Este trabajo fue fundada por el Institut Curie, París, Francia. La camilla mecánica fue diseñada por Damien Cuvelier (Institut Curie) y es fabricado por GREM (mecanique-grem.com). El patrón de PDMS fue desarrollado por Ammar Azioune (Universidad de Burdeos II).

1. La pasivación de la PDMS <ol><li> Corte un pedazo de PDMS aproximadamente 35 mm x 20 mm a partir de una hoja de pre-hechos (por ejemplo, GelPak, que se enumeran en la tabla de materias). </li><li> Quite la parte superior y las capas protectoras inferiores de plástico (si es necesario) y utilizar pinzas para colocar el PDMS en un plástico (no celular cultivo tratado) placa de Petri. </li><li> Lavar el PDMS con etanol al 70% durante 5 minutos en un rotador a 30 oscilaciones / min. </li><li> Seque la superficie por las corrientes de aire en él. </li><li> Iluminar con profunda UV (λ = 180 nm) durante 5 minutos a una distancia de las bombillas UV de unos 5 cm (ver hoja de Materiales para referencia lámpara; parámetros variarán para diferentes lámparas). </li><li> Preparar 200 l de solución de EDC / NHS para cada PDMS pieza (este debe estar preparado justo antes de su uso debido a que la reactividad de la solución se desintegra en cuestión de horas). Para 1 ml de 0,05 M MES + 0,5 M de tampón NaCl a pH 6,0, agregar 11,5 mg de sulfo-NHS y 19,2 mg de EDC. </li><li> Transferir elPDMS hojas de la placa de Petri a la tapa de la placa de Petri, que no ha sido iluminado. Esto asegurará que el entorno de los PDMS son muy hidrófobo y facilitar el siguiente paso. </li><li> Añadir la solución de EDC / NHS y se incuba durante 15 min a temperatura ambiente. </li><li> Enjuague EDC / NHS con agua. </li><li> Añadir la solución de PLL-g-PEG (0,5 mg / ml en HEPES 10 mM pH 8,6) y se incuba a partir de 3 horas a toda la noche a temperatura ambiente. </li><li> Enjuague el PLL-g-PEG con agua. PDMS pasivados (funcionalizadas con PLL-g-PEG) se pueden almacenar durante varios días a 4 ° C. </li></ol> 2. Patrones de la PDMS <ol><li> Tome una hoja de PDMS y colocarlo en una fotomáscara de cuarzo sintético que lleva las microfeatures para modelar (ver Figura 1). Coloque el lado de PDMS que lleva el PLL-g-PEG hacia el lado del cromo de la fotomáscara. </li><li> Iluminar durante 7 min a través de la fotomáscara a una distancia de las lámparas UV de aproximadamente 5 cm.</li><li> Añadir agua en la máscara + PDMS y pelar las PDMS lentamente fuera de la máscara. </li><li> Incubar con solución de fibronectina a 50 mg / ml en tampón de HEPES a pH 8,6 durante 1 hora a temperatura ambiente. Es posible utilizar otras proteínas ECM, pero éstos no se han probado con este protocolo. </li><li> Lavar con PBS. </li></ol> 3. Montaje del dispositivo <ol><li> Monte los PDMS previamente pasivado en el dispositivo de estiramiento (ver Discusión para solución de problemas). <ol><li> Una un lado del PDMS a la parte fija de la camilla. </li><li> Fijar el otro lado a la parte móvil de la camilla sin sujeción demasiado de la hoja de PDMS. </li></ol></li><li> Cortar un rectángulo de PDMS (22 mm x 19 mm) en una puñalada PDMS de espesor y cortar otro rectángulo interior con el fin de tener una piscina (o marco) que retendrá las células y los medios de comunicación (ver Figura 2). </li><li> Agregue grasa de silicona bajo este PDMS piscina y colóquelo en la parte superior de la hoja de PDMS para crear un medium retener piscina. La grasa permitirá que el deslizamiento de la piscina sobre la hoja de PDMS durante el estiramiento. </li></ol> 4. Modelar las celdas <ol><li> Separar las células de un 50% de confluencia matraz de cultivo con Versene. </li><li> Contar las células y resuspender en una concentración de 200.000 células / ml. </li><li> Añadir 1 ml de la suspensión celular en la piscina (ver la discusión para más detalles). </li><li> Deje que las células se unen a los patrones durante 10-30 min (dependiendo del tipo de célula, las células de RPE-1 se llevará a 10 min y las células HeLa 20 min). </li><li> Enjuague suavemente las células flotantes con medio equilibrada (equilibre medio en incubadora). </li><li> Deje que las células se extienden por unos pocos horas en los patrones (necesitará RPE-1 por lo menos 2 horas, las células HeLa necesitarán 3 h). </li><li> Añadir un cubreobjetos en la parte superior de la piscina para evitar la evaporación y medio de fuga en caso de que el PDMS se rompe. </li><li> Coloque el dispositivo en un microscopio invertido y empezar de imágenes. Evite el uso de ob inmersión en aceitetivos, ya que no va a funcionar debido a problemas de enfoque. </li></ol> 5. Extensión <ol><li> Gire el tornillo micrométrico, mientras que la corrección de la posición de etapa en el x-, y-, y el eje z (principalmente x). La posición de fase debe ser corregido para contrarrestar la ampliación de los PDMS y la pérdida de enfoque. (Consulte la sección &quot;Durante el tramo&quot; párrafo de la discusión). </li></ol>

<ol>
	<li>Vogel, V., Sheetz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+force+and+geometry+sensing+regulate+cell+functions.">Local force and geometry sensing regulate cell functions.</a> Nat. Rev. Mol. Cell Biol. 7, 265-275 (2006).</li><li>Terenna, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+mechanisms+redirecting+cell+polarity+and+cell+shape+in+fission+yeast.">Physical mechanisms redirecting cell polarity and cell shape in fission yeast.</a> Curr. Biol. 18 (22), 1748-1753 (2008).</li><li>Guillot, C., Lecuit, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+epithelial+tissue+homeostasis+and+morphogenesis.">Mechanics of epithelial tissue homeostasis and morphogenesis.</a> Science. 340 (6137), 1185-1189 (2013).</li><li>Bosveld, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+control+of+morphogenesis+by+Fat/Dachsous/Four-jointed+planar+cell+polarity+pathway.">Mechanical control of morphogenesis by Fat/Dachsous/Four-jointed planar cell polarity pathway.</a> Science. 336 (6082), 724-727 (2012).</li><li>Farhadifar, R., Roper, J. C., Aigouy, B., Eaton, S., Julicher, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+cell+mechanics,+cell-cell+interactions,+and+proliferation+on+epithelial+packing.">The influence of cell mechanics, cell-cell interactions, and proliferation on epithelial packing.</a> Curr. Biol. 17, 2095-2104 (2007).</li><li>Rauzi, M., Verant, P., Lecuit, T., Lenne, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+and+anisotropy+of+cortical+forces+orienting+Drosophila+tissue+morphogenesis.">Nature and anisotropy of cortical forces orienting Drosophila tissue morphogenesis.</a> Nat. Cell Biol. 10, 1401-1410 (2008).</li><li>Mitrossilis, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+single-cell+response+to+stiffness.">Real-time single-cell response to stiffness.</a> Proc. Natl. Acad. Sci. U.S.A. 107 (38), 16518-16523 (2010).</li><li>Irimia, D., Charras, G., Agrawal, N., Mitchison, T., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polar+stimulation+and+constrained+cell+migration+in+microfluidic+channels.">Polar stimulation and constrained cell migration in microfluidic channels.</a> Lab Chip. 12, 1783-1790 (2007).</li><li>Fink, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=External+forces+control+mitotic+spindle+positioning.">External forces control mitotic spindle positioning.</a> Nat. Cell. Biol. 13 (7), 771-778 (2011).</li><li>Azioune, A., Storch, M., Bornens, M., Th&#233;ry, M., Piel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+rapid+process+for+single+cell+micro-patterning.">Simple and rapid process for single cell micro-patterning.</a> Lab Chip. 9 (11), 1640-1642 (2009).</li><li>Azioune, A., Carpi, N., Tseng, Q., Th&#233;ry, M., Piel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+micropatterns:+A+direct+printing+protocol+using+deep+Uvs.">Protein micropatterns: A direct printing protocol using deep Uvs.</a> Methods Cell Biol. 97, 133-146 (2010).</li><li>Azioune, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+method+for+high-throughput+surface+patterning+of+deformable+substrates.">Robust method for high-throughput surface patterning of deformable substrates.</a> Langmuir. 27 (12), 7349-7352 (2011).</li><li>Carpi, N., Piel, M., Azioun, A., Cuvelier, D., Fink, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+on+silicon+elastomer+(PDMS)+with+deep+UVs.">Micropatterning on silicon elastomer (PDMS) with deep UVs.</a> Protoc. Exch. , (2011).</li><li>Sinha, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+respond+to+mechanical+stress+by+rapid+disassembly+of+caveolae.">Cells respond to mechanical stress by rapid disassembly of caveolae.</a> Cell. 144 (3), 402-413 (2011).</li></ol>

Los autores declaran no tener intereses financieros en competencia.

Aunque esta técnica ha sido utilizada en numerosas ocasiones y es sometido a pruebas exhaustivas, hay varios pasos críticos que pueden llevar a un experimento fallido. Acerca de los PDMS: Para este trabajo, GelPak, una hoja de PDMS delgada disponible en el mercado, se utilizó. Alternativamente hojas PDMS se puede lanzar directamente desde la mezcla de PDMS. Le recomendamos que utilice GelPak porque es más reproducible, y es menos probable que se rompa en compara...

Las células que componen un tejido en organismos superiores están sujetos a tensiones mecánicas y fuerzas de estiramiento próximos ya sea desde el ambiente externo o a partir de células que rodean 1,2. Las células deben adaptarse y resistir estas fuerzas a fin de mantener la integridad de los tejidos. Tales fuerzas son también importantes para tejidos morfogénesis durante el desarrollo 3,4. La aplicación de fuerzas mecánicas sobre las células cultivadas es una manera de imitar lo que podría suceder en un tejido, pero con un control cuantitativo e independiente de la forma celular y 5,6 deformación celular. Para ello, pueden utilizarse varias técnicas. Uno puede pulsar sobre las células (la célula entera o parte de ella), por ejemplo usando AFM o derivados de 7,8 o estirar el sustrato las células están creciendo en. El método descrito en este documento se demuestra cómo estirar un sustrato plano plateado con células. Esta técnica se desarrolló originalmente para evaluar el papel de las fuerzas ejercida sobre mcélulas de mamíferos itotic 9. Las células mitóticas mantenerse conectados al sustrato a través de fibras de retracción y estiramiento de la membrana ejercen una fuerza sobre las fibras, que a su vez provocaron la rotación del huso mitótico. El interés de la combinación de micropatrones adhesivas y estiramiento es para lograr un control independiente de las fuerzas y forma de las células individuales. Por ejemplo, es posible estirar una célula ovoide en una forma redonda perfectamente isotrópico, mientras que se aplica estiramiento uniaxial. Si las células no se entretejieron en micropatrones, estirado uniaxial resultados en la elongación celular, con la mayoría de células que tiene un eje largo alineado con el eje de estiramiento. Es entonces difícil separar el efecto de la alineación del eje largo y el efecto de la estiramiento aplicado a las células. El dispositivo es adecuado para cualquier imagen de células vivas, incluyendo la microscopía fluorescente lapso largo de tiempo, y las drogas se puede añadir durante el experimento. La profunda método micropatterning UVs 10se describe en detalles en Azioune et al. 11 Patrones de PDMS se describe en Azioune et al. 12 El presente estiramiento protocolo es una versión de vídeo de Carpi et al. 13

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GelPak</td>
			<td style="width:71px;">GelPak</td>
			<td style="width:119px;">PF-60-X4</td>
			<td style="width:167px;">Different thickness/stickiness are available. One alternative could be to cast your PDMS yourself.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Silicon grease</td>
			<td style="width:71px;">GE Bayer Silicones</td>
			<td style="width:119px;">Baysilone-Paste</td>
			<td>This one is biocompatible</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Stretching device</td>
			<td style="width:71px;">GREM m&#233;canique</td>
			<td style="width:119px;">Stretcher 2011</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">EDC (N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride)</td>
			<td style="width:71px;">Sigma</td>
			<td align="right" style="width:119px;">3450</td>
			<td>Stable 6 months at -20 &#176;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">NHS (N-Hydroxysulfosuccinimide sodium salt)</td>
			<td style="width:71px;">Sigma</td>
			<td align="right" style="width:119px;">56485</td>
			<td>Protect from humidity</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pll-g-peg (PLL(20)-g[3.5]-PEG(2) 20 mg)</td>
			<td style="width:71px;">SurfaceSolutions (Zurich)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Synthetic Quartz photomask</td>
			<td style="width:71px;">Toppan</td>
			<td style="width:119px;"></td>
			<td>Take standard binary photomask in Quartz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fibronectin from bovine plasma</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">F1141</td>
			<td></td>
		</tr>
	</tbody>
</table>

stretching micropatterned cells on a pdms membrane

Las fuerzas mecánicas ejercidas sobre las células y / o tejidos juegan un papel importante en numerosos procesos. Hemos desarrollado un dispositivo para estirar las células sembradas en placas sobre un polidimetilsiloxano (PDMS) de membrana, compatible con formación de imágenes. Esta técnica es reproducible y versátil. La membrana de PDMS se puede microestructurada con el fin de confinar las células o tejidos a una geometría específica. El primer paso es imprimir micropatrones en la membrana de PDMS con una técnica de UV profunda. La membrana de PDMS se monta entonces en una camilla mecánica. Una cámara está enlazado en la parte superior de la membrana con grasa biocompatible para permitir el deslizamiento durante el estiramiento. Las células se siembran y permitir que se extienda por varias horas en los micropatrones. La muestra puede ser estirada y no estirada varias veces con el uso de un tornillo micrométrico. Se tarda menos de un minuto para aplicar la recta en toda su extensión (en torno al 30%). La técnica presentada aquí no incluye un dispositivo motorizado, que es necesaria para unapplying ciclos repetidos de estiramiento de forma rápida y / o estiramiento controlado ordenador, pero esto puede ser implementado. El estiramiento de las células o el tejido puede ser de interés para las cuestiones relacionadas con las fuerzas de células, la respuesta celular al estrés mecánico o la morfogénesis de tejidos. Esta presentación en video le mostrará cómo evitar los problemas típicos que pueden surgir cuando se hace este tipo de aparentemente sencillo experimento.

<html> La técnica presentada en este protocolo de vídeo permite la aplicación de fuerzas sobre las fibras de retracción de las células de mamíferos de la mitosis. De hecho, durante la división celular, en la etapa mitótica, células de mamífero se retraen para tomar la forma de una esfera y dejar atrás los cables de actina delgadas rodeadas por una membrana que se unen al sustrato. Estos cables (fibras de retracción), son la memoria de la geometría de la célula antes de entrar en división. Haciendo micropat...

Watch this Scientific Journal Video about Stretching Micropatterned Cells on a PDMS Membrane at JoVE.com

Stretching Micropatterned Cells on a PDMS Membrane

Este manuscrito presenta una técnica para aplicar o liberar las fuerzas sobre las células o los tejidos adherentes utilizando estiramiento unidireccional.

El estiramiento células micropatterned en una membrana de PDMS

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

stretching-micropatterned-cells-on-a-pdms-membrane

Research

JoVE Journal

Bioengineering

15.2K vistas.  Institut Curie.  Este manuscrito presenta una técnica para aplicar o liberar las fuerzas sobre las células o los tejidos adherentes utilizando estiramiento unidireccional. 

Video: Stretching Micropatterned Cells on a PDMS Membrane

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Superficies micropatterned para estudiar las interacciones de ácido hialurónico con células de cáncer

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Investigación

Bioingeniería

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

Una mejor visualización y análisis cuantitativo de los efectos de drogas Uso de células micropatterned

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

Una Plataforma Novela Estiramiento para Aplicaciones en Celular y Tisular Mecanobiología

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

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 ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&amp;#949;-caprolactone) Electrospun Yarns

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

Optimización de unión de las células madre mesenquimales humanas en poli (ε-caprolactona) electrohiladas Hilos

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

La transfección de genes hacia células Esferoide sobre placas micropatterned Cultura para el trasplante de células genéticamente modificadas

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

Microfluidic genipina Deposición Técnica de Cultura extendido de micropatterned vasculares musculares Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

Construcción de Hojas modular de hidrogel para la arquitectura celular 3D a escala macro micropatterned

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

Generación de doble emulsión utilizando un flujo Focus Dispositivo de polidimetilsiloxano (PDMS) Co-axial

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

Tratamiento de las estructuras de ligamentos con suero acondicionado con ejercicio: Un modelo de ingeniería de tejidos traslacional

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...