Este trabajo fue apoyado financieramente por la Fundación Nacional Suiza para la Ciencia (NRP 62 - Materiales Inteligentes). También reconoció es el apoyo de B. Hanselmann, K. Kehl, U. Schütz y B. Leuthold.

1. La polimerización de plasma-iniciado <ol><li> Preparación de la solución de monómero. <ol><li> Disolver HEMA (100 ml; 0,718 mol) en 200 ml de agua y lavar 3 veces con hexano (100 ml) en un embudo de separación. Saturar la fase acuosa con cloruro de sodio y se extrae la HEMA con éter dietílico (50 ml). Se seca la fase orgánica sobre MgSO4 y se elimina el disolvente a vacío (100 mbar, 40 ° C). Destilar el HEMA bajo presión reducida (15 mbar; 99 ° C). </li><li> Prepare una solución 0,62 M en metanol de la HEMA-inhibidor libre producido en la Sección 1.1.1. Verter 30 ml de la solución en un matraz de una boca y eliminar el oxígeno por burbujeo de Ar a través de la solución durante 1 hora. </li></ol></li><li> Polimerización inducida en superficie. <ol><li> Coloque dos membranas de policarbonato lado de la otra en la cámara de plasma (Figura 2). Coloque el lado brillante de la membrana que apunta hacia la fase gaseosa. </li><li> Conecte la cámara de plasma de ahIGH de vacío (20 mbar) durante 5 min. Cerrar la válvula a la de vacío y abrir la otra válvula, que está conectado con argón y gas oxígeno y purgar la cámara con esta mezcla durante 2 h con 15 sccm de argón y oxígeno 2,5 sccm. </li><li> Iniciar el plasma y reducir la alimentación a la fuente deseada (por membrana de policarbonato: 12 W) y el tratamiento de las membranas durante 4 min con el plasma. Conectar la solución de monómero con la cámara mediante la apertura de la válvula correspondiente. Apague el plasma y evacuar la cámara. </li><li> Conectar la solución de monómero con la cámara mediante la apertura de la válvula correspondiente y se vierte la solución en la cámara. Asegúrese de que las membranas están cubiertos con la solución de monómero. Abrir la válvula conectado a la argón y almacenar la mezcla de reacción durante 12 horas a 20 ° C (sala de acondicionado). </li><li> Retire la solución de monómero. Se lavan las membranas con metanol en un baño de ultrasonidos durante 5 min. Repita el procedimiento de lavado con agua. </li><li> Secar la membrana envacío sobre tamices moleculares durante 2 horas. </li></ol></li></ol> 2. Modificación posterior de las membranas recubiertas <ol><li> Preparar una solución de spirobenzopyran 1 (Figura 1) (100 mg; 0,27 mmoles), N, N-diciclohexilcarbodiimida (DCC) (55 mg; 0,27 mmol) y dimetil aminopiridina (DMAP) (33 mg, 0,27 mmol) en terc-butylmetylether (TBME) (12 ml). </li><li> Coloque una barra agitadora protección y una rejilla de protección en un matraz de fondo redondo. Secar el matraz e inundar el matraz con argón. </li><li> Verter la solución en el matraz, seguido de la membrana recubierta. </li><li> Revuelva suavemente a temperatura ambiente durante 12 horas. </li><li> Eliminar la solución y se lava la membrana con terc-butylmetylether en un baño de ultrasonidos durante 5 min. Repita el procedimiento de lavado con etanol y agua. </li><li> Secar la membrana a vacío sobre tamices moleculares durante 2 horas. </li></ol> 3. Mediciones de la tensión superficial <ol><li&gt; Fix la membrana con una cinta de serie en una junta tórica de metal. Coloque una gota de agua nanopura (3,3 l) en la parte de la superficie de la membrana, que no está en contacto con la junta tórica. Mida el ángulo de contacto (CA) a 5 puntos diferentes de la membrana. </li><li> Para el ensayo de la estabilidad a largo plazo de las muestras, medir los ángulos de contacto en tres puntos diferentes de las membranas después de 0, 1, 2, 3, 7, 14, y 21 días. </li></ol> 4. Ensayos de permeabilidad de las membranas fotocrómicos <ol><li> Llenar la cámara del receptor de la célula de difusión de Franz con agua (12 ml). </li><li> Fijar la membrana en una célula de difusión de Franz. Asegúrese de que la membrana está en contacto con el agua de la cámara receptora. Llenar la cámara donante (la cámara en la parte superior de la membrana) con una solución acuosa de cafeína (20 mM; 3,0 ml). Irradiar la membrana desde la parte superior de la cámara donante con luz blanca (Figura 3). Recoger muestras (200 l) de la célula receptora; for membranas de policarbonato con bordes de la pista con un diámetro de poro de 200 nm, recoger muestras cada 10 min. </li><li> Repita el experimento como se describe en el paso 4.2. pero irradiar la membrana con luz UV (366 nm, 80 W / m 2) durante todo el ensayo de permeabilidad. </li><li> Determinación de las concentraciones de cafeína de las muestras recogidas. <ol><li> Trazar una curva de calibración con 15 concentraciones de cafeína diferentes (entre 0,05 mg / ml y 1,5 mM / l) utilizando un espectrómetro UV / VIS. Calibrar a 293 nm. </li><li> Determinar la concentración de cada una de las muestras recolectadas utilizando la curva de calibración. </li><li> Trazar la concentración determinada vs. el tiempo de las muestras recogidas. Hacer un ajuste lineal a través de los puntos y determinar? C de la pendiente. </li></ol></li></ol>

<ol>
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Los autores no tienen nada que revelar.

El proceso de plasma produce un gas de color púrpura, que es causada por argón ionizado. Un color anaranjado indicaría la presencia de nitrógeno no deseado de una fuga. El proceso de plasma no sólo formar radicales en la superficie sino también graba la membrana de 7,12. El exceso de ataque químico puede cambiar el diámetro de poro significativamente, lo que influiría en la permeabilidad de la membrana. Las condiciones de reacción controladas de la configuración presentada permiten la mejora de la ...

Modificación de plasma de materiales se ha convertido en un proceso importante en muchos campos industriales. Limpieza de superficies y funcionalización de superficies sin cambiar la propiedad mayor parte del material ha hecho que el tratamiento con plasma de un proceso esencial en la ciencia de superficies 1-8. El tratamiento con plasma de polímeros da como resultado la escisión del enlace homolítica. Esto conduce a un borde del material polimérico y a la formación de superficies ricas radicales. Mediante el uso de plasma que contiene moléculas de oxígeno, la superficie se vuelve rica en oxígeno y por lo tanto más hidrófilo 9-11. Sin embargo, la hidrofilicidad de las superficies no es estable en el tiempo 12. Con el fin de mejorar la estabilidad a largo plazo, la superficie tratada con plasma se puede modificar químicamente después de o durante el proceso de plasma 13-15. Este tratamiento se realiza normalmente mediante la adición de una especie de monómeros reactivos en la fase gaseosa durante el proceso de plasma; estos monómeros se polimerizan luegode los radicales creados de la superficie del polímero. Si el tratamiento químico se lleva a cabo con un monómero no volátil, el injerto de polímero tiene que tener lugar después de la modificación de plasma. Con el fin de realizar un injerto controlada después de que los radicales se forman en la superficie, una configuración de plasma se describe, que permite la polimerización inducida por plasma de la superficie-iniciada desde la superficie en solución bajo condiciones controladas 12,16. La presentación se centra en la modificación de las membranas de polímero pista filo 12,17. Mediante la modificación de la tensión superficial de estas membranas, la tasa de permeabilidad se puede variar 12. Este proceso limpio y rápido permite la creación de capas muy finas (&lt;5 nm), que cubren toda la superficie de la membrana sin cambiar la propiedad mayor de la membrana de polímero. Debido a la ribete durante el proceso de plasma, los diámetros de los poros de las membranas de la pista-filo aumentan ligeramente 12. La tasa de ribete es depending en el polímero y tiene un comportamiento lineal. Cuando se utiliza monómeros con grupos funcionales reactivos, los polímeros injertados se pueden funcionalizar adicionalmente. Esto se demuestra por la modificación posterior de una membrana de PHEMA-injertado con un ácido carboxílico de espiropirano funcionalizado. Esto resulta en una superficie fotocrómico, desde espiropirano se conoce para transformarse en una especie de merocianina cuando se irradia con luz UV. La forma espiropirano se puede restablecer mediante la irradiación de la forma merocianina con luz visible (Figura 1) 18,19. Puesto que la forma de merocianina es más polar que el estado de espiropirano, la tensión superficial del recubrimiento puede ser activado con la luz 20. El cambio en la tensión superficial influye en la resistencia a la permeabilidad de la membrana hacia soluciones acuosas. La puesta a punto la forma de realizar los ensayos de permeabilidad de estas membranas de luz sensible se mostrará y el cambio significativo en la resistencia a la permeabilidad (disminuir in resistencia a la permeabilidad en un 97%) se demostró. Tal membrana se puede integrar en una configuración de administración de fármacos o en los sistemas de detección inteligentes. <img alt="Figura 1" fo:content-width="5in" fo:src="/files/ftp_upload/51680/51680fig1highres.jpg" src="/files/ftp_upload/51680/51680fig1.jpg" /> Figura 1. Fotoisomerización de compuesto spirobenzopyran 1.

<table border="0" cellpadding="0" cellspacing="0" style="width:943px;" width="943">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Name of Reagent/ Equipment</td>
			<td style="width:284px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">2-Hydroxyethyl methacrylate, 97%</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">128635</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Hexane 99%</td>
			<td style="width:284px;">Biosolve</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Magnesium sulfate (MgSO4, anhydrous)</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Methanol, 99%&#160;</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">14262</td>
			<td>dried over molecular sieves</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">N,N-Dicylcohexylcarbodiimide, 99%</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Dimethyl aminopyridine, 99%</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">107700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Tert-butylmethylether, 98%</td>
			<td style="width:284px;">Fluka</td>
			<td style="width:119px;">306975</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Polycarbonate membrane</td>
			<td style="width:284px;">Whatman</td>
			<td style="width:119px;"></td>
			<td>Nanopore Track Etched (TE) (1.0 &#956;m, 0.2 &#956;m, 0.1 &#956;m, 50 nm, 30 nm and 15 nm pore diameter; 47 mm or 25 mm membrane diameter)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Caffeine (reagent plus)</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">C0750&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Franz diffusion cell (12 ml)</td>
			<td style="width:284px;">SES-Analysesysteme</td>
			<td style="width:119px;">6C010015</td>
			<td>15mm unjacheted Franz Cell, 12 ml Receptor volume, Flat ground, clear glass, stirbat and clamp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">UV-Lamp</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>UV irradiation (366 nm, 15 W/m2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">White light lamp</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>White light irradiation (500 W bulb)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">UV/Vis spectrophotometer</td>
			<td style="width:284px;">Varian 50Bio/50MPR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:373px;">Polyester membranes</td>
			<td style="width:284px;">Sterlitech</td>
			<td style="width:119px;">PET0225100</td>
			<td>Polyester Membrane Filters, 0.2 &#956;m pore diameter, 25 mmm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Polyvinylidene fluoride membranes</td>
			<td style="width:284px;">Millipore</td>
			<td style="width:119px;"></td>
			<td>PVDF Membranes Durapore (0.22 &#956;m pore diameter; 47 mm membrane diameter)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Argon (99.9995%)</td>
			<td style="width:284px;">Alphagaz</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Dressler Cesar RF Power Generator</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>Plasma chamber setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">MKS Multi Gas Controller 647C</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>Plasma chamber setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">MKS Mass-Flow controllers</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>Plasma chamber setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Vacuubrand RE 2.5 rotary vane vacuum pump</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>Plasma chamber setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Contact angle measurement device Kr&#252;ss G10</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Balances Mettler Toledo AB204-S and Mettler ME30</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of light-responsive membranes by a combined surface grafting and postmodification process

Con el fin de modificar la tensión superficial de las membranas de polímero de la pista-filo disponibles comerciales, se presenta un procedimiento de polimerización iniciada por la superficie. La polimerización de la superficie de la membrana es inducida por tratamiento de plasma de la membrana, seguido por reacción de la superficie de la membrana con una solución metanólica de metacrilato de 2-hidroxietilo (HEMA). Se presta especial atención a los parámetros de proceso para el tratamiento de plasma antes de la polimerización en la superficie. Por ejemplo, se estudia la influencia de la-tratamiento de plasma en diferentes tipos de membranas (por ejemplo, poliéster, policarbonato, fluoruro de polivinilideno). Además, la estabilidad dependiente del tiempo de las membranas injertado de superficie se muestra por las mediciones del ángulo de contacto. Cuando el injerto de poli (metacrilato de 2-hidroxietilo) (PHEMA) de esta manera, la superficie puede ser modificado adicionalmente mediante la esterificación del resto alcohol del polímero con una función de ácido carboxílico de la sustancia deseada.Por tanto, estas reacciones se pueden utilizar para la funcionalización de la superficie de la membrana. Por ejemplo, la tensión superficial de la membrana puede ser cambiada o una funcionalidad deseada como la capacidad de respuesta luz presentada puede ser insertado. Esto se demuestra mediante la reacción de PHEMA con un ácido carboxílico funcionalizado unidad spirobenzopyran lo que conduce a una membrana responde a la luz. La elección del disolvente juega un papel importante en la etapa de modificación posterior y se discute con más detalle en este documento. Las mediciones de la permeabilidad de dichas membranas funcionalizados se llevan a cabo usando una celda de Franz con una fuente de luz externa. Al cambiar la longitud de onda de la luz de lo visible a la gama UV, se observa un cambio de la permeabilidad de las soluciones acuosas de cafeína.

La velocidad de grabado puede ser seguido por un peso de la membrana después de diferentes períodos de tiempo. Como se puede ver en la figura 4, la velocidad de grabado sigue para el poliéster, fluoruro de polivinilideno, policarbonato y membranas de una velocidad de grabado lineal, que se puede determinar a partir de la pendiente de la correlación lineal entre el tiempo de grabado en comparación con la pérdida de masa. Como se muestra en la Figura 4, las membranas de pol...

Watch this Scientific Journal Video about Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process at JoVE.com

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

Un procedimiento de polimerización inducida por plasma se describe para la polimerización iniciada por superficie en las membranas poliméricas. Además modificación posterior del polímero injertado con sustancias fotocrómicas se presenta con un protocolo de la realización de mediciones de la permeabilidad de las membranas responde a la luz.

Preparación de Membranas Light responden por un injerto superficial combinado y proceso de modificación posterior

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

preparation-light-responsive-membranes-combined-surface-grafting

Research

JoVE Journal

Bioengineering

11.7K vistas.  Empa, Swiss Federal Laboratories for Materials Science and Technology.  Un procedimiento de polimerización inducida por plasma se describe para la polimerización iniciada por superficie en las membranas poliméricas. Además modificación posterior del polímero injertado con sustancias fotocrómicas se presenta con un protocolo de la realización de mediciones de la permeabilidad de las membranas responde a la luz. 

Video: Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail1,2. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another1-5, (2) can strongly influence the ability of tissues to interact with biomaterials6, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion7, but also by manipulation of key ECM molecules such as FN8-11 and 4) correlates with invasive potential of lung cancer12, fibrosarcoma13, brain tumor14 and prostate tumor cell lines15. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.

We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.

La medición de la cohesión agregado por tensiometría tejido de la superficie

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium.  We ...

Investigación

Bioingeniería

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Los andamios de autoinforme para el 3-Dimensional Cultivo Celular

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

Generación y Acrecentamiento de buques de ingeniería de tejidos en un modelo de ratón

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Adaptación del Proceso Electrohilado para proporcionar tres ambientes únicos para un Tri-capa<em&gt; In Vitro</em&gt; Modelo de la pared de la vía aérea

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

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 ...

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

Técnica robótica automatizada de dispensación de Orientación de la superficie de las células y Bioprinting

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

Síntesis de hidrogeles con propiedades antiincrustantes como membranas de purificación de agua

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and survival post-transplantation. This protocol aims to achieve surface engineering of pancreatic islets using an ultrathin heparin-incorporated starPEG (Hep-PEG) nanocoating. To generate the Hep-PEG nanocoating for pancreatic islet surface engineering, heparin succinimidyl succinate (Heparin-NHS) was first synthesized by modification of its carboxylate groups using N-(3-dimethylamino propyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The Hep-PEG mixture was then formed by crosslinking of the amino end-functionalized eight-armed starPEG (starPEG-(NH2)8) and Heparin-NHS. For islet surface coating, mouse islets were isolated via collagenase digestion and gradient purification using Histopaque. Isolated islets were then treated with ice cold Hep-PEG solution for 10 min to allow covalent binding between NHS and the amine groups of islet cell membrane. Nanocoating with the Hep-PEG incurs minimal alteration to islet size and volume and heparinization of the islets with Hep-PEG may also reduce instant blood-mediated inflammatory reaction during islet transplantation. This "easy-to-adopt" approach is mild enough for surface engineering of living cells without compromising cell viability. Considering that heparin has shown binding affinity to multiple cytokines, the Hep-PEG nanocoating also provides an open platform that enables incorporation of unlimited functional biological mediators and multi-layered surfaces for living cell surface bioengineering.

This protocol aims to achieve surface engineering of pancreatic islets using a heparin-incorporated starPEG nanocoating via pseudo-bioorthogonal chemistry between the N-hydroxysuccinimide groups of the nanocoating and the amine groups of islet cell membrane.

Ingeniería de superficies de islotes pancreáticos con un nanocobertura StarPEG heparinizada

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Detección de endotoxinas en las formulaciones Nano mediante ensayos de Limulus Amoebocyte Lysate (LAL)

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the wet spinning method, gelatin fibers are produced by smooth extrusion in a suitable coagulation medium. To increase the functional surface of these gelatin fibers and their ability to mimic the features of tissues, gelatin can be molded into a tube form by referring to this concept. Examined by in vitro and in vivo tests, the gelatin tubes demonstrate a great potential for application in tissue engineering. Acting as a suitable filling gap material, gelatin tubes can be used to substitute the tissue in the damaged area (e.g., in the nervous or cardiovascular system), as well as to promote regeneration by providing a direct replacement of stem cells and neural circuitry. This protocol provides a detailed procedure for creating a biomaterial based on a natural polymer, and its implementation is expected to greatly benefit the development of correlative natural polymers, which help to realize tissue regeneration strategies.

We developed and describe a protocol based on the wet spinning concept, for the construction of gelatin-based biomaterials used for the application of tissue engineering.

Proceso de moldeo basada en mojado-spinning de gelatina para la regeneración de los tejidos

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the ...