1. Aislamiento de condrocitos articulares primarios Cosecha 10-15 rebanadas completo de cartílago de espesor de las superficies articulares de las articulaciones de animales (por ejemplo, la articulación metacarpiana-falángica de vacas esqueléticamente maduros obtenidos de un matadero local). <ol><li> Colocar las rodajas de cartílago en un 100 mm placa de Petri y se incuba en 20 ml de 0,5% de proteasa en Ham F-12 (w / v) durante 2 horas a 37 ° C. Enjuague tres veces en medios de cultivo de Ham F-12, y se incuba con 20 ml de 0,15% de colagenasa A en los medios de cultivo de Ham F-12 durante la noche a 37 ° C. </li><li> Se filtra la suspensión celular a través de un filtro de malla 200 de pantalla en un plato limpio. Lavar el filtro con 5 ml de Ham F-12 y añadir al plato. Transferir la suspensión celular a un tubo cónico de 50 ml. Lavar la placa de Petri con 10 ml de Ham F-12 y añadir al tubo cónico. </li><li> Centrifugar el tubo a 600-800 rcf durante 6-8 min a temperatura ambiente. </li><li> Aspirar el sobrenadante y asegúrese de no molestar ªe pastilla de células y volver a suspender en 40 ml de medio de cultivo de Ham F-12. </li><li> Centrifugar el tubo a 600-800 rcf durante 6-8 min. </li><li> Repetir los pasos 1,4 y 1,5 veces más para un total de 3 veces. Antes de la centrifugación de la tercera vez, resuspender el precipitado a través de la agitación y obtener una alícuota de 500 l para el recuento celular. </li><li> Contar las células viables usando el método de exclusión con azul de tripano 9 con un hemocitómetro y un microscopio de luz invertida. </li><li> Aspirar el sobrenadante y volver a suspender el sedimento en un volumen de 12-F de Ham al doble de la densidad de siembra deseada (por ejemplo 20 x 10 6 células / ml para una concentración final de 10 x 10 6 células / ml después de la encapsulación). </li></ol> 2. Condrocito-agarosa hidrogel Encapsulación <ol><li> Calor 0,8 g autoclave Tipo VII de agarosa en 20 ml de PBS 1x estéril (pH 7,4) en una placa caliente ajustada a 120 ° C hasta que se disolvió. Una vez disuelto, reducir el calor a 60 ° C como temperaturas más altas will afectar la viabilidad celular. </li><li> En un tubo cónico de 50 ml, mezclar cuidadosamente volúmenes iguales de la suspensión celular con el doble de la concentración deseada de agarosa (por ejemplo, 4% de suspensión de agarosa para una final de 2% de hidrogel de agarosa). Evitar la creación de burbujas grandes, ya que pueden afectar a la viabilidad celular y la construcción de la vida de fatiga. </li><li> Cuidadosamente alícuota de 10 ml de la mezcla de condrocito-agarosa en un diámetro de 60 mm placa de Petri, evitando al mismo tiempo la creación de burbujas grandes. </li><li> Dejar reposar durante 30 minutos a temperatura ambiente hasta que gelificó. </li><li> Obtener el mayor número de condrocitos-agarosa muestras de hidrogel, según sea necesario (típicamente 20-30) usando un punzón de biopsia de 4 mm. </li><li> Mida y registre las dimensiones físicas (diámetro y altura) de muestras representativas utilizando un micrómetro. Las construcciones debe ser aproximadamente 5 mm de altura, descartar cualquier muestras obtenidas de una región donde la varianza altura de la muestra fue mayor que 2%. </li><li> Transferir las muestras a un nuevo 60 mm placa de Petri y suplementocon 10 ml de medio completo (20% por ejemplo, suero bovino fetal en medios de cultivo de Ham F-12 con 20 mM de HEPES, 100 g / ml de ácido ascórbico, y antibióticos / antimicóticos 2x). </li></ol> 3. La estimulación mecánica y radiomarcaje de condrocitos-agarosa hidrogeles <ol><li> Esterilizar en autoclave los componentes metálicos de la instalación de compresión. Esterilizar los componentes de plástico con 70% de etanol (durante la noche) y luz UV (por lo menos durante 15 min). </li><li> Para mantener las construcciones que se caiga, usando fórceps, lugar 12 anillos de plástico estériles de retención (con un diámetro interior mayor que el diámetro constructo) (n = 6, muestras y controles) en los pocillos de una placa de cultivo de 24 pocillos. </li><li> Utilizando pinzas, transferir cuidadosamente los hidrogeles de condrocitos-agarosa de la placa de Petri a la placa de 24-así, asegurando cada uno en un anillo de retención. </li><li> Transferir 400 l de medio completo (por ejemplo, 20% de FBS en los medios de cultivo de Ham F-12 con 20 mM de HEPES, 100 ug / ml de ácido ascórbico, y2x antibióticos / antimicóticos) a cada pocillo de muestra. </li><li> Montar la plataforma de compresión esterilizada (Figura 1) y fije las placas con tornillos de fijación. Una el equipo de perforación de compresión a la placa de cultivo de 24 pocillos. </li><li> Suelte y vuelva a sujetar-los tornillos de ajuste para establecer platina de hidrogel de contacto (cero estado de deformación). </li><li> Cargue la plataforma de compresión montado en un Mach-1 Testing System micromecánica. Plataforma segura para la etapa vertical mediante pasador de posicionamiento y bloqueo en su lugar con el tornillo de fijación. Retire la plantilla de interlineado. </li><li> Aplicar una carga de compresión dinámica en amplitud deseada (por ejemplo, 10% de amplitud de deformación sobre la base de la altura original de las muestras), la frecuencia (por ejemplo, 1 Hz) y la duración o el número de ciclos (por ejemplo, intervalos de tiempo de hasta 60 min). </li><li> Tras la finalización de la estimulación mecánica, sustituir la plantilla espaciamiento, afloje el tornillo de pasador de fijación localizar y eliminar la plataforma de compresión y la placa de cultivo de Mach-1 Testing System micromecánicos. </li&gt; <li> Radiomarcador hidrogel células constructos suplementando el medio con 5 Ci de isótopo deseado (por ejemplo, [3 H]-prolina etiqueta de proteínas para la síntesis de colágeno específico de condrocitos y [35 S]-azufre para la síntesis de proteoglicanos). </li><li> Incubar los constructos radiomarcados para 24 horas a 37 ° C, 5% CO 2 10. </li><li> Cosechar los constructos de hidrogel radiomarcados y enjuague 3 veces en 1X PBS (pH 7,4) para eliminar cualquier isótopo no incorporado. Digest muestras usando papaína (40 ug / ml en 20 mM de acetato de amonio, 1 mM de ácido ethyldiaminetetraacetic y 2 mM de ditiotreitol a 65 ° C durante 72 hr). </li><li> Ensayo para digerir el contenido de ADN mediante el ensayo PicoGreen tinte 11 y actividad incorporada isótopo, normalizado a contenido de ADN, por β-líquido de centelleo contando 12,13 digestión con papaína inmediatamente siguiente. </li></ol> Nota: La compra y venta de materiales radiactivos (isótopos y elementos de desechoque puedan haber estado en contacto con materiales radioactivos) debe seguir el institucional relevante (y / o gubernamentales) Políticas y procedimientos para la manipulación y eliminación de sustancias radiactivas. 4. Los resultados representativos Bovina condrocitos articulares-agarosa construcciones hidrogeles (2% de agarosa con 10 x 10 6 células / ml células encapsuladas) fueron estimuladas mecánicamente a una amplitud de 10% de deformación a la compresión a una frecuencia de 1 Hz durante 20 a 60 min (o 1.200 ciclos a 3.600 ) y se ensayó el contenido de ADN y la síntesis de matriz extracelular mediante la incorporación de radioisótopos. ADN y la síntesis de matriz extracelular se vio afectada de una manera dependiente de la dosis. Contenido de ADN mostraron una disminución significativa del 35% como resultado de 20 o 30 min de estimulación (p &lt;0,01), mientras que 60 min de estimulación no mostró ningún efecto (Figura 2). Cartílago específicos de la síntesis de colágeno y proteoglicanos (determinado por [3 H]-prolina y[35 S]-azufre incorporación, respectivamente) mostraron incrementos significativos de aproximadamente 60% ​​en respuesta a 20 o 30 min de estimulación (p &lt;0,01), con 60 min de estimulación que no presenta efecto observable (Figura 3). <img src="/files/ftp_upload/4229/4229fig1.jpg" alt="Figura 1" /> . Figura 1 Esquema de la plataforma de compresión dinámica hecha a la medida para estimular condrocitos agarosa construcciones de la izquierda:. Plataforma ensamblada Derecha:. Despiece que muestra los componentes individuales. <img src="/files/ftp_upload/4229/4229fig2.jpg" alt="Figura 2" /> Cambios Figura 2. En el contenido de ADN de condrocito-agarosa hidrogeles mecánicamente estimulados bajo una amplitud de deformación 10% de compresión a 1 Hz durante 20 a 60 min (media ± SEM, n = 6/grupo). Cambios Figura 3. En la síntesis de matriz extracelular (colágeno y proteoglicanos) de condrocito-agarosa hidrogeles mecánicamente estimulados bajo amplitud de la deformación a la compresión 10% a 1 Hz durante 20 a 60 minutos (media ± SEM, n = 6/grupo).

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<html> El método descrito para la aplicación de estímulos mecánicos controlados a la celda de semilla de agarosa hidrogeles permite la investigación directa en los efectos dinámicos de las fuerzas de compresión sobre el metabolismo de los condrocitos. El uso de la plataforma de pruebas de medida junto con los anillos de retención proporcionado restricción lateral para las construcciones para evitar problemas potenciales de la muestra de inflexión. El uso de platos de carga muerta ponderados fijados por los equi...

<table border="1"><tbody><tr><td> Nombre del reactivo o equipo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Ham F-12 </td><td> Thermo Fisher Scientific </td><td> SH3001002 </td><td></td></tr><tr><td> Colagenasa A </td><td> Sigma Aldrich Ltd. </td><td> C0130 </td><td></td></tr><tr><td> Proteasa </td><td> Sigma Aldrich Ltd. </td><td> P5147 </td><td></td></tr><tr><td> Suero Bovino Fetal </td><td> Sigma Aldrich Ltd. </td><td> F1051 </td><td></td></tr><tr><td> Ascorbato </td><td> Sigma Aldrich Ltd. </td><td> A4034 </td><td></td></tr><tr><td> Antibióticos / antimicóticos </td><td> Sigma Aldrich Ltd. </td><td> A5955 </td><td></td></tr><tr><td> HEPES </td><td> Bioshop Canada Ltd. </td><td> HEP001 </td><td></td></tr><tr><td> Trypan azul </td><td> Sigma Aldrich Ltd. </td><td> 93595 </td><td></td></tr><tr><td> Reichert Bright-Line Hemocitómetro </td><td> Hausser Scientific </td><td> 1490 </td><td></td></tr><tr><td> Quant-iT PicoGreen </td><td> Invitrogen </td><td> P7589 </td><td></td></tr><tr><td> La papaína de la papaya látex </td><td> Sigma Aldrich Ltd. </td><td> P3125 </td><td></td></tr><tr><td> Acetato de amonio </td><td> Sigma Aldrich Ltd. </td><td> A1542 </td><td></td></tr><tr><td> Ácido Ethyldiaminetetraacetic </td><td> Sigma Aldrich Ltd. </td><td> E9884 </td><td></td></tr><tr><td> DL-ditiotreitol </td><td> Sigma Aldrich Ltd. </td><td> 43819 </td><td></td></tr><tr><td> Bajo punto de fusión de agarosa, Tipo VII </td><td> Sigma Aldrich Ltd. </td><td> A9045 </td><td></td></tr> &lt;tr&gt; <td> Pantalla de malla (200) Filtrar </td><td> Sigma Aldrich Ltd. </td><td> S4145 </td><td></td></tr><tr><td> Mach-1 Tester micromecánico </td><td> Biomomentum Inc. </td><td> V500cs </td><td></td></tr><tr><td> Compresión Cargando Jig </td><td> Custom-construido </td><td></td><td> Producto similar podría ser suministrada por Biomomentum Inc. </td></tr><tr><td> Falcon 24 Placa de Cultura Bien </td><td> Thermo Fisher Scientific </td><td> B353047 </td><td></td></tr><tr><td> β-líquido contador de centelleo </td><td> Beckman Coulter </td><td> LS6500 </td><td></td></tr><tr><td> [3H] Proline </td><td> Perkin-Elmer </td><td> NET323005MC </td><td></td></tr><tr><td> [35S] Azufre </td><td> Perkin-Elmer </td><td> NEX041005MC </td><td></td></tr></tbody></table>

mechanical stimulation of chondrocyte-agarose hydrogels

El cartílago articular sufre de una capacidad limitada de reparación deteriorados por el insulto mecánico o degradados por la enfermedad, tal como la osteoartritis. Para remediar esta deficiencia, varias intervenciones médicas se han desarrollado. Uno de tales métodos es para recubrir la zona dañada con cartílago de tejido de ingeniería, sin embargo, la ingeniería de tejidos típicamente carece de las propiedades bioquímicas y la durabilidad de cartílago nativo, cuestionando su supervivencia a largo plazo. Esto limita la aplicación de la ingeniería de tejido cartilaginoso a la reparación de pequeños defectos focales, basándose en el tejido circundante para proteger el material implantado. Para mejorar las propiedades del tejido desarrollado, la estimulación mecánica es un método popular utilizado para mejorar la síntesis de la matriz extracelular cartilaginosa, así como las propiedades mecánicas resultantes de la ingeniería tisular. La estimulación mecánica se aplica fuerzas al tejido construye análoga a las experimentadas in vivo. Estase basa en la premisa de que el entorno mecánico, en parte, regula el desarrollo y mantenimiento de 1,2 tejido nativo. La forma más comúnmente aplicado de la estimulación mecánica en la ingeniería de tejido de cartílago es la compresión dinámica a cepas fisiológicos de aproximadamente 5-20% a una frecuencia de 1 Hz a 1,3. Varios estudios han investigado los efectos de la compresión dinámica y han demostrado que tienen un efecto positivo sobre el metabolismo de los condrocitos y la biosíntesis, en última instancia, afectar a las propiedades funcionales del tejido 4-8 desarrollado. En este trabajo, se expone el método para estimular mecánicamente condrocitos agarosa construcciones de hidrogel bajo compresión dinámica y analizar los cambios en la biosíntesis a través de ensayos bioquímicos y radioisótopos. Este método también puede ser fácilmente modificado para evaluar cualquier cambio potencialmente inducidos en respuesta celular como resultado de estímulos mecánicos.

Watch this Scientific Journal Video about Mechanical Stimulation of Chondrocyte-agarose Hydrogels at JoVE.com

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

La biosíntesis de la matriz extracelular cartilaginosa por los condrocitos pueden ser afectados por la aplicación de estímulos mecánicos. Este método describe la técnica de aplicación de dinámicas tensiones compresivas a los condrocitos encapsulados en construcciones en 3D y la evaluación de los cambios inducidos en el metabolismo de los condrocitos.

La estimulación mecánica de condrocitos-agarosa hidrogeles

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

mechanical-stimulation-of-chondrocyte-agarose-hydrogels

Research

JoVE Journal

Biology

11.6K vistas.  Queen's University. La biosíntesis de la matriz extracelular cartilaginosa por los condrocitos pueden ser afectados por la aplicación de estímulos mecánicos. Este método describe la técnica de aplicación de dinámicas tensiones compresivas a los condrocitos encapsulados en construcciones en 3D y la evaluación de los cambios inducidos en el metabolismo de los condrocitos.

Video: Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Ex vivo Mechanical Loading of Tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37&deg;C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

A new in vitro system for simultaneously loading four tendons in culture is described.

Ex vivo Carga mecánica del tendón

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

Investigación

Biología

Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain

The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated phenomena such as bone remodeling, muscular hypertrophy, and smooth muscle cell plasticity. However, the forces involved are often extremely complex and difficult to monitor and control in vivo. To better investigate the effects of mechanical forces on cells, we have developed an in vitro method for applying uniaxial cyclic tensile strain to adherent cells cultured on elastic membranes. This method utilizes a custom-designed bioreactor with a motorized cam-rotor system to apply the desired force. Here we present a step-by-step video protocol demonstrating how to assemble the various components of each "stretch chamber", including, in this case, a silicone membrane with micropatterned topography to orient the cells with the direction of the strain. We also describe procedures for sterilizing the chambers, seeding cells onto the membrane, latching the chamber into the bioreactor, and adjusting the mechanical parameters (i.e. magnitude and rate of strain). The procedures outlined in this particular protocol are specific for seeding human mesenchymal stem cells onto silicone membranes with 10 &micro;m wide channels oriented parallel to the direction of strain. However, the methods and materials presented in this system are flexible enough to accommodate a number of variations on this theme: strain rate, magnitude, duration, cell type, membrane topography, membrane coating, etc. can all be tailored to the desired application or outcome. This is a robust method for investigating the effects of uniaxial tensile strain applied to cells in vitro.

It is widely understood that mechanical forces in the body can influence cell differentiation and proliferation. Here we present a video protocol demonstrating the use of a custom-built bioreactor for delivering uniaxial cyclic tensile strain to stem cells cultured on flexible micropatterned substrates.

La estimulación mecánica de células madre utilizando tensión uniaxial cíclica

The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

La encapsulación celular en hidrogeles 3D para ingeniería de tejidos

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

Respuesta de células vivas a la estimulación mecánica estudiados por microscopía óptica integrada la Fuerza Atómica y

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Mecánica estimulación inducida por calcio Propagación de Ondas en monocapas de células: el ejemplo de las especies bovina células endoteliales corneales

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

Preparación de ADN-poliacrilamida reticulada hidrogeles

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. It has been discovered and rigorously studied using the patch-clamp technique for almost three decades. Its basic role of translating tension applied to the cell membrane into permeability response makes it a strong candidate to function as a mechanoelectrical transducer in artificial membrane-based biomolecular devices. Serving as building blocks to such devices, droplet interface bilayers (DIBs) can be used as a new platform for the incorporation and stimulation of MscL channels. Here, we describe a micropipette-based method to form DIBs and measure the activity of the incorporated MscL channels. This method consists of lipid-encased aqueous droplets anchored to the tips of two opposing (coaxially positioned) borosilicate glass micropipettes. When droplets are brought into contact, a lipid bilayer interface is formed. This technique offers control over the chemical composition and the size of each droplet, as well as the dimensions of the bilayer interface. Having one of the micropipettes attached to a harmonic piezoelectric actuator provides the ability to deliver a desired oscillatory stimulus. Through analysis of the shapes of the droplets during deformation, the tension created at the interface can be estimated. Using this technique, the first activity of MscL channels in a DIB system is reported. Besides MS channels, activities of other types of channels can be studied using this method, proving the multi-functionality of this platform. The method presented here enables the measurement of fundamental membrane properties, provides a greater control over the formation of symmetric and asymmetric membranes, and is an alternative way to stimulate and study mechanosensitive channels.

Bacterial mechanosensitive channels can be used as mechanoelectrical transducers in biomolecular devices. Droplet interface bilayers (DIBs), cell-inspired building blocks to such devices, represent new platforms to incorporate and stimulate mechanosensitive channels. Here, we demonstrate a new micropipette-based method of forming DIBs, allowing the study of mechanosensitive channels under mechanical stimulation.

Multifuncional, Micropipeta basada en el Método de Incorporación y la estimulación de Canales bacteriana mechanosensitive Ion en Gotita de interfaz Bilayers

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET Imaging en hidrogeles tridimensionales

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Evaluación de Dictyostelium discoideum respuesta a estimulación mecánica aguda

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

Hidrogeles de Alginato degradados proteolíticamente y microbiorreactores hidrófobos para encapsulación de oocitos porcinos

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...