1. Isolamento dei condrociti articolari primarie Harvest 10-15 completi fette cartilagine spessore dalle superfici articolari delle articolazioni degli animali (ad esempio il metacarpo-falangea di vacche scheletro ottenuta da un mattatoio locale). <ol><li> Cartilagine Mettere le fette in un piatto 100 millimetri Petri e incubare a 20 ml di 0,5% proteasi in Ham F-12 (w / v) per 2 ore a 37 ° C. Risciacquare tre volte di Ham F-12 terreni di coltura e incubare con 20 ml di 0,15% Collagenase A in Ham F-12 Mezzi di coltura durante la notte a 37 ° C. </li><li> Filtrare la sospensione cellulare attraverso un filtro 200 mesh in un piatto pulito. Lavare il filtro con 5 ml di Ham F-12 e aggiungere al piatto. Trasferire la sospensione cellulare in una provetta da 50 ml. Lavare la piastra Petri con 10 ml di Ham F-12 e aggiungere al tubo conico. </li><li> Centrifugare la provetta a 600-800 rcf per 6-8 minuti a temperatura ambiente. </li><li> Aspirare il supernatante, assicurando di non disturbare °e pellet e risospendere in 40 ml di Ham F-12 terreni di coltura. </li><li> Centrifugare la provetta a 600-800 rcf per 6-8 min. </li><li> Ripetere i passaggi 1,4 e 1,5 volte più per un totale di tre volte. Prima di centrifugazione per la terza volta, risospendere il pellet tramite agitazione e ottenere una aliquota 500 microlitri per la conta cellulare. </li><li> Contare le cellule vitali con il metodo di esclusione Trypan blu 9 con un emocitometro e un microscopio ottico invertito. </li><li> Aspirare il supernatante e risospendere il pellet in un volume di Ham F-12 supporti di raddoppiare la densità di semina desiderata (ad esempio 20 x 10 6 cellule / ml per una concentrazione finale di 10 x 10 6 cellule / ml dopo incapsulamento). </li></ol> 2. Condrociti-agarosio Hydrogel incapsulamento <ol><li> Calore 0,8 g autoclavato Type VII agarosio in 20 ml di PBS sterile 1x (pH 7,4) su una piastra calda impostata a 120 ° C fino a completa dissoluzione. Una volta sciolto, abbassare il calore a 60 ° C come temperature superiori will influenzare la vitalità delle cellule. </li><li> In una provetta da 50 ml, mescolare volumi uguali della sospensione cellulare con il doppio della concentrazione desiderata di agarosio (es. sospensione agarosio al 4% per un idrogel finale 2% agarosio). Evitare la formazione di bolle di grandi dimensioni in quanto possono influenzare la vitalità delle cellule e la costruzione di durata a fatica. </li><li> Un&#39;aliquota accuratamente 10 ml di agarosio-condrociti miscela in un piatto Petri diametro 60 mm, evitando la formazione di bolle grandi. </li><li> Lasciate riposare per 30 minuti a temperatura ambiente fino gelificata. </li><li> Ottenere il maggior numero di condrociti-agarosio campioni idrogel, se necessario (in genere 20-30) con una da 4 mm biopsia. </li><li> Misurare e registrare le dimensioni fisiche (diametro e altezza) di campioni rappresentativi con un micrometro. Costrutti deve essere di circa 5 mm di altezza, eliminare eventuali campioni ottenuti da una regione in cui la varianza altezza del campione è superiore al 2%. </li><li> Trasferire i campioni di un piatto dolce 60 millimetri Petri e integrarecon 10 ml di mezzi completi (ad esempio il 20% di siero fetale bovino in Ham F-12 mezzi di coltura con 20 mM HEPES, 100 pg / ml di acido ascorbico, e antibiotici / antimicotici 2x). </li></ol> 3. La stimolazione meccanica e radiomarcatura di condrociti-agarosio idrogel <ol><li> Autoclave i componenti metallici del rig compressione. Sterilizzare i componenti in plastica con il 70% di etanolo (durante la notte) e ai raggi UV (per almeno 15 minuti). </li><li> Per mantenere costrutti ribaltamento, mediante pinza, posto 12 anelli di plastica sterili fissaggio (con un diametro interno maggiore del diametro costrutto) (n = 6, campioni e controlli) nei pozzetti di una piastra a 24 pozzetti cultura. </li><li> Utilizzando pinze, trasferire accuratamente i condrociti-agarosio idrogel dalla capsula di Petri a 24 pozzetti, assicurando ognuno in un anello di ritegno. </li><li> Trasferire 400 ml di mezzi completi (ad esempio il 20% FBS in Ham F-12 terreni di coltura con 20 mM di HEPES, 100 mcg / ml di acido ascorbico, e2x antibiotici / antimicotici) in ciascun pozzetto. </li><li> Montare l&#39;impianto di compressione sterilizzato (Figura 1) e fissare le piastre con viti di fissaggio. Collegare l&#39;impianto di perforazione di compressione al 24 pozzetti piastra di coltura. </li><li> Rilasciare e ri-fissare le viti a stabilire contatto piastra-idrogel (zero stato di deformazione). </li><li> Caricare l&#39;impianto di compressione assemblati in un sistema di Mach-1 Test di micromeccanica. Rig sicuro allo stadio verticale tramite perno di posizionamento e bloccaggio in posizione con vite di fermo. Rimuovere la dima spaziatura. </li><li> Applicare al carico di compressione dinamica ampiezza desiderata (ampiezza di deformazione ad esempio 10% in base all&#39;altezza originale dei campioni), la frequenza (ad esempio 1 Hz) e durata o il numero di cicli (ad esempio, intervalli di tempo fino a 60 min). </li><li> Al termine della stimolazione meccanica, sostituire la maschera di distanza, allentare la vite di fermo posizionamento perno e rimuovere l&#39;impianto di compressione e piastra di coltura dal sistema-1 Mach Testing micromeccanica. </li&gt; <li> Radiotracciante cellula-idrogel costrutti completandola i media con 5 uCi dell&#39;isotopo desiderato (ad esempio, [3 H]-prolina label proteine ​​per condrociti specifica sintesi del collagene e [35 S] tenore di zolfo per la sintesi dei proteoglicani). </li><li> Incubare i costrutti radiomarcati per 24 hr a 37 ° C, 5% CO 2 10. </li><li> Raccogliere i costrutti idrogel radioattivi e lavare 3 volte in 1x PBS (pH 7.4) per rimuovere eventuale isotopo prive di personalità giuridica. Campioni digest usando papaina (40 mg / ml in 20 mM di acetato di ammonio, acido 1 mM ditiotreitolo ethyldiaminetetraacetic e 2 mM a 65 ° C per 72 ore). </li><li> Saggio digest per il contenuto di DNA per mezzo del test PicoGreen colorante 11 e l&#39;attività degli isotopi incorporato, normalizzato al contenuto di DNA, da β-liquido di scintillazione conteggio 12,13 digestione papaina subito dopo. </li></ol> Nota: L&#39;acquisto e lo smaltimento di materiali radioattivi (isotopi di rifiuti e oggettiche possano essere venuti in contatto con materiali radioattivi) deve seguire il istituzionale rilevante (e / o di governo) le politiche e le procedure per la manipolazione e lo smaltimento di sostanze radioattive. 4. Risultati rappresentativi Condrociti articolari bovini-agarosio costrutti idrogel (2% agarosio con 10 x 10 6 cellule / ml cellule incapsulate) sono stati stimolati meccanicamente ad una ampiezza di 10% di deformazione a compressione ad una frequenza di 1 Hz per 20 a 60 min (o 1.200 a 3.600 cicli ) e analizzati per il contenuto di DNA e la sintesi della matrice extracellulare per incorporazione radioisotopo. DNA e sintesi di matrice extracellulare è stato influenzato in un modo dipendente dalla dose. Contenuto di DNA hanno mostrato una diminuzione del 35% come risultato di 20 o 30 min di stimolazione (p &lt;0,01), mentre 60 min di stimolazione mostrato alcun effetto (Figura 2). Cartilagine-specifiche sintesi di collagene e proteoglicani (determinato da [3 H]-prolina e[35 S]-zolfo incorporazione, rispettivamente) mostra significativi aumenti di circa il 60% in risposta a 20 o 30 min di stimolazione (p &lt;0,01), con 60 min di stimolazione esibendo alcun effetto osservabile (Figura 3). <img src="/files/ftp_upload/4229/4229fig1.jpg" alt="Figura 1" /> . Figura 1 Schema della fuoriserie rig compressione dinamica per stimolare condrociti-agarosio costrutti sinistra:. Rig assemblato destra:. Esploso che mostra i singoli componenti. <img src="/files/ftp_upload/4229/4229fig2.jpg" alt="Figura 2" /> Modifiche Figura 2. Nel contenuto di DNA-agarosio condrociti idrogel stimolato meccanicamente sotto una ampiezza 10% di deformazione a compressione a 1 Hz per 20 a 60 minuti (media ± SEM, n = 6/group). Modifiche Figura 3. Nella sintesi della matrice extracellulare (collagene e proteoglicani) di condrociti-agarosio idrogel meccanicamente stimolato meno del 10% ampiezza di deformazione a compressione a 1 Hz per 20 a 60 minuti (media ± SEM, n = 6/group).

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<html> Il metodo descritto per l&#39;applicazione di stimoli meccanici controllati alla cella testa di serie agarosio idrogel permette l&#39;indagine diretta sugli effetti dinamici delle forze di compressione sul metabolismo dei condrociti. L&#39;uso del custom-test rig in combinazione con gli anelli di ritenuta previsto vincolo laterale per i costrutti di evitare potenziali problemi di campione ribaltamento. L&#39;uso di morti ponderati piani di carico garantiti da equipaggi set garantisce un contatto diretto con i cost...

<table border="1"><tbody><tr><td> Nome del reagente o attrezzature </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Ham F-12 </td><td> Thermo Fisher Scientific </td><td> SH3001002 </td><td></td></tr><tr><td> Collagenasi A </td><td> Sigma Aldrich Ltd. </td><td> C0130 </td><td></td></tr><tr><td> Proteasi </td><td> Sigma Aldrich Ltd. </td><td> P5147 </td><td></td></tr><tr><td> Siero fetale bovino </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> Gli antibiotici / antimicotici </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> Blu Trypan </td><td> Sigma Aldrich Ltd. </td><td> 93595 </td><td></td></tr><tr><td> Reichert Luminoso-Line Emocitometro </td><td> Hausser scientifico </td><td> 1490 </td><td></td></tr><tr><td> Quant-iT PicoGreen </td><td> Invitrogen </td><td> P7589 </td><td></td></tr><tr><td> Papaina da papaia lattice </td><td> Sigma Aldrich Ltd. </td><td> P3125 </td><td></td></tr><tr><td> Ammonio acetato </td><td> Sigma Aldrich Ltd. </td><td> A1542 </td><td></td></tr><tr><td> Acid Ethyldiaminetetraacetic </td><td> Sigma Aldrich Ltd. </td><td> E9884 </td><td></td></tr><tr><td> DL-Dithiothreitol </td><td> Sigma Aldrich Ltd. </td><td> 43819 </td><td></td></tr><tr><td> Basso punto di fusione agarosio, Type VII </td><td> Sigma Aldrich Ltd. </td><td> A9045 </td><td></td></tr> &lt;tr&gt; <td> Schermo Mesh (200) Filtro </td><td> Sigma Aldrich Ltd. </td><td> S4145 </td><td></td></tr><tr><td> Mach-1 Tester micromeccanici </td><td> Biomomentum Inc. </td><td> V500cs </td><td></td></tr><tr><td> Compressione Caricamento Jig </td><td> Custom-built </td><td></td><td> Prodotto similare potrebbe essere fornito da Biomomentum Inc. </td></tr><tr><td> Falcon 24 piastra di coltura Bene </td><td> Thermo Fisher Scientific </td><td> B353047 </td><td></td></tr><tr><td> β-liquido di scintillazione Contatore </td><td> Beckman Coulter </td><td> LS6500 </td><td></td></tr><tr><td> [3H] prolina </td><td> Perkin-Elmer </td><td> NET323005MC </td><td></td></tr><tr><td> [35S] Zolfo </td><td> Perkin-Elmer </td><td> NEX041005MC </td><td></td></tr></tbody></table>

mechanical stimulation of chondrocyte-agarose hydrogels

La cartilagine articolare soffre di una limitata capacità di riparazione, se danneggiato da insulto meccanico o degradato da malattie, come osteoartrite. Per rimediare a questa mancanza, diversi interventi medici sono stati sviluppati. Uno di questi metodi è quello di riaffiorare l&#39;area danneggiata con il tessuto-ingegnerizzato cartilagine, tuttavia, il tessuto ingegnerizzato manca in genere le proprietà biochimiche e la durata della cartilagine nativa, mettendo in discussione la sua sopravvivenza a lungo termine. Questo limita l&#39;applicazione di ingegneria tissutale cartilagine alla riparazione di piccoli difetti focali, basandosi sul tessuto circostante per proteggere il materiale impiantato. Per migliorare le proprietà del tessuto sviluppato, stimolazione meccanica è un metodo utilizzato per aumentare la sintesi di matrice extracellulare cartilaginea e le risultanti proprietà meccaniche del tessuto ingegnerizzato. Stimolazione meccanica applica forze al tessuto costrutti analoghi a quelli sperimentati in vivo. Questoè basato sulla premessa che l&#39;ambiente meccanico, in parte, regola lo sviluppo e la manutenzione di 1,2 tessuto nativo. La forma più comunemente applicato di stimolazione meccanica in ingegneria tissutale cartilagine è la compressione dinamica a ceppi fisiologiche di circa il 5-20% con una frequenza di 1 Hz 1,3. Diversi studi hanno studiato gli effetti della compressione dinamica e hanno mostrato di avere un effetto positivo sul metabolismo dei condrociti e biosintesi, infine influenzare le proprietà funzionali del tessuto sviluppato 4-8. In questo lavoro, ci illustrano il metodo per stimolare meccanicamente condrociti-agarosio costrutti idrogel in compressione dinamica e analizzare i cambiamenti nella biosintesi attraverso test biochimici e radioisotopi. Questo metodo può essere facilmente modificato per valutare eventuali cambiamenti potenzialmente indotti in risposta cellulare a seguito di stimoli meccanici.

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

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

La biosintesi di matrice extracellulare cartilaginea dai condrociti può essere influenzata da applicazione di stimoli meccanici. Questo metodo descrive la tecnica di applicazione di compressione dinamiche ceppi di condrociti incapsulati in costrutti 3D e la valutazione dei cambiamenti indotti nel metabolismo dei condrociti.

La stimolazione meccanica di condrociti-agarosio idrogel

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 Views.  Queen's University. La biosintesi di matrice extracellulare cartilaginea dai condrociti può essere influenzata da applicazione di stimoli meccanici. Questo metodo descrive la tecnica di applicazione di compressione dinamiche ceppi di condrociti incapsulati in costrutti 3D e la valutazione dei cambiamenti indotti nel metabolismo dei condrociti.

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 Carico meccanico del tendine

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

Ricerca

Biologia

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 stimolazione meccanica delle cellule staminali Utilizzando ciclica deformazione uniassiale

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.

Incapsulamento cellulare in idrogel 3D per l&#39;ingegneria tissutale

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.

Risposta cellulare allo stimolo meccanico vivere Studiato da Integrated ottica e microscopia a forza atomica

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 stimolazione meccanica indotta calcio Wave Propagation in monostrati cellulari: l&#39;esempio di bovini cellule endoteliali corneali

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.

Preparazione di DNA-reticolato poliacrilammide idrogel

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.

Multifunzionale, Metodo Micropipetta-based per la Costituzione e la stimolazione della batteriche meccanosensibili Ion Canali 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. ...

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 in idrogel tridimensionali

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.

Valutazione della risposta di Dictyostelium discoideum alla stimolazione meccanica acuta

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.

Idrogel alginato degradati proteolylylylyd e microbioreattori idrofobici per l'incapsulamento dell'Oocito Porcine

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