The authors would like to thank Eric Vasco for his assistance in the preparation of the PDMS substrates, Dr. Shiyong Wang in Dr. Marina Mata&#39;s lab at University of Michigan for helpful suggestions and training of the DRG isolation, and Dr. Mark Tuszynski and Dr. W. Marie Campana at UC San Diego for helpful suggestions and protocol for the SC isolation. This study was supported in part by the National Science Foundation (CBET 0941055 and CBET 1510895), the National Institute of Health (R21CA176854, R01GM089866, and R01EB014986).

All procedures for the isolation of the cells were approved by the Institutional Animal Care and Use Committee at Michigan State University.
1. Preparation of Pre-stretched Anisotropic Surface&#160;
<ol>
	<li>Mix a 10:1 solution of base and curing agent and pour the mixture into a tissue culture dish (12 cm diameter). Use 4,900 mg base and 490 mg curing agent for the total crosslinking mixture.</li>
	<li>Keep the gel mixture under vacuum for 20 min to remove air bubbles.</li>
	<li>Place the gel mixture in the ov...

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aligned+neurite+outgrowth+and+directed+cell+migration+in+self-assembled+monodomain+gels.">Aligned neurite outgrowth and directed cell migration in self-assembled monodomain gels.</a> Biomaterials. 35, 185-195 (2014).</li><li>Kidambi, S., Lee, I., Chan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+neuron/astrocyte+co-culture+on+polyelectrolyte+multilayer+films:+A+template+for+studying+astrocyte-mediated+oxidative+stress+in+neurons.">Primary neuron/astrocyte co-culture on polyelectrolyte multilayer films: A template for studying astrocyte-mediated oxidative stress in neurons.</a> Adv Funct Mater. 18, 294-301 (2008).</li><li>Xia, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+neurite+growth+of+rat+dorsal+root+ganglion+neurons+and+increased+colocalization+with+Schwann+cells+on+aligned+poly(methyl+methacrylate)+electrospun+nanofibers.">Directed neurite growth of rat dorsal root ganglion neurons and increased colocalization with Schwann cells on aligned poly(methyl methacrylate) electrospun nanofibers.</a> Brain Research. 1565, 18-27 (2014).</li><li>Smith, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretch+growth+of+integrated+axon+tracts:+Extremes+and+exploitations.">Stretch growth of integrated axon tracts: Extremes and exploitations.</a> Prog Neurobiol. 89, 231-239 (2009).</li><li>Mantuano, E., Jo, M., Gonias, S. L., Campana, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+Density+Lipoprotein+Receptor-related+Protein+(LRP1)+Regulates+Rac1+and+RhoA+Reciprocally+to+Control+Schwann+Cell+Adhesion+and+Migration.">Low Density Lipoprotein Receptor-related Protein (LRP1) Regulates Rac1 and RhoA Reciprocally to Control Schwann Cell Adhesion and Migration.</a> Journal of Biological Chemistry. 285, 14259-14266 (2010).</li><li>Kim, B., ET, K. P., Papautsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+stability+of+plasma+oxidized+PDMS+surfaces.">Long-term stability of plasma oxidized PDMS surfaces.</a> Conf Proc IEEE Eng Med Biol Soc. 7, 5013-5016 (2004).</li><li>Lopera, S., Mansano, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma-Based+Surface+Modification+of+Polydimethylsiloxane+for+PDMS-PDMS+Molding.">Plasma-Based Surface Modification of Polydimethylsiloxane for PDMS-PDMS Molding.</a> ISRN Polymer Science. 2012, (2012).</li><li>Chandra, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organosilicon Materials. , (2013).</li><li>Wu, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+poly(dimethylsiloxane)+surface+modification+to+control+cell+adhesion.">Simple poly(dimethylsiloxane) surface modification to control cell adhesion.</a> Surf Interface Anal. 41, 11-16 (2009).</li><li>Moore, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple-channel+scaffolds+to+promote+spinal+cord+axon+regeneration.">Multiple-channel scaffolds to promote spinal cord axon regeneration.</a> Biomaterials. 27, 419-429 (2006).</li><li>Clarke, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterned+methacrylate+polymers+direct+spiral+ganglion+neurite+and+Schwann+cell+growth.">Micropatterned methacrylate polymers direct spiral ganglion neurite and Schwann cell growth.</a> Hearing Res. 278, 96-105 (2011).</li><li>Pfister, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+transplantable+nervous+tissue+constructs+comprised+of+stretch-grown+axons.">Development of transplantable nervous tissue constructs comprised of stretch-grown axons.</a> J Neurosci Methods. 153, 95-103 (2006).</li></ol>

To induce axon alignment on pre-stretched surface, there are two critical steps: 1) the PDMS membrane must be flat and of homogenous thickness; and 2) glial cells must be removed from the DRG. After mixing the PDMS and crosslinker and curing in an oven, the crosslinked PDMS gel should be kept on a flat bench top and handled carefully to avoid any tilting. The oxygen plasma treatment of the PDMS membrane should be followed within 6 hr by PLL coating, since the hydrophilicity of the surface (required for cell attachment) a...

In nerve injuries, the proximal and distal nerve stumps are often prevented from direct realignment of nerve fascicles due to the lesion site 1-2. Normally, axon tracts are composed of highly ordered and aligned bundles of axons, which form complex networks of connectivity. However, nerve regeneration is a chaotic process due to poorly organized axon alignment 3-4. Therefore, to generate a sufficient number of regenerating axons that bridge the lesion site, it is necessary to induce well organized axonal alignment. Additionally, demyelination accompanies nerve injuries due to death of the myelinating cells at the injury site. Since demyelination strongly impairs the conductive capacity of surviving axons, treatments targeting demyelination or promoting remyelination are significant for functional recovery after nerve injury 5. Thus the goal of this protocol is to illustrate an engineering approach that addresses these two issues of nerve regeneration.
Surface anisotropy, which is defined as a difference, when measured along different axes, in a material&#39;s physical or mechanical properties, has been applied to influence cell alignment, growth, and migration 6-7. In addition to topography, there are other methods to induce anisotropy. Previously, we investigated surface anisotropy induced by mechanical static pre-stretch of poly-dimethyl-siloxane (PDMS) membrane. The theory of &#34;small deformation super imposed on large&#34; predicted that the effective stiffness the cells sense in the stretched direction differs from the perpendicular direction, and this difference in effective stiffness is due to surface anisotropy 8. Mesenchymal stem cells (MSCs) cultured on a pre-stretched PDMS membrane are able to sense the anisotropy by actively pulling the surface and as a result, align in the pre-stretched direction 9. Similarly, an anisotropic surface arising from a mechanical pre-stretched surface affects the alignment, as well as growth and myelination of dorsal root ganglion (DRG) axons 10. Here we provide a protocol for inducing surface anisotropy on a static pre-stretched PDMS substrate to enhance axon regeneration 10.
To elicit axon alignment, topological features with desired patterns, reported to provide contact guidance through aligned fibers and channels 6,11-12, were demonstrated to facilitate axon alignment 11,13. However, reported techniques for inducing axon alignment through topological features, such as fibers, channels and patterning, were unable to lengthen and increase the thickness of the axons. In contrast, gradual mechanical stretching led to axon alignment in the stretch direction with longer and thicker axons that increased with the magnitude of the stretch 14. However, incorporating a powered motor device in vivo is not feasible. In contrast, static pre-stretched induced anisotropy is less complicated and can be more readily incorporated into future scaffold designs for in vivo applications.
In this protocol, a static pre-stretched cell culture system is used to induce surface anisotropy without topological features. The pre-stretched culture system is composed of a PDMS membrane, a stretchable frame and a stretching stage, whereupon the membrane is fixed onto the frame and a predetermined stretch magnitude is applied on the stretching stage. Freshly isolated DRG neurons cultured on the pre-stretched surface for up to 21 days are monitored for axon alignment and thickness. Subsequently, Schwann cells (SCs) co-cultured with the aligned axons are monitored for myelination. By incorporating pre-stretch induced surface anisotropy we were able to enhance cell alignment-differentiation and axon alignment-growth of MSCs and DRG neurons 9-10, respectively.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Polydimethylsiloxane &#65288;PDMS)</td>
			<td>Dow Corning</td>
			<td>SYLGARD 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium 1X</td>
			<td>GibcoBRL</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement 50X</td>
			<td>GibcoBRL</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax-I 100X</td>
			<td>GibcoBRL</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumax-I</td>
			<td>GibcoBRL</td>
			<td>11020-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Nerve Growth Factor-7S</td>
			<td>Invitrogen</td>
			<td>13290-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>GibcoBRL</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA/1mM EDTA</td>
			<td>GibcoBRL</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Trevigen</td>
			<td><a href="http://www.trevigen.com/item/2/7/64/220/Cultrex_PolyLLysine/">3438-100-01</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma</td>
			<td>p-6407</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoro-2 deoxy-uridine</td>
			<td>Sigma</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma</td>
			<td>U3003</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td>Invitrogen</td>
			<td>14170-112</td>
			<td>Isolation Buffer</td>
		</tr>
		<tr>
			<td>Type I Collagenase</td>
			<td>Worthington</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11885</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat inactivated Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH30080.03</td>
			<td></td>
		</tr>
		<tr>
			<td>BPE</td>
			<td>Clonetics</td>
			<td>CC-4009</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Calbiochem</td>
			<td>344270</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone chamber</td>
			<td>Greiner bio-one</td>
			<td>FlexiPERM ConA</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaning/etching system</td>
			<td>March Instruments</td>
			<td>PX-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Thy 1.1 antibody</td>
			<td>Sigma- Aldrich</td>
			<td>M7898</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Complement</td>
			<td>Sigma- Aldrich</td>
			<td>S-7764</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard growth medium</td>
			<td></td>
			<td></td>
			<td>For 500 ml Neurobasal Medium 1X, add 10 ml of B-27 50X, 5 ml of Glutamax-I 100X,</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>2.5 ml of Penicillin/Streptomycin (Penn/Strep), 1 ml of Albumax-I, and 1 &#956;l of NGF-- 7S (50 ug/ml).</td>
		</tr>
		<tr>
			<td>FDU and Uridine stock solution</td>
			<td></td>
			<td></td>
			<td>FDU 100mg in 10ml of ddw (10mg/ml), filter in the hood and divided in 500ul aliquots and store at -20 &#186;C.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Uridine 5g in 166.7ml of ddw (33mg/ml), filter in hood, divide in 200ul aliquots and store at -20 &#186;C.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Take 61.5ul of FDU (10mg/ml) and 20.5ul of Uridine(33mg/ml), and add 4918ul of ddw to a final stock concentration,</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>then divide in 1 ml aliquots and store at -20 &#186;C.</td>
		</tr>
	</tbody>
</table>

an approach to enhance alignment and myelination of dorsal root ganglion neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

The pre-stretched cell culture system promoted DRG axon alignment 10. DRG neurons were cultured onto pre-stretched and unstretched surfaces for 12 days. The axons were stained for &#946;-III-tubulin to demonstrate their alignment. Figure 2 compares axon orientation on the pre-stretched and unstretched PDMS substrates after 12 days of culture. The DRG axons aligned parallel to the stretched direction, whereas they showed random alignment and formed an interconne...

Watch this Scientific Journal Video about An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons at JoVE.com

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

The overall goal of this protocol is to enhance axon alignment and myelination of dorsal root ganglion neurons using a static, pre-stretched cell culture system. This method can help answer key question in the nerve tissue engineering field, such as axon alignment and thickness growth. The main advantage of this technique is that this pre-stretched method not only enhances axon alignment but also promotes myelination.To begin this procedure, pour the mixture of base and curing agent into a tissue culture dish. Keep the gel mixture under vacuum for 20 minutes to remove air bubbles. Then place the gel mixture in the oven at 60 degrees Celsius for overnight curing.After the PDMS membrane has cured, treat the surface with oxygen plasma in the plasma cleaning and etching system for three minutes. Subsequently, cut a piece of rectangular membrane from the dish. Fix it onto a stretching frame with the oxygen-treated side facing up.Then place the frame on a stretching stage and evenly stretch it by turning the knob on the stage until it reaches 10%elongation in the longer axis. Next, fix the stretch by tightening the screws on the frame. After that, remove the frame from the stage.Ensure the membrane surface is dry and free of dust before placing a silicone chamber onto the membrane. Allow the sticky side of the chamber to attach tightly to the membrane. Then sterilize the surface with UV for 10 minutes.Afterward, add one milliliter of PLL to the PDMS surface in the chamber within six hours of plasma treatment. Next, incubate it for two hours at 37 degrees Celsius prior to seeding the DRG neurons to enhance cell attachment. In this step, prepare the standard grow medium for the DRG neurons.Prior to isolation, autoclave the isolation equipment and filter reagents. Add five milliliters of isolation buffer into one well of a six-well plate and place the plate on ice. Then prepare 10 milliliters of dissociation medium by adding one milliliter of collagenase A to nine milliliters of 0.05%Trypsin-EDTA.Filter the solution with a 0.22 micrometer filter and place it on ice. Afterward, sacrifice 10 to 12 five to seven days old SD rats and cut away the skin overlying the spinal cord from the back of each one. Remove any excess tissue around the spine.Under a surgical magnifier, make the first incision from the neck then make one cut along the spine on both sides with scissors. Detach the spine from the body of the pup and remove excess muscles. Then cut along the long axis of the spine and use tweezers to open the spine completely to extract the spinal cord.Next, remove the ganglion from the bone pocket. Trim the nerve roots and transfer the ganglia to the ice-cold isolation buffer. Collect approximately 10 to 16 DRGs from both sides.Repeat the dissection procedure for the rest of the pups. Now, transfer the DRGs with isolation buffer into a sterile 15-milliliter tube. Let the tissue settle to the bottom of the tube and gently remove the isolation buffer on top.Then add 10 milliliters of dissociation medium into the tube. Incubate the dissected tissues in the dissociation medium in a 37-degree Celsius water bath for one hour while shaking the tube every five to 10 minutes. After the chemical dissociation, centrifuge the ganglia at four degrees Celsius for five minutes.After five minutes, remove the supernatant, resuspend the pellet in 10 milliliters of standard growth medium and vortex it. Subsequently, centrifuge the dissociated cells once again. Then remove the supernatant, resuspend the pellet in 12 milliliters of standard growth medium and vortex it.Prior to seeding the cells, remove the PLL solution from the chamber. Rinse the PDMS surface with sterile water and air-dry it. After resuspending the cells, let the suspension sit for one to two minutes to allow the debris to settle to the bottom of the tube.Next, add 1.5 milliliters of cell suspension into each stretched chamber and incubate it at 37 degrees Celsius with 5%CO2. To eliminate glial cells at one day in vitro, add 10 microliters or 15 microliters of the FDU-U mixture stock solution to each well. After seven hours, replace this medium with fresh standard growth medium and place the pre-stretched culture device in a 37-degree Celsius incubator in 5%CO2.During the cell culture period, change the medium every two days by replacing half the spent medium with fresh medium. In this procedure, remove the culture medium from the Schwann cells and add five milliliters of 0.05%Trypsin-EDTA into the flask. Incubate the cells at 37 degrees Celsius in 5%CO2 for two to three minutes.Check the cells under the optical microscope using a 10X objective to see if they lift up from the flask. Then add five milliliters of culture medium to the cells in the flask and mix. Subsequently, add the cell suspension to a 15-milliliter centrifuge tube and centrifuge at 20 degrees Celsius for five minutes.Then remove the supernatant and resuspend the pellet in seven milliliters of standard DRG growth medium. Following this, transfer 10 microliters of the cell suspension into a 0.5-milliliter microcentrifuge tube. Mix it with 10 microliters of Trypan blue and then count the cell number with a hemocytometer using a 10X objective.Add the DRG growth medium to dilute the cell suspension to 5, 000 cells per milliliter. Next, remove 0.5 milliliters of the medium from the DRG culture in the stretched chamber and add 0.5 milliliters of Schwann cell suspension to the DRG culture. Culture the cells for one week at 37 degrees Celsius in 5%CO2.Change the medium every two days by replacing half of the spent medium with fresh standard growth medium. After culturing on the stretched and unstretched surfaces for two weeks, purified Schwann cells were added to the chamber and cultured for another week. Shown here is the beta-III tubulin staining for axons on the stretched surface.And this is the overlay of beta-III tubulin and P0 staining on the stretched surface, suggesting the Schwann cells are attached to and likely myelinating the axons. This image shows the beta-III tubulin staining for axons on the unstretched surface. And the overlay of beta-III tubulin and P0 staining on the unstretched surface suggests that the Schwann cells are not attached to the axons.Once mastered, this technique can be done in three to four hours if it is performed properly. While attempting this procedure, it is important to remember to keep the PDMS membrane flat and of homogeneous thickness. Following this procedure, atomizers like pre-stretched microchannels can be performed in order to answer additional questions like axon lens and Schwann cells migration.After its development, this technique will pave the way for researchers in the field of tissue engineering to explore the role of surface anisotropy on nerve regeneration in spinal cord and sciatic nerve. After watching this video, you should have a good understanding of how to induce axon alignment and myelination using pre-stretched device.

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JoVE Journal

Neuroscience

8.4K Views.  Michigan State University. The overall goal of this protocol is to enhance axon alignment and myelination of dorsal root ganglion neurons using a static, pre-stretched cell culture system. This method can help answer key question in the nerve tissue engineering field, such as axon alignment and thickness growth. The main advantage of this technique is that this pre-stretched method not only enhances axon alignment but also promotes myelination.To begin this procedure, pour the mixture of base and curing agent in...