Esta investigación fue parcialmente apoyado por la Fundación Nacional de Ciencia (NSF 1227359), WVU programa EPSCoR financiado por la National Science Foundation (EPS-1003907), oficina WVU ADVANCE patrocinado por la Fundación Nacional de Ciencia (1.007.978), y WVU PSCoR, respectivamente. El trabajo se realizó en microfabricación WVU compartido investigación (instalaciones para salas blancas) y Microfluidic Investigación Celular Integral de Laboratorio Chip (microchip Lab) de la Universidad de West Virginia. La imagen confocal se realizó en WVU Fondo de imágenes del microscopio.

1. La fotolitografía Fabricación de Fotoresinas Molde Maestro El siguiente protocolo muestra el proceso para la fabricación de los microcanales con diámetros entre 30-60 micras. Para obtener un microcanal con un diámetro más pequeño (menos de 30 micras), un solo giro de recubrimiento de resina fotosensible se necesita. <ol><li> Transferir la resina fotosensible reflujo de la nevera a 4 ° C para la sala limpia 24 horas antes de su uso y deje que se caliente a temperatura ambiente. </li><li> Limpiar una oblea de silicio y hornear durante una hora a 150 ° C para permitir que se deshidrate. La deshidratación se ayudar a la adhesión a la resina fotosensible sustrato de silicio. </li><li> Spin-capa de la primera capa de resina fotosensible usando la siguiente receta: </li></ol><table border="1"><tbody><tr><td> Paso </td><td> Tiempo (segundos) </td><td> Velocidad (rpm) </td><td> Aceleración </td></tr><tr><td> 1 </td><td> 2 &lt;/ Td&gt; <td> 300 </td><td> alto </td></tr><tr><td> 2 </td><td> 10 </td><td> 0 </td><td></td></tr><tr><td> 3 </td><td> 3 </td><td> 300 </td><td> alto </td></tr><tr><td> 4 </td><td> 60 </td><td> 600 </td><td> alto </td></tr><tr><td> 5 </td><td> 10 </td><td> 500 </td><td> alto </td></tr><tr><td> 6 </td><td> 10 </td><td> 600 </td><td> alto </td></tr></tbody></table><ol start="4"><li> A continuación, cocer la oblea en una placa calefactora con una temperatura de 110 ° C durante 90 seg. Después de la cocción suave el espesor de la resina fotosensible será 20-30 micras. </li><li> Girar capa una segunda capa de resina fotosensible siguiendo la misma receta utilizada para la primera capa. </li><li> Hornear suave de la oblea de nuevo colocándolo en una placa caliente con una temperatura de 110 ° C durante 90 seg. Después de esto hornear suave el espesor de la resina fotosensible será 40-60 micras. </li><li> Generar patrones maestros positivos mediante la exposición de la photorESIST a la luz ultravioleta con una dosis de exposición de 14.500 mJ / cm 2 a través de una máscara de la película. </li><li> Diluir el desarrollador con agua desionizada (DI) agua (1:2 (v / v)). Enjuague la oblea repetidamente en la solución hasta que el patrón está completamente desarrollado. Luego lave la oblea con agua desionizada y séquelo con gas nitrógeno. </li><li> Reflujo: Lugar de la oblea en una placa caliente con una temperatura de 120 ° C durante 4 min y cubierta con una placa de Petri de vidrio para evitar la evaporación del disolvente. Retire la oblea de la placa de cocción y deje que se enfríe a temperatura ambiente. El espesor de la resina fotosensible después del reflujo, será de 50-60 micras. </li></ol> 2. Fabricación litografía blanda de red de microcanales PDMS <ol><li> Preparar la solución de polidimetilsiloxano (PDMS) en la relación en peso de 10:01 (base: agente de curado) y se mezcla a fondo usando un mezclador planetario centrífuga. </li><li> Echad la solución de PDMS sobre la reflowed molde matriz fotosensible. Coloque el PDMS fundido en un desecador durante 15 minutos para desgasificar. Utilice gas nitrógeno para eliminar cualquier burbuja restantes si es necesario. </li><li> Hornear el PDMS en un horno a una temperatura de 60 ° C durante 3 horas para permitir que se cure. A continuación, retire la capa de PDMS curado del molde maestro. </li><li> Use un punzón afilado para crear orificios de entrada / salida haciendo agujeros en la red de canales. Limpiar la superficie de la PDMS utilizando gas nitrógeno. </li><li> Tratar dos capas de PDMS con plasma de oxígeno durante 30 segundos en el interior de un limpiador de plasma a una presión de funcionamiento de 4,5 x 10 -2 Torr y una tasa de flujo de oxígeno de 3,5 m 3 / min. A continuación, alinee las superficies de la PDMS manualmente bajo un microscopio óptico. Utilice una gota de agua si es necesario para un mejor control de la alineación. </li><li> Hornear el dispositivo en un horno a 60 ° C durante 30 minutos para conseguir una unión permanente. </li></ol> 3. Cultura HUVEC y siembra en el chip <ol><li> Cultura las HUVEC con medio de cultivo con L-glutamina suplementado con 10% de suero bovino fetal (FBS). Para thSe han usado pasajes experimento E entre 2 y 5. </li><li> Una vez que las HUVEC son confluentes, contar las células y la cosecha de ellos por primera enjuagar las células con solución salina tamponada con HEPES (HEPES-BSS), y a continuación, tratar las células con tripsina / EDTA y se incuba durante 2-6 min a 37 ° C. Después de la tripsinización es completa, neutralizar la tripsina / EDTA con tripsina solución neutralizante. Centrifugar y suspender las células en medio de cultivo con 8% de dextrano para recogerlos. El dextrano se utiliza para aumentar la viscosidad media para ayudar en la siembra de células y una mejor unión. </li><li> Se trata el dispositivo con plasma de oxígeno durante 5 min con una presión de funcionamiento de 4,5 x 10 -2 Torr y una tasa de flujo de oxígeno de 3,5 m 3 / min. A continuación, cargue el dispositivo con agua DI y tratamiento con luz UV durante 8 horas en una campana de bioseguridad laminar para la esterilización. </li><li> Un día antes de que las células están listas, lave el aparato con 1x tampón fosfato salino (PBS 1x) después cubra con fibronectina (100 mg / ml, dilbuido con 1x PBS) y se incuba en el refrigerador a 4 ° C durante la noche. </li><li> Después de que el revestimiento de la fibronectina, lave el aparato con 1x PBS y luego cargar con medio de cultivo. Incubar el dispositivo a una temperatura de 37 ° C durante 15 min. </li><li> Cargar las células HUVEC en medio de cultivo 8% de dextrano con una concentración de células de 3-4 x 10 6 células / ml. Coloque una gota de 20 l de células a una entrada del dispositivo y la inclinación de introducir las células en el canal microfluídico. Después de 15-20 min las células comienzan a adherirse a las paredes laterales de los canales. Girar el dispositivo cada 15 minutos para crear una distribución más uniforme de las células. Si es necesario, la carga adicional se puede realizar. </li></ol> 4. Configuración de perfusión a largo plazo Después de 5-6 horas de cultivo estático, las HUVECs adjuntos comenzarán a extenderse plenamente. Configure la perfusión con un sistema de bomba de jeringa con mando a distancia, con un flujo constante de 10 l / h. La perfusión se puede ajustar fo un caudal mayor, y pueden durar un período de tiempo entre los 4 días a 2 semanas. 5. Tinción de células y caracterización Microscopio <ol><li> Cuando las células alcanzan la confluencia en el interior del dispositivo, en primer lugar, lávese el dispositivo con 1x PBS para eliminar completamente el medio. A continuación, cargar el dispositivo con colorante rojo diluido con diluyente (4 l-1 ml). Cargar el medio de contraste similar al procedimiento de carga de las células. Incubar el dispositivo en la oscuridad durante 5 min a temperatura ambiente, y luego lavar el dispositivo con medio de cultivo para detener la tinción. Largo de incubación del medio de contraste puede causar toxicidad celular y disadhesion. </li><li> Cargar el dispositivo con colorante azul diluido en PBS 1x (2 gotas por ml). Incubar en la oscuridad durante 5 min a temperatura ambiente, luego lavar bien el dispositivo con 1x PBS. </li><li> Examinar la tinción de las células bajo un microscopio óptico invertido. Si la tinción era bueno, cargue los microcanales con un medio de fijación (3,5% de paraformaldehído diluido con 1x PBS), a continuación, sumerjael dispositivo de fijación de medio y cubrir completamente con papel de aluminio. Guarde el dispositivo en el refrigerador a una temperatura de 4 ° C para evitar que el dispositivo se seque y photobleaching. El dispositivo fijo está ahora listo para la formación de imágenes confocal, que puede ser realizado por un microscopio confocal de barrido láser. </li></ol>

Los autores declaran que no tienen intereses financieros en competencia.

<html> 1. Fabricación del molde maestro Uno de los principios rectores para el diseño y la morfometría vascular se conoce como la ley de Murray 16, que establece que la distribución de los diámetros de los vasos a lo largo de la red se rige por consideraciones de energía mínimo. También establece que el cubo de los diámetros de un vaso principal en una bifurcación es igual a la suma de los cubos de los diámetros de los vasos hija ( <img alt="Ecuación 1" fo:content-width="0...

Los microvasos, como una parte del sistema de circulación, median las interacciones entre la sangre y los tejidos, apoyar las actividades metabólicas, definir microambiente del tejido, y desempeñan un papel crítico en muchas condiciones patológicas y de salud. Recapitulación de microvasos funcionales in vitro podría proporcionar una plataforma para el estudio de fenómenos vasculares complejas. Sin embargo, convencional en los ensayos de microvasos in vitro, tales como ensayos de migración de células endoteliales, ensayos de formación de tubos endoteliales, y ensayos de anillos aórticos de rata y de ratón, no son capaces de recrear la microvasos in vivo con respecto a la geometría tridimensional (3D) y de control de flujo continuo 1-8. Estudios de microvasos utilizando modelos animales y en ensayos in vivo, tales como ensayo de angiogénesis corneal, ensayo de angiogénesis de membrana corioalantoidea de pollo, y el ensayo de tapón de Matrigel, son más tiempo, alta en costo, desafiando con respecto a la observación y cuantificaciones, yplantear cuestiones éticas 1, 9-13. Los avances en la microfabricación y tecnologías de chips de microfluidos han permitido una gran variedad de conocimientos sobre ciencias biomédicas, mientras reduciendo el alto costo y la complejidad asociados con los animales de experimentación y en estudios in vivo 14, como las condiciones biológicas controladas fácilmente y firmemente y entornos fluidos dinámicos, que no tendrían sido posible con las técnicas convencionales macroescala. A continuación, presentamos un método para construir un endotelizado microcanales-on-a-chip que imita la geometría 3D de in vivo microvasos y se ejecuta bajo flujo de perfusión continua controlada mediante el uso de la combinación de la técnica de fotolitografía reflowable fotosensible, litografía blanda, y microfluidos.

<table border="1">
	<tbody>
		<tr>
			<td>Reagent/Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflow Photoresist</td>
			<td>AZ Electronic Materials</td>
			<td>AZP4620</td>
			<td></td>
		</tr>
		<tr>
			<td>Developer</td>
			<td>AZ Electronic Materials</td>
			<td>AZ 400K</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS</td>
			<td>Dow Corning Corporation</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>MCDB 131 Culture Medium</td>
			<td>Invitrogen</td>
			<td>10372-019</td>
			<td></td>
		</tr>
		<tr>
			<td>NacBlue Nuclei Staining</td>
			<td>Invitrogen</td>
			<td>H1399</td>
			<td></td>
		</tr>
		<tr>
			<td>PKH Red Stain</td>
			<td>Sigma</td>
			<td>MINI26 and PKH26GL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Gibco</td>
			<td>PHE0023</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Invitrogen</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffered Saline Solution</td>
			<td>Lonza</td>
			<td>CC-5024</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Invitrogen</td>
			<td>25300-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin Neutralizing Solution</td>
			<td>Lonza</td>
			<td>CC-5002</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS Curing Agent</td>
			<td>Dow Corning Corporation</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary Human Umbilical Vein Endothelial Cells</td>
			<td>Lonza</td>
			<td>CC-2517</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Lonza</td>
			<td>14-501F</td>
			<td></td>
		</tr>
		<tr>
			<td>Diluent C</td>
			<td>Sigma</td>
			<td>CGLDIL</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst33342</td>
			<td>Invitrogen, Molecular Probes</td>
			<td>R37605</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Sigma</td>
			<td>95771</td>
			<td></td>
		</tr>
		<tr>
			<td>3.5% Paraformaldehyde</td>
			<td>Electron Microscopy Science</td>
			<td>15710-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spinner</td>
			<td>Laurell Technologies Corporation</td>
			<td>WS-400BZ-6NPP/LITE</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>BelArt Products</td>
			<td>999320237</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Pump System</td>
			<td>Harvard Apparatus</td>
			<td>PHD Ultra</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar Biosafety Hood</td>
			<td>Thermo Scientific</td>
			<td>1300 Series A2</td>
			<td></td>
		</tr>
		<tr>
			<td>Planetary Centrifugal Mixer</td>
			<td>Thinky</td>
			<td>ARE-310</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp Oven</td>
			<td>Fisher Scientific</td>
			<td>13-246-516GAQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Microscope</td>
			<td>Zeiss</td>
			<td>Invertoskop 40C</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-32G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hotplate</td>
			<td>Barnstead/Thermolyne Cimarec</td>
			<td>SP131635</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Confocal Microscope</td>
			<td>Zeiss</td>
			<td>LSM 510</td>
			<td></td>
		</tr>
	</tbody>
</table>

procedure for the development of multi-depth circular cross-sectional endothelialized microchannels-on-a-chip

Los esfuerzos se han centrado en el desarrollo de ensayos in vitro para el estudio de microvasos porque en estudios con animales in vivo son más que consume tiempo, es caro, y la observación y cuantificación son muy desafiante. Sin embargo, convencional en los ensayos de microvasos in vitro tienen limitaciones cuando se representa en microvasos in vivo con respecto a la geometría tridimensional (3D) y proporcionar flujo de fluido continuo. Usando una combinación de la técnica de fotolitografía reflowable fotosensible, litografía blanda, y microfluidos, hemos desarrollado un multi-profundidad endotelializadas transversales circulares microcanales-on-a-chip, que imita a la geometría 3D en vivo microvasos y se ejecuta bajo perfusión continua controlada flujo. Un fotoprotector reflowable positivo se utilizó para fabricar un molde principal con una red de microcanales de sección transversal semicircular. Por la alineación y unión de los dos (PDMS) microcanales polidimetilsiloxano replicated del molde maestro, se creó una red de microcanales cilíndrica. Los diámetros de los microcanales pueden ser bien controlados. Además, las células primarias endoteliales de la vena umbilical humana (HUVEC) sembradas dentro del chip mostraron que las células se alineaban en la superficie interior de los microcanales de acuerdo con la perfusión controlada duradera para un período de tiempo entre 4 días a 2 semanas.

Nuestro enfoque para la fabricación de la red de microcanales de múltiples profundidad imita las complejas geometrías 3D de microvasos in vivo, en el que los microcanales han redondeado las secciones transversales 15. Además, los diámetros de los canales de ramificación matrices y los canales de memoria de aproximadamente obedecen la ley de Murray para mantener el flujo de fluido a un nivel requerido para que la resistencia total del canal es baja y velocidades de flujo son más uniforme en tod...

Watch this Scientific Journal Video about Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip at JoVE.com

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

Una plataforma de micro-canales-on-a-chip fue desarrollado por la combinación de la técnica de fotolitografía reflowable fotosensible, litografía blanda, y microfluidos. La plataforma de microcanales endotelizado imita la geometría tridimensional (3D) de microvasos in vivo, se ejecuta bajo flujo de perfusión continua controlada, permite la formación de imágenes de alta calidad y en tiempo real y se puede aplicar para la investigación microvascular.

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, ...

procedure-for-development-multi-depth-circular-cross-sectional

Research

JoVE Journal

Bioengineering

13.8K vistas.  West Virginia University.  Una plataforma de micro-canales-on-a-chip fue desarrollado por la combinación de la técnica de fotolitografía reflowable fotosensible, litografía blanda, y microfluidos. La plataforma de microcanales endotelizado imita la geometría tridimensional (3D) de microvasos in vivo, se ejecuta bajo flujo de perfusión continua controlada, permite la formación de imágenes de alta calidad y en tiempo real y se puede aplicar para la investigación microvascular. 

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

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

Procedimiento para la Ingeniería de pulmón

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

Investigación

Bioingeniería

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

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 ...

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

Procedimiento para descelularización de corazón porcino por perfusión coronaria retrógrada

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

Los métodos para la caracterización de la Co-desarrollo de la biopelícula y Hábitat Heterogeneidad

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Sólido lípidos nanopartículas (GLC) Aplicaciones para intracelular de focalización

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

Caracterización cualitativa de la fracción acuosa de hidrotérmico Licuefacción de algas mediante 2D cromatografía de gases con espectrometría de masas de tiempo de vuelo

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

Desarrollo de un Modelo Organotípicos multicelular tridimensional de la mucosa intestinal humanas cultivadas en condiciones de microgravedad

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

Montaje y Seguimiento de Desarrollo Comunitario Microbiano dentro de una Plataforma de Matriz de Micropedulos

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Standard Test Method ASTM D 7998-19 for the Cohesive Strength Development of Wood Adhesives

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the adhesive cure, and the effect of adhesive penetration on the wood interphase; thus, normal testing of a neat adhesive film is generally not useful. Most tests of wood adhesive bond strength are slow, laborious, can be strongly influenced by the wood and do not provide information on the kinetics of cure. Test method ASTM D 7998-19, however, can be used for fast evaluation of the strength of wood bonds. The use of a smooth, uniform, and strong wood surface, like maple face-veneer, and sufficient bonding pressure reduces the adhesion and wood strength effects on bond strength. This method has three main applications. The first is to provide consistent data on bond strength development. The second is to measure the dry and wet strengths of bonded lap shear samples. The third is to better understand the adhesive heat resistance by quickly evaluating thermal sensitivity and distinguishing between thermal softening and thermal degradation.

We present a procedure, ASTM D7998-19, for a rapid and more consistent evaluation of both dry and wet strength of adhesive bonds on wood. The method can also be used to provide information on strength development as a function of temperature and time or strength retention up to 250 &#176;C.

Método de prueba estándar ASTM D 7998-19 para el desarrollo de la resistencia cohesiva de adhesivos para madera

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the ...

Asymmetrical Flow Field-Flow Fractionation for Sizing of Gold Nanoparticles in Suspension

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and size distribution of nanoparticles is of utmost importance for various applications. The size range is also important, as it defines the most &#8220;active&#8221; component of a nanoparticle dose.
Asymmetrical Flow Field-Flow Fractionation (AF4) is a powerful technique for sizing of particles in suspension in the size range of approximately 1&#8211;1000 nm. There are several ways to derive size information from an AF4 experiment. Besides coupling AF4 online with size-sensitive detectors based on the principles of Multi-Angle Light Scattering or Dynamic Light Scattering, there is also the possibility to correlate the size of a sample with its retention time using a well-established theoretical approach (FFF theory) or by comparing it with the retention times of well-defined particle size standards (external size calibration).
We here describe the development and in-house validation of a standard operating procedure (SOP) for sizing of an unknown gold nanoparticle sample by AF4 coupled with UV-vis detection using external size calibration with gold nanoparticle standards in the size range of 20&#8211;100 nm. This procedure provides a detailed description of the developed workflow including sample preparation, AF4 instrument setup and qualification, AF4 method development and fractionation of the unknown gold nanoparticle sample, as well as the correlation of the obtained results with the established external size calibration. The SOP described here was eventually successfully validated in the frame of an interlaboratory comparison study highlighting the excellent robustness and reliability of AF4 for sizing of nanoparticulate samples in suspension.

This protocol describes the use of Asymmetrical Flow Field-Flow Fractionation coupled with UV-vis detection for the determination of the size of an unknown gold nanoparticle sample.

Fraccionamiento flujo de campo de flujo asimétrico para dimensionamiento de nanopartículas de oro en suspensión

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and ...