Apoyado por el NIH subvención HD052670.

1. El recubrimiento de placas con las proteínas ECM <ol><li> En condiciones estériles, mezclar 120 g de laminina con 12 ml de PBS estéril 1X frío por placa. </li><li> Tomar Bioflex placas sin tratar y añadir 2 ml de la solución a cada pocillo (concentración final de laminina es 2 mg / cm 2). Alternativamente, otras proteínas ECM podrían ser utilizados, tales como colágeno-1 [10 g / cm 2], la fibronectina [5 g / cm 2], vitronectina [0,5 g / cm 2] o elastina [10 g / cm 2]. La técnica de revestimiento es similar a lo que se ha descrito para laminina-recubrimiento. Asegúrese de que los pozos están completamente cubiertas por la solución. </li><li> Envolver las placas con plástico y poner a 4 ° C sobre una superficie plana durante la noche para permitir la laminina para adsorber al fondo de los pocillos. En estas condiciones, las placas pueden ser almacenados por lo menos una semana al tiempo que conserva una buena función ECM. </li><li> Al día siguiente, bajo condiciones estériles, lavar los pocillos 3 veces con PBS 1X. </li><li&gt; Añadir 1 ml de BSA al 1% en PBS 1X a cada pocillo y se incuba a 37 ° C durante 1 hora para no bloquear sitios de unión específicos en las membranas. </li><li> Lavar las placas 3 veces con PBS 1X para eliminar las proteínas no adsorbidas. </li><li> Mantener las placas a 37 ° C en DMEM hasta que las células se aíslan de paso 2,14. </li></ol> 2. Aislamiento de pulmón fetal roedores células de tipo II epiteliales El día antes del aislamiento que las tazas de pantalla con diferentes mallas de nylon de tamaño autoclave y listo. <ol><li> Obtener los pulmones del feto a partir de disolución-embarazada de ratón / rata en la E17-19 de la gestación. Transferir el tejido en un tubo cónico de 50 ml que contenía medio DMEM y lo coloca en hielo. </li><li> Hacer tampón de digestión en un tubo cónico de 50 ml: (10 ml). Para este volumen, el tejido pulmonar de aproximadamente 20 fetos de ratón / rata se utiliza. 8,5 ml de DMEM 0,5 ml de 500 mM de pH 7,4 HEPES 5 mg de colagenasa 1 5 mg de colagenasa 1A 1 ml de suero de pollo, inactivado por calor </li><li>Mezclar bien hasta que todos los componentes se disuelven y el filtro a través de un filtro de 0,2 micras jeringa dentro de la campana estéril. Se coloca el tubo cónico que contenía el tampón de digestión en un baño de agua a 37 ° C. </li><li> Eliminar el medio DMEM del tubo cónico de paso 2,1 y transferir el tejido en una placa de Petri estéril. </li><li> Picar los pulmones bien con tijeras estériles o cuchilla de afeitar para hacer piezas de tejido inferior a 1 mm y transferir el tejido en el tubo cónico que contenía el tampón de digestión pre-calentado desde el paso 2,3. </li><li> Digerir el tejido pipeteando arriba y abajo: 100 veces con 10 ml de pipeta 100 veces con 5 ml de pipeta 100 veces con pipeta Pasteur </li><li> Centrifugar el homogenado a 1300 rpm durante 5 minutos a temperatura ambiente. </li><li> Retirar con cuidado el sobrenadante por aspiración y volver a suspender el precipitado que contiene las células en 15 ml de DMEM más FBS al 20%. </li><li> Saca las copas de pantalla con 100 -, 30 - y 15 micras y mallas de nylon º lugarem en estériles de 150 ml vasos. </li><li> Añadir homogenado de células a partir del paso 2.8 y secuencialmente filtrar a través de 100 -, 30 -, y 15 micras tazas de malla. </li><li> Recoge agrupadas sin filtrar las células de los años 30 - y las mallas de 15 micras después de varios lavados con DMEM más FBS al 10% para facilitar la filtración de células no epiteliales. </li><li> Desechar el filtrado de la malla 15-micras que contiene principalmente los fibroblastos. </li><li> Purificar nuevas células de tipo II mediante la incubación de las células de paso 2,11 cm en 75-2 matraces durante 30 min (approximatelly10 ml de suspensión de células por matraz). </li><li> Recoger el sobrenadante y se centrifuga a 1.300 rpm durante 5 minutos a temperatura ambiente para precipitar las células. </li><li> Retire con cuidado el sobrenadante por aspiración y vuelva a suspender el pellet que contiene las células en DMEM libre de suero. El volumen de resuspensión depende de la cantidad de tejido procesado. Aproximadamente, se placa el equivalente de células obtenidas de un feto en cada pocillo (2 ml) a fin de Achivíspera entre 60-70% de confluencia el día siguiente. </li><li> Placa de suspensión de la celda en Bioflex seis placas recubiertas con laminina y fibronectina. </li></ol> 3. Aislamiento de fibroblastos fetales roedores pulmonares <ol><li> Siga los mismos pasos descritos anteriormente para las células de tipo II de aislamiento de hasta 2,11. </li><li> Tomar el filtrado de la malla 15-micrones (sin lavado previo; volumen aproximado es de 10-15 ml) y la placa a 75 cm de 2 a 37 ° C durante 30-60 minutos para permitir que los fibroblastos de adherirse. </li><li> Aspirar el sobrenadante y reemplazar con DMEM libre de suero y se incuba durante la noche. </li><li> Al día siguiente, cosechar las células con 0,25% (peso / volumen) de tripsina en 0,4 mM EDTA y la placa de ellas en placas recubiertas previamente con fibronectina Bioflex. </li></ol> 4. Sistema experimental para proporcionar la estimulación mecánica a las células pulmonares <ol><li> De semillas de las células (alrededor del 50% confluente) en placas de cultivo de Bioflex en DMEM libre de suero (2 ml / pocillo) y dejar que ellos adhieren y SPREanuncio por lo menos durante 6h antes de los experimentos. En general nuestro laboratorio mantiene células en cultivo de alrededor de 24 horas antes de la aplicación de la tensión mecánica. Si un número determinado de células son necesarios para los experimentos, después del aislamiento, las células de tipo II son mantenidos en DMEM libre de suero de 75 cm 2 frascos y al día siguiente, las células se tripsinizaron, se contaron y sembraron entonces. </li><li> Al día siguiente, los medios se sustituyen con fresco DMEM libre de suero (2 ml / pocillo) y las placas Bioflex están montados en una unidad Flexcell FX-5000 Cepa. Monocapas no debe ser mayor del 80% confluentes antes de la iniciación de los experimentos. </li><li> Cepa Equibiaxial se aplica a las membranas. El régimen de deformación varía en función de simulación de fuerzas mecánicas in vivo (véase la discusión). </li><li> Las células cultivadas en no-estirados membranas cultivadas en paralelo se utilizan como controles. </li><li> Al final de los experimentos, las monocapas se pueden procesar para analizar los cambios en la expresión génica (por ejemplo la proteína C surfactante)por la abundancia de proteínas PCR en tiempo real, por Western blot, etc Además, las monocapas se puede fijar para los experimentos de inmunocitoquímica. Para esta técnica, después de la fijación, las membranas de silastic se cortan a partir de las placas y se montaron en portaobjetos de vidrio utilizando 10-20 l de agua como un agente de montaje antes de permeabilización e incubación con anticuerpos. El sobrenadante puede utilizarse también para investigar la presencia de factores de crecimiento, citoquinas, etc </li></ol> 5. Los resultados representativos Figura 1 y Figura 2 muestra representativa de contraste de fase fotografías de fetos de ratón E18 células tipo II aislado utilizando la técnica descrita en este manuscrito. La Figura 3 demuestra que la tensión mecánica induce la diferenciación de las células de tipo II fetales epiteliales utilizando la proteína surfactante-C como un marcador. <img src="/files/ftp_upload/3543/3543fig1.jpg" /> Figura 1. Repsentante de contraste de fase fotografía tomada justo después de aislamiento que muestra el aspecto de los fetos se agruparon las células tipo II. <img src="/files/ftp_upload/3543/3543fig2.jpg" /> Figura 2. E18 fetales células de tipo II epiteliales se aislaron como se describe aquí y chapada en placas recubiertas con Bioflex laminina. La fotografía fue tomada el día siguiente después de no estirado las células fueron fijadas en paraformaldehído. La pureza de las células se determinó que era de 90 ± 5% por análisis microscópico de la morfología de las células epiteliales y inmunotinción para SP-C. <img src="/files/ftp_upload/3543/3543fig3.jpg" /> Figura 3. Fetal las células tipo II epiteliales fueron expuestos a la cepa cíclico de 5% a 40 ciclos por minuto durante 16 horas. Una mancha) del Norte de la proteína surfactante C (SP-C) que muestra que la expresión del ARNm cepa induce la diferenciación de células de tipo II utilizando diferentes sustratos ECM . Signos + / - representan la exposición o no a la sentrenar, respectivamente. Los datos se presentan como media + / - SEM, n = 3; * P &lt;0,05 B) Imágenes de fluorescencia inmunocitoquímica demuestra la SP-C los niveles de proteína (verde) en las células tipo II fetales expuestos o no (control) a una tensión mecánica.. Los núcleos se tiñeron con DAPI (azul). Bar, 10 m. C) los resultados de Western blot a partir de tres experimentos que demuestran que el estiramiento mecánico aumenta la proteína SP-C. N = 3; * p &lt;0,05.

<ol>
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Lung. Cell. Mol. Physiol. 291, L820-L827 (2006).</li><li>Sanchez-Esteban, J., Wang, Y., Filardo, E. J., Rubin, L. P., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+{beta}1,+{alpha}6,+and+{alpha}3+contribute+to+mechanical+strain-induced+differentiation+of+fetal+lung+type+II+epithelial+cells+via+distinct+mechanisms.">Integrins {beta}1, {alpha}6, and {alpha}3 contribute to mechanical strain-induced differentiation of fetal lung type II epithelial cells via distinct mechanisms.</a> Am. J. Physiol. Lung. Cell. Mol. Physiol. 290, L343-L350 (2006).</li><li>Wang, Y., Maciejewski, B. S., Soto-Reyes, D., Lee, H. 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En este manuscrito, se describe un método para aislar células tipo II fetales epiteliales y fibroblastos y exponerlos a la estimulación mecánica utilizando el aparato de tensión Flexcell. Hemos utilizado esta técnica para evaluar la diferenciación de las células epiteliales 1,2 y el estudio de los receptores y las vías de señalización activadas por tramo 3-9. Además, este método también se puede utilizar para investigar las respuestas de células inducidas por lesión mecánica 1...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D5648</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase 1</td>
			<td>Sigma</td>
			<td>C0130</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase 1A</td>
			<td>Sigma</td>
			<td>C9891</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chicken serum</td>
			<td>Sigma</td>
			<td>C5405</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Screen cups</td>
			<td>Sigma</td>
			<td>CD1-1KT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Syringe filters</td>
			<td>Fisher Scientific</td>
			<td>09-754-25</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>100 micron nylon mesh</td>
			<td>Small Parts, INC</td>
			<td>CMN-100-D</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>30 micron mesh</td>
			<td>Small Parts, INC</td>
			<td>CMN-30-D</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 micron mesh</td>
			<td>Dynamic Aqua-Supply Ltd.</td>
			<td>NTX15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagen-1</td>
			<td>Collagen Biomed</td>
			<td>PC0701</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma</td>
			<td>F1141</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vitronectin</td>
			<td>Sigma</td>
			<td>V-0132</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Elastin</td>
			<td>Sigma</td>
			<td>E-6402</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bioflex plate</td>
			<td>Flexcell International</td>
			<td>BF-3001U</td>
			<td>Uncoated</td>
		</tr>
		<tr>
			<td>Flexcell Strain Unit</td>
			<td>Flexcell International</td>
			<td>FX-5000</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

an experimental system to study mechanotransduction in fetal lung cells

Las fuerzas mecánicas generadas en el útero por la repetición como la respiración, los movimientos y por la distensión del fluido son fundamentales para el desarrollo normal de los pulmones. Un componente clave del desarrollo de los pulmones es la diferenciación de las células alveolares de tipo II epiteliales, la principal fuente de surfactante pulmonar. Estas células también participan en la homeostasis de los líquidos en la luz alveolar, la defensa del huésped, y la reparación de lesiones. Además, las células del parénquima pulmonar distal puede estar directamente expuestos a la extensión exagerada durante la ventilación mecánica después del nacimiento. Sin embargo, los mecanismos moleculares y celulares mediante el cual las células pulmonares detectan los estímulos mecánicos para influir en el desarrollo pulmonar y promover la lesión pulmonar no se conocen. Aquí, se proporciona un método simple y de alta pureza para aislar las células de tipo II y fibroblastos de pulmón fetal de roedores. A continuación, se describe un sistema in vitro, la Unidad de tensión Flexcell, para proporcionar la estimulación mecánica de las células fetales, simulando las fuerzas mecánicas deel desarrollo pulmonar fetal o lesión pulmonar. Este sistema experimental constituye una excelente herramienta para investigar los mecanismos moleculares y celulares en las células del pulmón fetal expuestos a estirar. Con este enfoque, nuestro laboratorio ha identificado varios receptores y las proteínas de señalización que participan en mecanotransducción en el desarrollo pulmonar fetal y la lesión pulmonar.

Watch this Scientific Journal Video about An Experimental System to Study Mechanotransduction in Fetal Lung Cells at JoVE.com

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Las fuerzas mecánicas juegan un papel clave en el desarrollo pulmonar y lesión pulmonar. A continuación, describimos un método para aislar roedores pulmonares fetales células tipo II epiteliales y fibroblastos y exponerlos a la estimulación mecánica mediante un<em&gt; In vitro</em&gt; Sistema.

Un sistema experimental para estudiar Mecanotransducción en células de pulmón fetal

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

an-experimental-system-to-study-mechanotransduction-fetal-lung

Research

JoVE Journal

Bioengineering

12.9K vistas.  Alpert Medical School of Brown University. Las fuerzas mecánicas juegan un papel clave en el desarrollo pulmonar y lesión pulmonar. A continuación, describimos un método para aislar roedores pulmonares fetales células tipo II epiteliales y fibroblastos y exponerlos a la estimulación mecánica mediante un In vitro Sistema.

Video: An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

El crecimiento del axón Tramo: El Mecanotransducción de crecimiento neuronal

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

Investigación

Bioingeniería

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Diseño de un biorreactor de presión cíclica de la Ex Vivo Estudio de las válvulas aórtica del corazón

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1. The main difficulty of this assay is the perceived cost ($25-100K) and technical expertise required for implementing a laser ablation system2,3. However, solid-state pulse lasers of modest costs (&lt;$10K) can provide robust performance for laser ablation in transparent preparations where target axons are "close" to the tissue surface. Construction and alignment of a system can be accomplished in a day. The optical path provided by light from the focused condenser to the ablation laser provides a convenient alignment guide. An intermediate module with all optics removed can be dedicated to the ablation laser and assures that no optical elements need be moved during a laser ablation session. A dichroic in the intermediate module allows simultaneous imaging and laser ablation. Centering the laser beam to the outgoing beam from the focused microscope condenser lens guides the initial alignment of the system. A variety of lenses are used to condition and expand the laser beam to fill the back aperture of the chosen objective lens. Final alignment and testing is performed with a front surface mirrored glass slide target. Laser power is adjusted to give a minimum size ablation spot (&lt;1um). The ablation spot is centered with fine adjustments of the last kinematically mounted mirror to cross hairs fixed in the imaging window. Laser power for axotomy will be approximately 10X higher than needed for the minimum ablation spot on the target slide (this may vary with the target you use). Worms can be immobilized for laser axotomy and time-lapse imaging by mounting on agarose pads (or in microfluidic chambers4). Agarose pads are easily made with 10% agarose in balanced saline melted in a microwave. A drop of molten agarose is placed on a glass slide and flattened with another glass slide into a pad approximately 200 um thick (a single layer of time tape on adjacent slides is used as a spacer). A "Sharpie" cap is used to cut out a uniformed diameter circular pad of 13mm. Anesthetic (1ul Muscimol 20mM) and Microspheres (Chris Fang-Yen personal communication) (1ul 2.65% Polystyrene 0.1 um in water) are added to the center of the pad followed by 3-5 worms oriented so they are lying on their left sides. A glass coverslip is applied and then Vaseline is used to seal the coverslip and prevent evaporation of the sample.

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Laser axotomy followed by time-lapse imaging is a sensitive way to assay the effects of mutations in C. elegans on axon regeneration. A high quality, but inexpensive, laser ablation system can be easily added to most microscopes. Time lapse imaging over 15 hours requires careful immobilization of the worm.

La construcción de un láser de bajo presupuesto axotomía Sistema para el Estudio de la regeneración de axones en C. elegans</em

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1.  The main difficulty of this assay ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

La luminiscencia de energía de resonancia de transferencia para estudiar cambios conformacionales en proteínas de membrana expresado en células de mamífero

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

Estudios electrotaxis de pulmón células de cáncer utilizando un Multicanal Dual-campo eléctrico chip de microfluidos

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Generación de células epiteliales de las vías respiratorias del ratón ESC-Obtenido usando Descelularizado pulmón andamios

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

An Experimental Protocol for Assessing the Performance of New Ultrasound Probes Based on CMUT Technology in Application to Brain Imaging

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ultrasound (US) probes. Particularly, a more realistic analysis with application-specific imaging targets can be extremely valuable to assess the expected performance of US probes in their potential clinical field of application.
The experimental protocol presented in this work was purposely designed to provide an application-specific assessment procedure for newly-developed US probe prototypes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology in relation to brain imaging.
The protocol combines the use of a bovine brain fixed in formalin as the imaging target, which ensures both realism and repeatability of the described procedures, and of neuronavigation techniques borrowed from neurosurgery. The US probe is in fact connected to a motion tracking system which acquires position data and enables the superposition of US images to reference Magnetic Resonance (MR) images of the brain. This provides a means for human experts to perform a visual qualitative assessment of the US probe imaging performance and to compare acquisitions made with different probes. Moreover, the protocol relies on the use of a complete and open research and development system for US image acquisition, i.e. the Ultrasound Advanced Open Platform (ULA-OP) scanner.
The manuscript describes in detail the instruments and procedures involved in the protocol, in particular for&#160;the calibration, image acquisition and registration of US and MR images. The obtained results prove the effectiveness of the overall protocol presented, which is entirely open (within the limits of the instrumentation involved), repeatable, and covers the entire set of acquisition and processing activities for US images.

The development of new ultrasound (US) probes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology requires an early realistic assessment of imaging capabilities. We describe a repeatable experimental protocol for US image acquisition and comparison with magnetic resonance images, using an ex vivo bovine brain as an imaging target.

Un protocolo Experimental para evaluar el desempeño de nuevas sondas de ultrasonido basado en la tecnología CMUT en aplicación a la proyección de imagen de cerebro

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Nanotubos de ADN como una herramienta versátil para el estudio de polímeros Semiflexible

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Un Orbital agitación cultivo de células mamíferas en vasos en forma de O para producir agregados uniforme

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.