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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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'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 "small deformation super imposed on large" 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.

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Protokół

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 

1. 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.
2. Keep the gel mixture under vacuum for 20 min to remove air bubbles.
3. Place the gel mixture in the oven for overnight curing at 60 °C.
4. After the PDMS membrane cures, treat the surface with oxygen plasma using a plasma cleaning/etching system for 3 min at 165 mTorr and 65 sccm flow of O₂.
5. Cut a piece of rectangular membrane (5 × 3.5 cm) from the dish, while being cognizant which side is oxygen-treated, make sure the oxygen-treated side is facing up and fix onto a stretching frame. Place the frame on a stretching stage and evenly stretch by turning the knob on the stage until it reaches 10% elongation (or some other pre-determined stretch; the elongation can be directly read from the stretching stage) in the longer axis ⁹. Fix the stretch by tightening the screws on the frame.
6. Remove the frame from the stage. Ensure the membrane surface is dry and free of dust, then place a silicone chamber onto the membrane allowing the sticky side of the chamber to attach tightly to the membrane.
   Note: The silicone chamber provides a well for retaining the cell medium on the PDMS membrane. The device design is shown in Figure 1.
7. Sterilize the surface with UV for 10 min.
8. Add 1 ml Poly-L-Lysine (PLL) (0.01% in Phosphate Buffered Saline) to the PDMS surface within the chamber, within 6 hr of plasma treatment and incubate for 2 hr at 37 °C prior to seeding the DRG neurons. This enhances cell attachment.

2. Isolation of DRG

1. Prepare standard growth medium for the DRG neurons.
2. Prior to the isolation, autoclave the isolation equipment and filter reagents. Add 5 ml of isolation buffer into one well of a 6-well plate, and place the plate on ice. Prepare 10 ml of dissociation medium by adding 1 ml of collagenase A (500 U/ml) to 9 ml of 0.05% Trypsin-EDTA (1 mg/ml), filter the solutions with a 0.22 µm filter and place on ice.
3. Use ten to twelve 5 - 7 days' old Sprague-Dawley rats for the isolation. Spray the pups with 75% isopropanol, and sacrifice by decapitation.
4. Cut away the skin overlying the spinal cord from the back and remove any excess tissue around the spine. Under a surgical magnifier with the surgical spot lights on, make the first incision from the neck and then one cut along the spine on both sides with scissors. Detach the spine from the body of the pups and remove the excess muscles. Then, use scissors to cut along the long axis of the spine and use tweezers to open the spine completely and extract the spinal cord.Remove the tip from a pipette to create a larger opening for transferring the DRGs with isolation buffer into a sterile 15 ml tube.
5. Using a fine pair of tweezers, remove the ganglion from the bone pocket and collect approximately 10 – 16 DRG from both sides. Trim the nerve roots and transfer the ganglia to the ice-cold isolation buffer.
6. Remove the tip from a pipette to create a larger opening for transferring the DRGs with isolation buffer into a sterile 15 ml tube. After the chemical dissociation, centrifuge the ganglia at 900 x g and 4 °C for 5 min.
7. Let the tissues settle to the bottom of the tube and gently remove the isolation buffer on top and add 10 ml of dissociation medium into the tube.
8. Incubate the dissected tissues in the dissociation medium in a 37 °C water bath for 1 hr while shaking the tube every 5 - 10 min.
9. After the chemical dissociation, centrifuge the ganglia at 900 x g and 4 °C for 5 min.
10. Remove the supernatant and re-suspend the pellet in 10 ml standard growth media and vortex.
11. Centrifuge the dissociated cells as indicated in section 2.9 once again.
12. Remove the supernatant, re-suspend the pellet in 12 ml of standard growth media and vortex.

3. Culture of DRG on Pre-stretched Surface

1. Prior to seeding the cells, remove the PLL solutions from the chamber and rinse the PDMS surface with sterile water and air dry.
2. After re-suspending the cells, let the suspension set for 1 - 2 min to allow the debris to settle to the bottom of the tube. Add 1.5 ml of cell suspension into each stretch chamber and incubate at 37 °C at 5% CO₂.
3. To eliminate glial cells, at 1 day in vitro (DIV), add 10 µl (or 15 µl) mixture of fluoro-2 deoxy-uridine and uridine (FDU-U) stock solution (See Table of Materials) to each well. After 7 hr, replace this medium with fresh standard growth medium and place the pre-stretched culture device in a 37 °C incubator with 5% CO₂.
4. During the cell culture period (2 - 3 weeks), change the media every two days by replacing half the spent medium with fresh medium. Note: After culturing on the stretched and unstretched surfaces for 2 weeks, purified SCs are added to the chamber and cultured for another week (step 4.7).

4. Co-culture of Schwann Cells (SCs) with DRG Neurons on Pre-stretched Surface

1. Isolate SCs as described by Dr. Campana's group previously ¹⁵.
   1. Briefly, isolate the SCs from a 1 day old pup. Collect and dissociate the sciatic nerves both chemically (Trypsin-EDTA and Collagenase A) and mechanically (18 gauge needle/10 ml syringe) ¹⁵.
   2. Seed the dissociated cells into poly-D-lysine (PDL) coated T25 flask and culture at 37 °C at 5% CO₂. On day 5, purify the cells through antibody selection using Anti-thy 1.1 antibody and Rabbit Complement and subculture the purified cells to passage 2 - 4. Use culture medium containing Dulbecco's Modified Eagle Medium (DMEM), 10% Fetal bovine serum (FBS), 1% Penicillin/Streptomycin (Penn/Strep), 21 µg/ml Bovine Pituitary Extract (BPE), and 4 µM Forskolin.
2. Remove the culture medium from the SCs, and add 5 ml 0.05% Trypsin-EDTA into the flask. Incubate the cells at 37 °C at 5% CO₂ for 2 - 3 min.
3. Check cells under the optical microscope using a 10X objective to see if they lift up from the flask, then add 5 ml of culture medium and mix with the cells in the flask.
4. Add the cell suspension to a 15 ml centrifuge tube and centrifuge at 200 x g and 20 °C for 5 min.
5. Remove the supernatant and resuspend the pellet in 7 ml standard DRG growth media.
6. Take 10 µl of cell suspension into a 0.5 ml microcentrifuge tube, mix with 10 µl of trypan blue, and then count the cell number using a hemocytometer under optical microscopy using a 10X objective. Dilute the cell suspension to 5,000 cells per ml by adding DRG growth media.
7. Remove 0.5 ml of medium from the DRG culture in the stretched chamber and add 0.5 ml of SC suspension to the DRG culture.
8. Culture the cells for 1 week at 37 °C and 5% CO_2. Change the media every two days by replacing half the spent medium with fresh standard growth medium for DRG. After 1 week co-culture, process the cells by immunohistochemistry ¹⁰.

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Wyniki

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 β-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...

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Dyskusje

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...

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Materiały

Name	Company	Catalog Number	Comments
Neurobasal Medium 1x	GibcoBRL	21103-049
B27 Supplement 50x	GibcoBRL	17504-044
Glutamax-I 100x	GibcoBRL	35050-061
Albumax-I	GibcoBRL	11020-021
Nerve Growth Factor-7S	Invitrogen	13290-010
Penicillin-streptomycin	GibcoBRL	15140-122
0.05% Trypsin-EDTA/1 mM EDTA	GibcoBRL	25300-054
Poly-L-Lysine	Trevigen	3438-100-01
Poly-D-Lysine	Sigma	p-6407
Fluoro-2 deoxy-uridine	Sigma	F0503
Uridine	Sigma	U3003
Hank’s Balanced Salt Solution (HBSS)	Invitrogen	14170-112	Isolation Buffer
Type I Collagenase	Worthington	LS004196
DMEM	Gibco	11885
Heat inactivated Fetal Bovine Serum	Hyclone	SH30080.03
BPE	Clonetics	CC-4009
Forskolin	Calbiochem	344270
Silicone chamber	Greiner bio-one	FlexiPERM ConA
Plasma cleaning/etching system	March Instruments	PX-250
Anti-Thy 1.1 antibody	Sigma- Aldrich	M7898
Rabbit Complement	Sigma- Aldrich	S-7764
Standard growth medium			For 500 ml Neurobasal Medium 1x, add 10 ml of B-27 50x, 5 ml of Glutamax-I 100x, 2.5 ml of Penicillin/Streptomycin (Penn/Strep), 1 ml of Albumax-I, and 1 μl of NGF-- 7S (50 μg/ml).
FDU Uridine stock solution			FDU 100 mg in 10 ml of ddH2O (10 mg/ml), filter in the hood and divided in 500 μl aliquots and store at -20 ºC. Uridine 5 g in 166.7 ml of ddH2O (33 mg/ml), filter in hood, divide in 200 μl aliquots and store at -20 ºC. Take 61.5 μl of FDU (10 mg/ml) and 20.5 μl of Uridine(33 mg/ml), and add 4,918 μl of ddH2O to a final stock concentration, then divide in 1 ml aliquots and store at -20 ºC.