1. Isolation of Primary Articular Chondrocytes
Harvest 10-15 full thickness cartilage slices from the articular surfaces of animal joints (e.g. the metacarpal-phalangeal joint of skeletally mature cows obtained from a local abbatoir).
<ol>
 <li>Place cartilage slices in a 100 mm Petri dish and Incubate in 20 ml of 0.5% protease in Ham's F-12 (w/v) for 2 hr at 37 &deg;C. Rinse three times in Ham's F-12 culture media, and incubate with 20 ml of 0.15% Collagenase A in Ham's F-12 culture media overnight at 37 &deg;C.</li>
 <li>Filter the cell suspension through a 200 mesh screen filter into a clean dish. Wash the filter with 5 ml of Ham's F-12 and add to the dish. Transfer the cell suspension to a 50 ml conical tube. Wash the Petri dish with 10 ml of Ham's F-12 and add to the conical tube.</li>
 <li>Centrifuge the tube at 600-800 rcf for 6-8 min at room temperature.</li>
 <li>Aspirate the supernatant, ensuring not to disturb the cell pellet and re-suspend in 40 ml of Ham's F-12 culture media.</li>
 <li>Centrifuge the tube at 600-800 rcf for 6-8 min.</li>
 <li>Repeat steps 1.4 and 1.5 twice more for a total of 3 times. Prior to centrifuging for the third time, resuspend the pellet via agitation and obtain a 500 &mu;l aliquot for cell counting.</li>
 <li>Count viable cells using the Trypan blue exclusion method9 with a hemacytometer and an inverted light microscope.</li>
 <li>Aspirate the supernatant and re-suspend the pellet in a volume of Ham's F-12 media to double the desired seeding density (e.g. 20 x 106 cells/ml for a final concentration of 10 x 106 cells/ml after encapsulation).</li>
</ol>
2. Chondrocyte-agarose Hydrogel Encapsulation
<ol>
 <li>Heat 0.8 g autoclaved Type VII agarose in 20 ml of sterile 1x PBS (pH 7.4) on a hot plate set to 120 &deg;C until dissolved. Once dissolved, lower the heat to 60 &deg;C as higher temperatures will affect cell viability.</li>
 <li>In a 50 ml conical tube, thoroughly mix equal volumes of the cell suspension with double the desired concentration of agarose (e.g. 4% agarose suspension for a final 2% agarose hydrogel). Avoid the creation of large bubbles as they can affect cell viability and construct fatigue life.</li>
 <li>Carefully aliquot 10 ml of the chondrocyte-agarose mixture into a 60 mm diameter Petri dish, while avoiding the creation of large bubbles.</li>
 <li>Let stand for 30 min at room temperature until gelled.</li>
 <li>Obtain as many chondrocyte-agarose hydrogel samples as needed (typically 20-30) using a 4 mm biopsy punch.</li>
 <li>Measure and record the physical dimensions (diameter and height) of representative samples using a micrometer. Constructs should be approximately 5 mm in height, discard any samples obtained from a region where the sample height variance was greater than 2%.</li>
 <li>Transfer samples to a fresh 60 mm Petri dish and supplement with 10 ml of complete media (e.g. 20% fetal bovine serum in Ham's F-12 culture media with 20 mM HEPES, 100 &mu;g/ml ascorbic acid, and 2x antibiotics/antimycotics).</li>
</ol>
3. Mechanical Stimulation and Radiolabelling of Chondrocyte-agarose Hydrogels
<ol>
 <li>Autoclave the metallic components of the compression rig. Sterilize the plastic components with 70% ethanol (overnight) and UV light (for at least 15 min).</li>
 <li>To keep constructs from falling over, using forceps, place 12 sterile plastic retaining rings (with an inner diameter larger than the construct diameter) (n=6, samples and controls) within the wells of a 24-well culture plate.</li>
 <li>Using forceps, carefully transfer the chondrocyte-agarose hydrogels from the Petri dish to the 24-well plate, securing each one in a retaining ring.</li>
 <li>Transfer 400 &mu;l of complete media (e.g. 20% FBS in Ham's F-12 culture media with 20 mM HEPES, 100 &mu;g/ml ascorbic acid, and 2x antibiotics/antimycotics) to each sample well.</li>
 <li>Assemble the sterilized compression rig (Figure 1) and secure the platens with set screws. Attach the compression rig to the 24-well culture plate.</li>
 <li>Release and re-secure the set screws to establish platen-hydrogel contact (zero strain state).</li>
 <li>Load the assembled compression rig into a Mach-1 Micromechanical Testing System. Secure rig to the vertical stage via locating pin and lock in place with set screw. Remove the spacing jig.</li>
 <li>Apply dynamic compressive loading at desired amplitude (e.g. 10% strain amplitude based on the original height of the samples), frequency (e.g. 1 Hz) and duration or number of cycles (e.g. time intervals up to 60 min).</li>
 <li>Upon the completion of mechanical stimulation, replace the spacing jig, loosen the locating pin set screw and remove the compression rig and culture plate from the Mach-1 Micromechanical Testing System.</li>
 <li>Radiolabel cell-hydrogel constructs by supplementing the media with 5 &mu;Ci of desired isotope (e.g. [3H]-proline protein label for chondrocyte-specific collagen synthesis and [35S]-sulfur for proteoglycan synthesis).</li>
 <li>Incubate the radiolabeled constructs for 24 hr at 37 &deg;C, 5% CO210.</li>
 <li>Harvest the radiolabeled hydrogel constructs and rinse 3 times in 1x PBS (pH 7.4) to remove any unincorporated isotope. Digest samples using papain (40 &mu;g/ml in 20 mM ammonium acetate, 1 mM ethyldiaminetetraacetic acid and 2 mM dithiothreitol at 65 &deg;C for 72 hr).</li>
 <li>Assay digest for DNA content using the PicoGreen dye assay11 and incorporated isotope activity, normalized to DNA content, by &beta;-liquid scintillation counting12,13 immediately following papain digestion.</li>
</ol>
Note: The purchase and disposal of radioactive materials (waste isotopes and items that may have come into contact radioactive materials) must follow the relevant institutional (and/or governmental) policies and procedures for the safe handling and disposal of radioactive substances.
4. Representative Results
Bovine articular chondrocyte-agarose hydrogels constructs (2% agarose with 10 x 106 cells/ml encapsulated cells) were mechanically stimulated at an amplitude of 10% compressive strain at a frequency of 1 Hz for 20 to 60 min (or 1,200 to 3,600 cycles) and assayed for DNA content and extracellular matrix synthesis by radioisotope incorporation. DNA and extracellular matrix synthesis was affected in a dose-dependent manner. DNA content exhibited a significant 35% decrease as a result of 20 or 30 min of stimulation (p&lt;0.01), whereas 60 min of stimulation exhibited no effect (Figure 2). Cartilage-specific collagen and proteoglycan synthesis (determined by [3H]-proline and [35S]-sulfur incorporation, respectively) exhibited significant increases of approximately 60% in response to 20 or 30 min of stimulation (p&lt;0.01), with 60 min of stimulation exhibiting no observable effect (Figure 3).
<img src="/files/ftp_upload/4229/4229fig1.jpg" alt="Figure 1" /> 
 Figure 1. Schematic of the custom-built dynamic compression rig for stimulating chondrocyte-agarose constructs. Left: Assembled rig. Right: Exploded view showing individual components.
<img src="/files/ftp_upload/4229/4229fig2.jpg" alt="Figure 2" /> 
 Figure 2. Changes in DNA content of chondrocyte-agarose hydrogels mechanically stimulated under a 10% compressive strain amplitude at 1 Hz for 20 to 60 min (mean &plusmn; SEM, n=6/group).
<img src="/files/ftp_upload/4229/4229fig3.jpg" alt="Figure 3" /> 
 Figure 3. Changes in extracellular matrix synthesis (collagen and proteoglycans) of chondrocyte-agarose hydrogels mechanically stimulated under 10% compressive strain amplitude at 1 Hz for 20 to 60 min (mean &plusmn; SEM, n=6/group).

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No conflicts of interest declared.

The described method for applying controlled mechanical stimuli to cell-seeded agarose hydrogels allows for the direct investigation into the effects of dynamic compressive forces on chondrocyte metabolism. The use of the custom-testing rig in conjunction with the retaining rings provided lateral constraint for the constructs to avoid potential problems of sample tipping. The use of dead-weighted loading platens secured by sets crews ensures direct contact with constructs despite potential differences in sample height. R...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent or equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Ham's F-12</td>
 <td>Thermo Fisher Scientific</td>
 <td>SH3001002</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase A</td>
 <td>Sigma Aldrich Ltd.</td>
 <td>C0130</td>
 <td></td>
 </tr>
 <tr>
 <td>Protease</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>Antibiotics/antimycotics</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 blue</td>
 <td>Sigma Aldrich Ltd.</td>
 <td>93595</td>
 <td></td>
 </tr>
 <tr>
 <td>Reichert Bright-Line Hemacytometer</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>Papain from papaya latex</td>
 <td>Sigma Aldrich Ltd.</td>
 <td>P3125</td>
 <td></td>
 </tr>
 <tr>
 <td>Ammonium Acetate </td>
 <td>Sigma Aldrich Ltd.</td>
 <td>A1542</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethyldiaminetetraacetic Acid </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>Low Melting Point Agarose, Type VII</td>
 <td>Sigma Aldrich Ltd.</td>
 <td>A9045</td>
 <td></td>
 </tr>
 <tr>
 <td>Mesh Screen (200) Filter</td>
 <td>Sigma Aldrich Ltd.</td>
 <td>S4145</td>
 <td></td>
 </tr>
 <tr>
 <td>Mach-1 Micromechanical Tester</td>
 <td>Biomomentum Inc.</td>
 <td>V500cs</td>
 <td></td>
 </tr>
 <tr>
 <td>Compression Loading Jig</td>
 <td>Custom-built</td>
 <td></td>
 <td>Similar product could be supplied by Biomomentum Inc.</td>
 </tr>
 <tr>
 <td>Falcon 24 Well Culture Plate</td>
 <td>Thermo Fisher Scientific</td>
 <td>B353047</td>
 <td></td>
 </tr>
 <tr>
 <td>&beta;-Liquid Scintillation Counter</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] Sulfur</td>
 <td>Perkin-Elmer</td>
 <td>NEX041005MC </td>
 <td></td>
 </tr>
 </tbody>
</table>

mechanical stimulation of chondrocyte-agarose hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

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

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

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

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11.6K Views.  Queen's University. The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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