1. Isolement des chondrocytes articulaires primaires Récolte 10-15 tranches complètes de cartilage épaisseur des surfaces articulaires des articulations des animaux (par exemple, l&#39;articulation métacarpo-phalangienne du squelette vaches matures obtenus à partir d&#39;un abattoir local). <ol><li> Tranches de cartilage dans un plat de Pétri de 100 mm et incuber dans 20 ml de protéase 0,5% de Ham F-12 (p / v) pendant 2 heures à 37 ° C. Rincer trois fois dans les médias Ham F-12 de la culture et incuber avec 20 ml de collagénase 0,15% A dans les médias Ham culture F-12 pendant la nuit à 37 ° C. </li><li> Filtrer la suspension de cellules à travers un filtre de 200 mesh écran dans un plat propre. Laver le filtre avec 5 ml de Ham F-12 et ajouter au plat. Transférer la suspension de cellules dans un tube de 50 ml conique. Laver la boîte de Pétri avec 10 ml de Ham F-12 et ajouter dans le tube conique. </li><li> Centrifuger le tube à 600-800 rcf pendant 6-8 min à température ambiante. </li><li> Aspirer le surnageant, en veillant à ne pas déranger ee culot de cellules et remettre en suspension dans 40 ml de milieu de culture de Ham F-12. </li><li> Centrifuger le tube à 600-800 rcf pendant 6-8 min. </li><li> Répétez les étapes 1,4 et 1,5 fois plus pour un total de 3 fois. Avant de centrifugation pour la troisième fois, reprendre le culot par une agitation et d&#39;obtenir une aliquote de 500 ul pour le comptage des cellules. </li><li> Compter les cellules viables en utilisant la méthode d&#39;exclusion du bleu trypan 9 avec un hématimètre et d&#39;un microscope optique inversé. </li><li> Aspirer le surnageant et remettre en suspension le culot dans un volume de Ham F-12 médias de doubler la densité de semis souhaitée (par exemple 20 x 10 6 cellules / ml pour une concentration finale de 10 x 10 6 cellules / ml après encapsulation). </li></ol> 2. Encapsulation Hydrogel chondrocytes agarose <ol><li> Heat 0,8 g autoclave de type VII agarose dans 20 ml de PBS 1x stérile (pH 7,4) sur une plaque chauffante réglée à 120 ° C jusqu&#39;à dissolution. Une fois dissous, baisser le feu à 60 ° C, des températures plus élevées will la viabilité des cellules. </li><li> Dans un tube conique de 50 ml, à fond mélanger des volumes égaux de la suspension de cellules avec le double de la concentration souhaitée de l&#39;agarose (par exemple une suspension de 4% d&#39;agarose d&#39;un hydrogel d&#39;agarose 2% finale). Eviter la formation de grosses bulles, car ils peuvent affecter la viabilité des cellules et de construire la fatigue. </li><li> Soigneusement aliquote de 10 ml du mélange chondrocytes agarose dans un plat de Pétri de diamètre 60 mm, tout en évitant la création de grosses bulles. </li><li> Laisser reposer pendant 30 min à température ambiante jusqu&#39;à gélifié. </li><li> Obtenir autant d&#39;échantillons d&#39;hydrogel chondrocytes agarose selon les besoins (en général 20-30) à l&#39;aide de 4 mm biopsie. </li><li> Mesurer et noter les dimensions physiques (diamètre et hauteur) d&#39;échantillons représentatifs à l&#39;aide d&#39;un micromètre. Constructions doit être d&#39;environ 5 mm de hauteur, rejeter tous les échantillons obtenus à partir d&#39;une région où la hauteur de la variance d&#39;échantillonnage est supérieure à 2%. </li><li> Transférer les échantillons dans un plat de 60 mm de Pétri frais et supplémentavec 10 ml de milieu complet (par exemple, 20% de sérum de veau foetal dans les médias Ham culture F-12 avec 20 mM d&#39;HEPES, 100 d&#39;acide ascorbique pg / ml, et les antibiotiques 2x / antimycotiques). </li></ol> 3. La stimulation mécanique et radiomarquage de chondrocytes-agarose hydrogels <ol><li> Autoclaver les composants métalliques de la plate-forme de compression. Stériliser les composants en plastique avec de l&#39;éthanol à 70% (la nuit) et de la lumière UV (pendant au moins 15 minutes). </li><li> Pour garder les constructions de tomber, en utilisant une pince, place 12 anneaux en plastique stériles de retenue (avec un diamètre intérieur plus grand que le diamètre construction) (n = 6, échantillons et contrôles) dans les puits d&#39;une plaque de culture de 24 puits. </li><li> En utilisant des pinces, transférer soigneusement les hydrogels chondrocytes-agarose de la boîte de Pétri à la plaque de 24 puits, la sécurisation chacun dans un anneau de retenue. </li><li> Transfert 400 pi de milieu complet (par exemple 20% de FBS dans les médias Ham culture F-12 avec 20 mM d&#39;HEPES, 100 pg / ml d&#39;acide ascorbique et2x antibiotiques antimycosiques /) pour chaque échantillon. </li><li> Monter la plate-forme de compression stérilisés (Figure 1) et fixer les plaques avec vis de réglage. Fixez la plate-forme de compression de la plaque de culture de 24 puits. </li><li> Relâchez et re-fixer les vis de réglage pour établir plateau-hydrogel de contact (zéro l&#39;état de contrainte). </li><li> Chargez la plate-forme de compression monté dans un interféromètre de Mach-1 Système de test micromécanique. Plate-forme sécurisée pour la phase verticale par goupille de positionnement et de verrouillage en place avec vis de réglage. Retirez le gabarit d&#39;espacement. </li><li> Appliquer une charge de compression dynamique à l&#39;amplitude désirée (p.ex. l&#39;amplitude de contrainte à 10% en fonction de la hauteur d&#39;origine des échantillons), la fréquence (par exemple, 1 Hz) et la durée ou le nombre de cycles (par exemple des intervalles de temps allant jusqu&#39;à 60 minutes). </li><li> À la fin de la stimulation mécanique, remplacer le gabarit d&#39;espacement, desserrer la vis goupille de positionnement et en retirer l&#39;appareil de compression et plaque de culture de la Mach-1 Système de test micromécanique. </li&gt; <li> Radiomarqueur cellules d&#39;hydrogel constructions en complétant les médias avec 5 pCi de l&#39;isotope désiré (par exemple [3 H]-proline étiquette protéines pour la synthèse du collagène chondrocyte spécifique et [35 S]-soufre pour la synthèse de protéoglycanes). </li><li> Incuber les constructions radiomarqués pour 24 heures à 37 ° C, 5% CO 2 10. </li><li> Récolter les constructions d&#39;hydrogel radiomarqués et rincer 3 fois dans du PBS 1X (pH 7,4) pour éliminer toute isotope non constituée en société. Digérer les échantillons en utilisant la papaïne (40 pg / ml dans 20 mM d&#39;acétate d&#39;ammonium, 1 mM d&#39;acide ethyldiaminetetraacetic et 2 mM de dithiothréitol à 65 ° C pendant 72 heures). </li><li> Essai Digest pour la teneur en ADN en utilisant le test PicoGreen colorant 11 et l&#39;activité des isotopes incorporé, normalisée par rapport à la teneur en ADN, en β-scintillation liquide compter 12,13 digestion par la papaïne qui suit immédiatement. </li></ol> Remarque: L&#39;achat et l&#39;élimination des matières radioactives (isotopes de déchets et des articlesqui ont pu entrer en contact avec des matières radioactives) doit suivre les dispositions institutionnelles pertinentes (et / ou gouvernementale) des politiques et procédures pour la manipulation et l&#39;élimination des substances radioactives. 4. Les résultats représentatifs Chondrocytes articulaires bovins-agarose constructions hydrogels (2% d&#39;agarose à 10 x 10 6 cellules / ml de cellules encapsulées) ont été stimulées mécaniquement à une amplitude de la contrainte de compression de 10% à une fréquence de 1 Hz pendant 20 à 60 min (1.200 à 3.600 ou cycles ) et analysés pour leur teneur d&#39;ADN et de synthèse de la matrice extracellulaire par incorporation de radio-isotopes. ADN et la synthèse de la matrice extracellulaire a été touchée d&#39;une manière dose-dépendante. La teneur en ADN a montré une diminution significative de 35% en raison de 20 ou 30 minutes de stimulation (p &lt;0,01), alors que 60 min de stimulation montré aucun effet (Figure 2). Cartilage spécifique synthèse du collagène et des protéoglycanes (déterminée par la [3 H]-proline et[35 S]-soufre constitution, respectivement) a montré une augmentation significative d&#39;environ 60% en réponse à 20 ou 30 minutes de stimulation (p &lt;0,01), avec 60 minutes de stimulation présentant aucun effet observable (Figure 3). <img src="/files/ftp_upload/4229/4229fig1.jpg" alt="Figure 1" /> . Figure 1 Schéma de l&#39;appareil de mesure intégré compression dynamique pour stimuler chondrocytes agarose constructions gauche:. Banc assemblé droite:. Vue éclatée montrant les composants individuels. <img src="/files/ftp_upload/4229/4229fig2.jpg" alt="Figure 2" /> Changements Figure 2. Teneur en ADN des chondrocytes stimulés mécaniquement hydrogels d&#39;agarose sous une amplitude de déformation à la compression de 10% à 1 Hz pendant 20 à 60 min (moyenne ± SEM, n = 6/groupe). Changements Figure 3. Dans la synthèse de la matrice extracellulaire (collagène et de protéoglycanes) de chondrocytes stimulés mécaniquement hydrogels d&#39;agarose sous amplitude de déformation à la compression de 10% à 1 Hz pendant 20 à 60 min (moyenne ± SEM, n = 6/groupe).

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<html> La méthode décrite pour appliquer des stimuli mécaniques contrôlées à la cellule tête de série agarose hydrogels permet d&#39;enquêter directement sur les effets des dynamiques des forces de compression sur le métabolisme des chondrocytes. L&#39;utilisation de la plate-forme de mesure de test en même temps que les bagues de retenue prévu contrainte latérale pour les constructions d&#39;éviter d&#39;éventuels problèmes de basculement échantillon. L&#39;utilisation de plateaux de chargement morts po...

<table border="1"><tbody><tr><td> Le nom du réactif ou de l&#39;équipement </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> Ham F-12 </td><td> Thermo Fisher Scientific </td><td> SH3001002 </td><td></td></tr><tr><td> Collagénase A </td><td> Sigma Aldrich Ltd </td><td> C0130 </td><td></td></tr><tr><td> Protéase </td><td> Sigma Aldrich Ltd </td><td> P5147 </td><td></td></tr><tr><td> Fetal Bovine Serum </td><td> Sigma Aldrich Ltd </td><td> F1051 </td><td></td></tr><tr><td> Ascorbate </td><td> Sigma Aldrich Ltd </td><td> A4034 </td><td></td></tr><tr><td> Antibiotiques / antifongiques </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> Bleu trypan </td><td> Sigma Aldrich Ltd </td><td> 93595 </td><td></td></tr><tr><td> Reichert Bright-ligne Hémacytomètre </td><td> Hausser scientifique </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 papaïne extraite de la papaye latex </td><td> Sigma Aldrich Ltd </td><td> P3125 </td><td></td></tr><tr><td> Acétate d&#39;ammonium </td><td> Sigma Aldrich Ltd </td><td> A1542 </td><td></td></tr><tr><td> Acide Ethyldiaminetetraacetic </td><td> Sigma Aldrich Ltd </td><td> E9884 </td><td></td></tr><tr><td> DL-dithiothréitol </td><td> Sigma Aldrich Ltd </td><td> 43819 </td><td></td></tr><tr><td> Faible point de fusion d&#39;agarose, de type VII </td><td> Sigma Aldrich Ltd </td><td> A9045 </td><td></td></tr> &lt;tr&gt; <td> Moustiquaire (200) Filtre </td><td> Sigma Aldrich Ltd </td><td> S4145 </td><td></td></tr><tr><td> Mach-1 Testeur micromécanique </td><td> Biomomentum Inc </td><td> V500cs </td><td></td></tr><tr><td> Chargement de compression Jig </td><td> Fabriqués sur mesure </td><td></td><td> Produit similaire pourrait être fourni par Biomomentum Inc </td></tr><tr><td> Falcon 24 Plaque Culture Bien </td><td> Thermo Fisher Scientific </td><td> B353047 </td><td></td></tr><tr><td> compteur à scintillation β-liquide </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] Soufre </td><td> Perkin-Elmer </td><td> NEX041005MC </td><td></td></tr></tbody></table>

mechanical stimulation of chondrocyte-agarose hydrogels

Le cartilage articulaire souffre d&#39;une capacité limitée de réparation lorsqu&#39;il a été endommagé par l&#39;insulte mécanique ou dégradés par la maladie, comme l&#39;arthrose. Pour remédier à cette lacune, plusieurs interventions médicales ont été développées. Une de ces méthodes consiste à resurfacer la zone endommagée avec génie tissulaire du cartilage, mais l&#39;ingénierie tissulaire n&#39;a généralement les propriétés biochimiques et la durabilité du cartilage natif, remettant en question sa survie à long terme. Cela limite l&#39;application de l&#39;ingénierie tissulaire du cartilage à la réparation de petits défauts de coordination, en s&#39;appuyant sur les tissus environnants pour protéger le matériau implanté. Pour améliorer les propriétés du tissu développé, la stimulation mécanique est une méthode courante utilisée pour augmenter la synthèse de la matrice extracellulaire du cartilage ainsi que les propriétés mécaniques résultantes de l&#39;ingénierie tissulaire. La stimulation mécanique applique des forces sur le tissu construit analogue à ceux rencontrés in vivo. Cetteest basé sur la prémisse que l&#39;environnement mécanique, en partie, de réglementer le développement et la maintenance de 1,2 tissu natif. La forme la plus couramment utilisée dans la stimulation mécanique de l&#39;ingénierie des tissus cartilagineux est la compression dynamique des déformations physiologiques de l&#39;ordre de 5-20% à une fréquence de 1 Hz 1,3. Plusieurs études ont examiné les effets de la compression dynamique et ont montré qu&#39;elle ait un effet positif sur le métabolisme des chondrocytes et la biosynthèse, en influant finalement sur ​​les propriétés fonctionnelles de l&#39;4-8 tissu développé. Dans cet article, nous illustrons la méthode pour stimuler mécaniquement les constructions d&#39;hydrogel chondrocytes agarose sous compression dynamique et analyser les changements dans la biosynthèse par des dosages biochimiques et radio-isotope. Cette méthode peut également être facilement modifié pour évaluer les changements potentiellement induits dans la réponse cellulaire à la suite de stimuli mécaniques.

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

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

La biosynthèse de la matrice extracellulaire du cartilage par les chondrocytes peuvent être affectés par l&#39;application de stimuli mécaniques. Cette méthode décrit la technique d&#39;application dynamiques contraintes de compression de chondrocytes encapsulés dans des constructions 3D et l&#39;évaluation des changements induits dans le métabolisme des chondrocytes.

La stimulation mécanique des chondrocytes-agarose hydrogels

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

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11.6K vues.  Queen's University. La biosynthèse de la matrice extracellulaire du cartilage par les chondrocytes peuvent être affectés par l'application de stimuli mécaniques. Cette méthode décrit la technique d'application dynamiques contraintes de compression de chondrocytes encapsulés dans des constructions 3D et l'évaluation des changements induits dans le métabolisme des chondrocytes.

Vidéo: 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 Chargement mécanique du 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 ...

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Biologie

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 stimulation mécanique des cellules souches en utilisant cyclique uniaxiale souche

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.

Encapsulation Cellulaire dans les hydrogels 3D pour l&#39;ingénierie tissulaire

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.

Réponse Live Cell à la stimulation mécanique étudiée par microscopie à force atomique et optique intégré

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.

La stimulation mécanique induite par le calcium propagation des ondes dans des monocouches de cellules: l&#39;exemple de bovins cellules endothéliales de la cornée

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.

Préparation de l&#39;ADN réticulé polyacrylamide hydrogels

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.

Multifonctionnel, Micropipette à base Méthode de constitution et la stimulation de la société Bacterial mécanosensible Canaux ioniques en gouttelettes interface 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 hydrogels en trois dimensions

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.

Évaluation de Dictyostelium discoideum réponse à la Stimulation mécanique aiguë

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.

Hydrogels d’Alginate et microbioréacteurs hydrophobes protéolytically dégradés pour l’encapsulation des oocytes porcins

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