hausser Scientific Co.</td><td>3200</td><td></td></tr><tr><td>Gel loading tips</td><td>VWR</td><td>37001-152</td><td></td></tr><tr><td>18 G Blunt Fill Needle</td><td>BD&amp;nbsp;</td><td>305180</td><td></td></tr><tr><td>20 G Stainless Steel Dispensing Needle</td><td>McMaster-Carr</td><td>75165A123</td><td></td></tr><tr><td>Tygon 1/32&amp;rdquo; ID, 3/32&quot; OD Silicon Tubing</td><td>Cole-Parmer</td><td>EW-95702-00</td><td></td></tr><tr><td>1/16&quot; Tube-to-tube Coupling</td><td>McMaster-Carr</td><td>5116K165</td><td></td></tr><tr><td>90&amp;deg; Elbow Connectors, Tube-to-Tube</td><td>McMaster-Carr</td><td>5121K901</td><td></td></tr><tr><td>Luer Lock Coupling (Female, 1/16&quot; ID)</td><td>McMaster-Carr</td><td>51525K211</td><td></td></tr><tr><td>Plastic Forceps, with Jaw Grips</td><td>Electron Microscopy Services</td><td>72971</td><td></td></tr><tr><td>Dual Syringe Pump</td><td>Harvard Apparatus</td><td>70-4505</td><td></td></tr><tr><td>5 ml Polystyrene Round-bottom tube</td><td>Fisher Scientific</td><td>14-959-2A</td><td></td></tr><tr><td>&lt;strong&gt;Device Analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>F8775</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A8806-5G</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T-9284</td><td></td></tr><tr><td>Rabbit anti-hCD31</td><td>Abcam</td><td>ab32457</td><td>1:25 working dilution</td></tr><tr><td>FITC conjugated anti-von Willebrand Factor antibody</td><td>Abcam</td><td>ab8822</td><td>1:100 working dilution</td></tr><tr><td>Goat anti-rabbit 568 secondary antibody</td><td>Thermofisher Scientific</td><td>A-11011</td><td>1:100 working dilution</td></tr><tr><td>Hoescht</td><td>Thermofisher Scientific</td><td>H1399</td><td>Resuspended in DMSO</td></tr><tr><td>Sodium cacodylate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C0250</td><td>To make 0.2 M cacodylate buffer</td></tr><tr><td>Ethanol</td><td>VWR International</td><td>BDH1164-4LP</td><td></td></tr><tr><td>40 kDa FITC-conjugated Dextran</td><td>Sigma-Aldrich</td><td>FD40S&amp;nbsp;</td><td></td></tr><tr><td>&lt;strong&gt;Additional Culture Reagents&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CHIR-99021</td><td>Selleck Chem</td><td>S2924</td><td>Small molecule GSK-3 inhibitor</td></tr><tr><td>Human recombinant VEGF</td><td>Peprotech</td><td>100-20</td><td></td></tr><tr><td>Human recombinant bFGF</td><td>Peprotech</td><td>AF-100-18B</td><td></td></tr></tbody></table>

micropatterning and assembly of 3d microvessels

En plataformas in vitro para estudiar las células endoteliales y la biología vascular se limitan en gran medida a la cultura de células endoteliales 2D, el flujo cámaras con polímero o sustratos a base de vidrio, y ensayos de formación de tubo a base de hidrogel. Estos ensayos, mientras informativa, no se recapitulan la geometría del lumen, la matriz extracelular adecuada, y la proximidad multicelular, que juegan un papel clave en la modulación de la función vascular. Este manuscrito describe un método de moldeo por inyección para generar vasos diseñado con diámetros del orden de 100 micras. Los microvasos se fabrican mediante la siembra de células endoteliales en un canal microfluídico incrustado dentro de un tipo nativo I colágeno hidrogel. Mediante la incorporación de las células del parénquima dentro de la matriz de colágeno antes de la formación de canal, microambientes específicos de tejido pueden ser modeladas y estudiadas. modulaciones adicionales de propiedades y medios hidrodinámico composición permiten el control de la función vascular compleja dentro del microambiente deseada.Esta plataforma permite el estudio de la contratación perivascular de células, las interacciones sangre-endotelio, respuesta de flujo, y las interacciones de tejido microvascular. microvasos ingeniería ofrecen la posibilidad de aislar la influencia de los componentes individuales de un nicho vascular y precisamente controlan sus químicos, biológicos, mecánicos y propiedades para estudiar la biología vascular tanto en la salud y la enfermedad.

La plataforma recipiente ingeniería crea microvasculatura funcional incrustado dentro de un tipo natural de colágeno I de la matriz y permite el control ajustado del medio ambiente celular, biofísicas y bioquímicas in vitro. Para fabricar los microvasos de ingeniería, las células endoteliales de la vena umbilical humana (HUVECs) se sometieron a perfusión a través de la red de microfluidos colágeno embebido en donde se unen para formar un lumen de patentes y el endotelio...

Watch this Scientific Journal Video about Micropatterning and Assembly of 3D Microvessels at JoVE.com

Micropatterning and Assembly of 3D Microvessels

Este manuscrito presenta un método de moldeo por inyección para diseñar microvasos que recapitular las propiedades fisiológicas de endotelio. El proceso basado en microfluidos crea redes vasculares 3D patente con condiciones tailorable, tales como gradientes bioquímicos de flujo, composición celular, la geometría, y. Se describe el proceso de fabricación y los ejemplos de posibles aplicaciones.

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 ...

micropatterning-and-assembly-of-3d-microvessels

Research

JoVE Journal

Bioengineering

11.7K vistas.  University of Washington.  Este manuscrito presenta un método de moldeo por inyección para diseñar microvasos que recapitular las propiedades fisiológicas de endotelio. El proceso basado en microfluidos crea redes vasculares 3D patente con condiciones tailorable, tales como gradientes bioquímicos de flujo, composición celular, la geometría, y. Se describe el proceso de fabricación y los ejemplos de posibles aplicaciones. 

Video: Micropatterning and Assembly of 3D Microvessels

Process of Making Three-dimensional Microstructures using Vaporization of a Sacrificial Component

Vascular structures in natural systems are able to provide high mass transport through high surface areas and optimized structure. Few synthetic material fabrication techniques are able to mimic the complexity of these structures while maintaining scalability. The Vaporization of a Sacrificial Component (VaSC) process is able to do so. This process uses sacrificial fibers as a template to form hollow, cylindrical microchannels embedded within a matrix. Tin (II) oxalate (SnOx) is embedded within poly(lactic) acid (PLA) fibers which facilitates the use of this process. The SnOx catalyzes the depolymerization of the PLA fibers at lower temperatures. The lactic acid monomers are gaseous at these temperatures and can be removed from the embedded matrix at temperatures that do not damage the matrix. Here we show a method for aligning these fibers using micromachined plates and a tensioning device to create complex patterns of three-dimensionally arrayed microchannels. The process allows the exploration of virtually any arrangement of fiber topologies and structures.

The Vaporization of a Sacrificial Component (VaSC) process is used to fabricate microvascular structures. This procedure uses sacrificial poly(lactic) acid fibers to form hollow microchannels with precise 3D geometric positioning provided by laser micromachined guide plates.

Proceso de fabricación microestructuras tridimensionales mediante vaporización de un componente de sacrificio

Vascular structures in natural systems are able to provide high mass transport through high surface areas and optimized structure. Few synthetic material ...

Investigación

Ingeniería

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

Un dispositivo de microfluidos con los patrones de Surco para el estudio del comportamiento celular

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

Biología

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Microfabricación de chip del tamaño de andamios para el cultivo de células en tres dimensiones

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal microfluidic device. W/O microdroplets have recently been used in powerful methods that enable miniaturized chemical experiments. Therefore, developing a versatile method to yield monodisperse W/O microdroplets is needed. We have developed a method for generating monodisperse W/O microdroplets based on a capillary-based centrifugal axisymmetric co-flowing microfluidic device. We succeeded in controlling the size of microdroplets by adjusting the capillary orifice. Our method requires equipment that is easier-to-use than with other microfluidic techniques, requires only a small volume (0.1-1 &#181;l) of sample solution for encapsulation, and enables the production of hundreds of thousands number of W/O microdroplets per second. We expect this method will assist biological studies that require precious biological samples by conserving the volume of the samples for rapid quantitative analysis biochemical and biological studies.

Here, we demonstrate a simple production method for size-controllable, monodisperse, water-in-oil (W/O) microdroplets using a capillary-based centrifugal microfluidic device. This method requires only a small sample volume and enables high-yield production. We expect this method will be useful for rapid biochemical and cellular analyses.

Dispositivo de microfluidos centrífuga basada en capilar para la Formación Tamaño-controlable de monodisperso microgotas

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal ...

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

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Lípidos bicapa vesículas Generación Usando Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology. 
This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence. 
The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

We present a technology that uses capillarity-assisted assembly in a microfluidic platform to pattern micro-sized objects suspended in a liquid, such as bacteria and colloids, into prescribed arrays on a polydimethylsiloxane substrate.

Modelado de microorganismos y micropartículas a través del ensamblaje secuencial asistido por capilaridad

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

Guiada por imagen, fabricación basada en láser de Redes de microfluidos derivados-vascular

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Un método versátil de Patterning proteínas y células

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

Control de la geometría celular a través de micropatterning asistido por láser infrarrojo

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...