This work was funded by NSF CAREER CBET-0747615. The authors would like to thank Drs. Douglas H. Smith and David F. Meaney for their mentorship and support.

1. Overview of The Axon Stretch Growth Bioreactor System
<ol>
	<li>Two predominant approaches have been utilized to apply experimental forces to neurons. In the first approach, forces are applied to the entire neuron (Lu, Franze et al. 2006; Chetta, Kye et al. 2010; Lindqvist, Liu et al. 2010). In the second approach, forces are applied directly to the axon by pulling on the growth cone. Using glass needles, this latter approach has been used to model formation of new axon (Bernal, Pullarkat et al. 2007; O'Toole, Lamoureux et al. 2008; Lamoureux, Heidemann et al. 2010), to identify force thresholds for elongation and retraction (Dennerll, Lamoureux et al. 1989; Zheng, Lamoureux et al. 1991; Lamoureux, Zheng et al. 1992), and to analyze neurotransmitter clustering in axon terminals (Siechen, Yang et al. 2009). A unique tissue engineering extension of this method was developed to produce large nerve constructs using an automated Axon Stretch Growth (ASG) bioreactor system (Iwata, Browne et al. 2006; Pfister, Iwata et al. 2006). Recently, we developed a miniaturized version of the bioreactor system to study ASG microscopically in real time (Loverde, Ozoka et al. 2011). Here, we detail the 9-day protocol (table 1) we currently use to prepare bioreactors and perform ASG routinely.</li>
	<li>Overall Operation: The ASG bioreactor system is comprised of three main components, 1) a bioreactor chamber with independent lanes (elongated wells) where neurons are cultured and stretched, 2) an automated linear motion table to apply stretching forces and 3) a step motor drive controller with software to control stretch growth (figure 1).</li>
	<li>Briefly, fabrication of bioreactor prototypes was performed in a machine shop using a vertical milling machine (Bridgeport, Elmira, NY). For biocompatibility, ease of sterilization and durability, the internal bioreactor components were machined from 3/8" polyetheretherketone (PEEK). Transparent polycarbonate was used for lids to allow for light microscopy and viewing of cultures. Corrosion resistant 316 stainless steel screws and hardware complete the assembly (McMaster-Carr, Elmhurst, IL).</li>
	<li>The bioreactor chamber is comprised of a stretching frame that forms 3 lanes, an adjustable towing block that manipulates cells across the lanes, and protruding towing rods for external manipulation (figure 2). Neuronal cultures are plated onto disposable Aclar towing culture substrates supported by the towing block (figures 2 &amp; 3). A disposable coverslip stationary substrate attaches to the bottom of the stretching frame and spans all 3 lanes. Prior to stretching, plated neurons must extend axonal processes from the towing substrate onto the stationary substrate via growth cone extension. The population of axons that bridge the substrates will subsequently undergo stretch by controlling displacement of the towing block.</li>
	<li>The automated linear motion table consists of a stepper motor (HT23-397, Applied Motion Products, Watsonville, CA) and linear motion table (MIPS-2-10-1.0mm, Servo Systems, Monteville, NJ) mounted to a delrin alignment table. The bioreactor chamber is seated within the table parallel to the linear motion table. The towing rods extending from the bioreactor chamber are fastened to the linear motion table using an adaptor.</li>
	<li>The step motor drive controller (Si 2035, Applied Motion Products) is programmed using the included Si programmer software to control manipulation of the towing substrates. Axonal stretch is applied in a stepwise fashion by taking a series of small displacement steps spaced by dwell times (Pfister, Iwata et al. 2004; Pfister, Iwata et al. 2006). This computer-controlled system provides the ability to program customized profiles for ASG, and is essential for continuous experimentation over several days to weeks.</li>
</ol>
2. Preparation of Bioreactor Chamber
<ol>
	<li>Prepare the disposable towing and stationary culture substrates for neuronal culture:
	<ol>
		<li>Towing culture substrates are cut from 8.5" x 11" sheets of Aclar film (33C 2.0 mil, Structure Probe, West Chester, PA). Using a sharp blade cut the substrates to approximately 0.5 x 2.5cm, or slightly shorter than the width of the lanes to allow at least 1-2mm clearance on both sides.</li>
		<li>Lightly sand the lower 1/3rd of the towing culture substrates on both sides using fine 1200-grit sandpaper (McMaster Carr). Sanding of the towing substrates facilitates growth of axons from the towing substrates onto the coverslip stationary substrate.</li>
		<li>Cut a 5 x 7cm piece of Aclar or use a No. 1 glass coverslip to serve as the stationary substrate (#4865-1, Brain Research Labs, Newton, MA).</li>
		<li>Clean the culture substrates with a dilute Alconox solution and rinse thoroughly with purified dH2O.</li>
		<li>Sterilize the culture substrates by immersion in 70% ethanol for 30 minutes. Allow the substrates to air dry within a sterile tissue culture hood.</li>
	</ol>
	</li>
	<li>Clean the bioreactor chamber with dilute Alconox and autoclave sterilize within an autoclave container. Immediately after autoclaving, transfer to a sterile hood and allow to air dry.</li>
	<li>Continuing within the sterile hood, glue the culture substrates to the bioreactor using Silicon RTV (Dow Corning #732, McMaster Carr) and sterile cotton tipped swabs (McMaster Carr):
	<ol>
		<li>With the towing block legs in their full upright position, glue the towing culture substrates to the towing block legs at the non-sanded portion. Avoid contact with the sanded culture surface.</li>
		<li>Glue the stationary culture substrate to the bottom of the bioreactor chamber.</li>
		<li>Remove excess glue and air pockets by gently depressing a dry swab against the glued substrates.</li>
		<li>Since Silicone RTV leaches acetic acid that is toxic to neurons, the bioreactor is left to dry under UV light within the hood for 2 full days prior to introducing neuronal cultures.</li>
	</ol>
	</li>
	<li>Lower the towing block legs to achieve a 2-3mm overlap between the tips of the sanded towing substrates and the stationary substrate. Crucial attention must be paid to the size of the overlap, if it is too large, the tips of the towing substrates may deflect off of the stationary substrate; reducing the number of axons that span the overlap (figure 3).</li>
	<li>Position the towing block at the starting position, with the towing rods retracted inside the bioreactor. Tighten the immobilization screws to prevent movement of the towing rods prior to stretch growth.</li>
</ol>
3. Neuronal Cultures
<ol>
	<li>Pool 1mL of 10 &mu;g/mL high molecular weight poly-d-lysine (cat# 354210, BD, Bedford, MA) in serum-free media at the substrate interface area of each lane. Allow the solution to adhere undisturbed for 1 hour at room temperature. Rinse gently 3x with dH2O, followed by a final rinse with culture media. Pipetting should be performed at the back of the lanes, farthest the substrate interface.</li>
	<li>Isolate Dorsal Root Ganglia explants (DRGs) from an E16 rat pup. Using a stereomicroscope, dilute the explants into drops using petri dishes. Collect 3-4 explants using a 100 &mu;L pipette and plate onto the edge of the sanded towing culture substrate. Care must be taken to plate the explants at the edge of the towing substrate using a small puddle of media. Up to 1mL of culture media per lane is sufficient to prevent evaporation for several hours while limiting movement of the explants. Media formulation: Neurobasal with B-27 + 0.5mM L-Glutamine (Invitrogen, Carlsbad, CA), 1% FBS-HI (Hyclone, Waltham, MA), 2.5g/L D-Glucose (G-7528, Sigma, St. Louis, MO), 20ng/mL NGF (13290-010, Invitrogen) and 20 &mu;M FdU + 20 &mu;M Uridine mitotic inhibitors (F-0503, U-3003, Sigma).</li>
	<li>Attach the lid and transfer the bioreactor to an incubator for 1 hour or until cells adhere.</li>
	<li>Fill the bioreactor with culture media at the point farthest the explants to avoid dislodgment. Incubate the bioreactor for a minimum of 5 days while neurons extend axonal processes onto the stationary substrate.</li>
</ol>
4. Axon Stretch Growth
<ol>
	<li>The bioreactor undergoes final preparation procedures inside a sterile hood:
	<ol>
		<li>Fill reservoirs within the bioreactor with phosphate buffered saline to humidify the chamber and limit evaporation of culture media. In our system, the hollowed enclosure walls serve as reservoirs. Alternatively, small petri dish lids may be placed on either side of the towing block atop the culture lanes.</li>
		<li>Replace the culture media and fill the lanes to capacity. Pipetting should be performed at the back of the lanes, farthest the substrate interface.</li>
	</ol>
	</li>
	<li>Seat the bioreactor within the automated linear motion table. If the experiment is to be run within an incubator, it must not be humidified due to potential corrosion of the automated linear motion table.</li>
	<li>Fasten the towing rod adaptor to the towing rods.</li>
	<li>Using Si Programmer software jog the stage of the linear motion table to align with the towing rod adaptor and fasten it to the stage. For convenience, prepare Si Programmer sequences in advance in order to manipulate the stage in specific increments.</li>
	<li>Loosen the immobilization screws on the bioreactor chamber to allow free movement of the towing block.</li>
	<li>Stretch is applied in a stepwise fashion by initiating a series of small displacement steps spaced by dwell times (Pfister, Iwata et al. 2006). Our paradigm starts by taking 2 &mu;m steps every 172 seconds, resulting in a net 1mm stretch over 24 hours. After one day, the stretch rate can be ramped by increasing the displacement or decreasing the dwell time (table 2).</li>
	<li>Once ASG is initiated, media changes are typically not required. Over time, however, culture media generally turns acidic and is evident by a yellowish change in color. If further experimentation is required, old media is never fully drained, but instead only partially changed in order to minimize trauma to floating axons.</li>
</ol>
5. Representative Results:
Axonal processes can undergo remarkably rapid and robust stretch growth. Initially, the process begins with a period of slow stretching (&le; 1mm/day) that consists of small, infrequent displacements. Within the first 24 hours of stretching, mechanotransduction of neuronal growth pathways occurs, whereby neurons begin addition to the axon cylinder. Within 24 hours of continuous ASG, axons show an increasing tolerance to greater and more frequent displacements. In general, axons can withstand an increase in the stretch rate of 1mm/day every 12-24 hours (Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006). Increasing the stretch rate too soon, however, may lead to more rapid growth of select axons but will also lead to pathological occlusion that causes disconnection.
Stretch-growing axons have the tendency to form bundles, resembling the architecture of fascicles. Utilizing current protocols, the central, stretch grown portion of axon bundles have no adhesions to the culture substrate. Only the initially adhered, proximal and distal segments of stretch-growing axons remain attached to the culture substrates. Accordingly, the central portion of stretch grown axons float freely, and are sensitive to disruption due to handling.
For a variety of reasons, some axons cannot grow at the applied stretch rate. For example, a DRG neuron with two axonal processes, both of which are undergoing stretch, may not be able to translate sufficient protein and grow at the applied stretch rate. Axons that cannot accommodate the applied stretch will thin following Poisson's effect. Subsequent stretch will lead to occlusion of the axons, inhibiting assembly, leading to pathological disconnection. The majority of axons, however, are able to undergo ASG successfully and only a small percentage of axons undergo this pruning-like process.
<img alt="Figure 1" src="/files/ftp_upload/2753/2753fig1.jpg" /> 
Figure 1. Axon Stretch Growth Bioreactor System. (A) Bioreactor culture chamber &amp; Automated linear motion table, (B) Step motor drive controller and Si Programming software.
<img alt="Figure 2" src="/files/ftp_upload/2753/2753fig2.jpg" /> 
Figure 2. Bioreactor Culture Chamber. This cartoon depicts the bioreactor chamber from the top with the lid removed. The position of the towing block reflects the end stage of axon stretch growth. Stretch grown axons can be seen in bundles within the culture lanes.
<img alt="Figure 3" src="/files/ftp_upload/2753/2753fig3.jpg" /> 
Figure 3. Culture Substrate Plating Interface. This cartoon depicts the components of the towing mechanism within each lane of the bioreactor from side view. (Top) Correct overlap of the towing and stationary culture substrates. (Bottom) Excessive overlap of the towing and stationary substrates causes the tip of the towing substrate to curl.
Movie 1. Attachment of Culture Substrates to Bioreactor Chamber. <a href="https://www.jove.com/files/ftp_upload/2753/movie1.mov">Click here to watch video</a>
Movie 2. Plating of DRG Explants onto Towing Substrates. <a href="https://www.jove.com/files/ftp_upload/2753/movie2.mov">Click here to watch video</a>
Movie 3. SiProgrammer Usage. <a href="https://www.jove.com/files/ftp_upload/2753/movie3.mov">Click here to watch video</a>
Movie 4. Growth Cone Extension onto Stationary Substrate. <a href="https://www.jove.com/files/ftp_upload/2753/movie4.mov">Click here to watch video</a>
Movie 5. Axon Stretch Growth. <a href="https://www.jove.com/files/ftp_upload/2753/movie5.mov">Click here to watch video</a>
<table border="1">
	<tbody>
		<tr>
			<td>Day</td>
			<td>Step</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Sterilization &amp; Drying</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Gluing &amp; Assembly</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Coatings &amp; Neuronal Culture Plating</td>
		</tr>
		<tr>
			<td>9</td>
			<td>Stretch Growth Start</td>
		</tr>
	</tbody>
</table>
Table 1. Experiment Schedule.
<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>Time [hr]</td>
			<td>Stretch Rate [mm/day]</td>
			<td>Dwell Time [s]</td>
			<td>Total Length [mm]</td>
			<td>Total Stretch Time [days]</td>
		</tr>
		<tr>
			<td>Pretension</td>
			<td>24</td>
			<td>1</td>
			<td>172.8</td>
			<td>0</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Stretch</td>
			<td>24</td>
			<td>1</td>
			<td>172.8</td>
			<td>1</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Stretch</td>
			<td>24</td>
			<td>2</td>
			<td>86.4</td>
			<td>3</td>
			<td>3</td>
		</tr>
		<tr>
			<td>Stretch</td>
			<td>24</td>
			<td>3</td>
			<td>57.6</td>
			<td>6</td>
			<td>4</td>
		</tr>
		<tr>
			<td>Stretch</td>
			<td>24</td>
			<td>4</td>
			<td>43.2</td>
			<td>10</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Stretch</td>
			<td>24</td>
			<td>5</td>
			<td>34.6</td>
			<td>15</td>
			<td>6</td>
		</tr>
	</tbody>
</table>
Table 2. Stretch Rate Schedule. All stretch steps are 2 &mu;m in displacement (10 step motor steps = 2 &mu;m stretch).

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The ex-vivo mechanical elongation of neuronal cells is patented under US Patents #6264944 and #7429267.

Two critical steps should be observed during preparation of bioreactors. First, an optimal overlap at the substrate interface is needed to ensure that axons can cross onto the stationary substrate. Aclar that is excessively curled or otherwise imperfect should not be used (figure 3). To optimize the overlap, confirm that the towing substrate is sanded evenly and contacts the stationary substrate uniformly over a 2-3mm long contact patch. The overlap should be optimized prior to each experiment by carefully adjusting the height of the towing legs.
Second, while providing for attachment of neurons, substrate coatings sustain considerable sheering forces caused by displacement of the bioreactor and contractile tension of the axons (Heidemann and Buxbaum 1990; Pfister, Iwata et al. 2004; Loverde, Ozoka et al. 2011). Substrates should be rinsed thoroughly with sterilized water both prior to and after lysine coating. Coatings should be applied from freshly thawed aliquots and spread as evenly as possible. Importantly, the substrates should not be moved or otherwise disturbed during the adhesion period. In subsequent steps, avoid contact with the substrates during plating, and pipet all solutions from the far end of the lanes away from the substrate interface.
Tolerances in the connections of each bioreactor component can manifest in the form of slack. During the initial period of ASG, slack in the system is evident as movement of the automated linear motion table occurs without movement of the towing block. Slack can vary per experiment, but is typically &lt;1mm in our experience. For this reason, a "pre-tension" slack elimination phase of 1mm/day, for one day, precedes the ASG schedule during which the bioreactor parts engage and begin movement of the towing substrates.
Troubleshooting may be required if the displacement step of the automated linear motion table does not match the displacement of the towing block after the pre-tension phase is complete. Asynchronous, inaccurate displacements of the towing block are associated with 'stiction' or static friction within the towing hardware and flexing of the adaptor. To prevent these issues from occurring, movement of the towing block should be checked by hand, after assembly, for smooth near-effortless movement. If binding occurs, the towing assembly should be freed prior to experimentation. Sufficient stiffness of the adaptor is also necessary to assure accurate, synchronous displacements are not overcome by stiction of the towing hardware.

<table border="1">
	<tbody>
		<tr>
			<td>Name of component</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>PolyEtherEtherKetone (PEEK)</td>
			<td>McMaster-Carr, Elmhurst, IL</td>
			<td>8504K69</td>
			<td>Custom made bioreactor chamber</td>
		</tr>
		<tr>
			<td>Polycarbonate</td>
			<td>McMaster-Carr, Elmhurst, IL</td>
			<td>8574K28</td>
			<td>Custom made bioreactor chamber lid</td>
		</tr>
		<tr>
			<td>Stepper motor</td>
			<td>Applied Motion Products, Watsonville, CA</td>
			<td>HT23-397</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Linear motion table</td>
			<td>Servo Systems, Montville, NJ</td>
			<td>MIPS-2-10-1.0mm</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Step motor drive controller</td>
			<td>Applied Motion Products, Watsonville, CA</td>
			<td>Si2035</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aclar</td>
			<td>Structure Probe Inc., West Chester, PA</td>
			<td>1859</td>
			<td>Towing culture substrates</td>
		</tr>
		<tr>
			<td>No. 1 coverslip</td>
			<td>Brain Research Labs, Newton, MA</td>
			<td>4865-1</td>
			<td>Stationary culture substrate</td>
		</tr>
		<tr>
			<td>Silicon RTV</td>
			<td>McMaster-Carr, Elmhurst, IL</td>
			<td>7587A37</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cotton-tipped swabs</td>
			<td>McMaster-Carr, Elmhurst, IL</td>
			<td>7074T62</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>BD, Bedford, MA</td>
			<td>354210</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

axon stretch growth: the mechanotransduction of neuronal growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

Watch this Scientific Journal Video about Axon Stretch Growth: The Mechanotransduction of Neuronal Growth at JoVE.com

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

axon-stretch-growth-the-mechanotransduction-of-neuronal-growth

Research

JoVE Journal

Bioengineering

16.7K Views.  New Jersey Institute of Technology. A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

Video: Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their extracellular matrix (ECM) environment. Eukaryotic cells constantly sense their local microenvironment through surface mechanosensors to transduce physical changes of ECM into biochemical signals, and integrate these signals to achieve specific changes in gene expression. Interestingly, physicochemical and mechanical parameters of the ECM can couple with each other to regulate cell fate. Therefore, a key to understanding mechanotransduction is to decouple the relative contribution of ECM cues on cellular functions.
Here we present a detailed experimental protocol to rapidly and easily generate biologically relevant hydrogels for the independent tuning of mechanotransduction cues in vitro. We chemically modified polyacrylamide hydrogels (PAAm) to surmount their intrinsically non-adhesive properties by incorporating hydroxyl-functionalized acrylamide monomers during the polymerization. We obtained a novel PAAm hydrogel, called hydroxy-PAAm, which permits immobilization of any desired nature of ECM proteins. The combination of hydroxy-PAAm hydrogels with microcontact printing allows to independently control the morphology of single-cells, the matrix stiffness, the nature and the density of ECM proteins. We provide a simple and rapid method that can be set up in every biology lab to study in vitro cell mechanotransduction processes. We validate this novel two-dimensional platform by conducting experiments on endothelial cells that demonstrate a mechanical coupling between ECM stiffness and the nucleus.

We present a new polyacrylamide hydrogel, called hydroxy-PAAm, that allows a direct binding of ECM proteins with minimal cost or expertise. The combination of hydroxy-PAAm hydrogels with microcontact printing facilitates independent control of many cues of the natural cell microenvironment for studying cellular mechanostransduction.

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...