A Technique for Harvesting and Dissociating Dorsal Root Ganglia for Neuronal Cultures

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Experimental Set-up

1. Prior to starting tissue and cell harvest, check that all the tools are sterile. If necessary, autoclave a pair of sharp surgical scissors, a pair of very fine forceps, and a pair of fine standard forceps. Also, as appropriate, sterilize each substrate before cell seeding using UV sterilization, ethanol exposure, or steam sterilization.
2. Preparation of media and stock solutions for neuron harvest and dissociation
   1. Prepare Bottenstein and Sato's (BS) medium, by adding 1% penicillin-streptomycin (PS) and 1% N2 supplement to Ham's F12 medium. Calculate the final volume needed as 500 µl per well (if using a 24-well plate).
   2. Prepare collagenase IV stocks in Ham's F12 medium at the concentration of 1.25% wt/v. Filter-sterilize the solution and store at -20 °C as 200 µl aliquots.
   3. Prepare bovine pancreatic trypsin stocks in Ham's F12 medium at the concentration of 2.5% wt/v, filter-sterilize and store at -20 °C as 200 µl aliquots.
   4. Prepare the nerve growth factor (NGF) stock solution at the concentration of 5 µg/mL in a filter-sterilized 1 mg/ml fatty acid-free bovine serum albumin (BSA) solution in F12 medium and store at -20 °C as 200 µl aliquots. Do not filter the NGF after reconstitution.
3. In case no surface modification has been performed, coat coverslips/plates with poly-D-lysine (0.1 mg/ml for 15 min at room temperatre [RT]) and/or laminin (2-10 µg/cm² for 2 hr at 37 °C) to support neuron attachment and neurite outgrowth as appropriate.
4. Always warm up media in a water bath at 37 °C before using.

2. Harvest and Dissociation of Dorsal Root Ganglia (DRG) Neurons

1. Terminate the rat by cervical dislocation and decapitation. Shave the rat and lift the skin to expose the spinal column. Using sharp scissors, excise the spinal column taking extra care of the confined organs and blood vessels. Transfer the spinal column in a biological safety cabinet (class II) using a Petri dish and remove any dorsal part.
2. Divide the spinal column in half along the longitudinal axis using sterile and sharp surgical scissors to expose the cord tissue. At this point, it is helpful to cut the spinal column in two smaller segments below the level of the rib cage, to make it easier to handle during the DRG neuron harvest. Using fine forceps, gently remove all the cord tissue, paying attention to not pull and remove the DRG roots. This way the DRG and roots will be exposed within the vertebral canals, still encased in the column. Observe DRG as white filaments coming out directly from the canals.
3. Pull out the entire DRG root (not just the DRG) from the vertebral canals by using very fine forceps, going deep into the vertebral canals and paying attention not to damage the roots of the ganglia. Transfer the DRG into a small petri dish (60 mm²) containing 3-4 ml of Ham's F12 medium supplemented with 1% PS. If using different animals, use separate dishes.
4. Under a dissecting microscope, clean the DRG of any excess of nerve roots surrounding the ganglia using sterile forceps and a scalpel to reduce glial cell contamination. Transfer the DRG into a small petri dish (35 mm²) with 1.8 ml of fresh F12 medium.
5. Add 200 µl of 1.25% wt/v collagenase type IV stock solution (final concentration of 0.125%) and incubate the DRG at 37 °C, 5% CO₂ for 1 hr. Carefully aspirate the medium with a glass pipette, paying attention not to aspirate or damage the DRG. Add fresh F12 medium supplemented with 0.125% wt/v collagenase type IV enzyme and incubate for 1 hr as previously described.
6. Aspirate the medium and gently wash the DRG with F12 medium. Add then 1.8 ml of F12 medium and 200 µl of trypsin (final concentration of 0.25% wt/v) and incubate at 37 °C, 5% CO₂ for 30 min.
7. Remove trypsin and add 1 ml of F12 medium supplemented with 500 µl of fetal bovine serum (FBS) to arrest the enzymatic reaction. Aspirate the medium and gently wash the DRG with F12 medium for three times to remove traces of serum.
8. Add 2 ml of fresh F12 medium and carefully transfer the DRG with the medium into a 15 ml tube using a glass pipette. Gently dissociate the DRG neurons by pipetting up and down (about 8-10 times) with the glass pipette (plastic tips can be used as alternative).
9. Allow the pellet to settle at the bottom of the tube and collect the medium in a new tube. Add 2 ml of fresh F12 medium to the tube containing the pellet and repeat the mechanical dissociation with the glass pipette. Repeat this step until the suspension becomes homogeneous (about 3-4 times) and collect all the dissociated DRG in the new tube. This method reduces the stress deriving from the mechanical dissociation and improves the viability of the neurons.
10. Filter the resulting homogenized suspension into a new 15 ml tube using a 100 µm cell strainer to remove un-dissociated neurons and other debris. At this stage, it might be convenient to first filter the cell suspension into a 50 ml tube and then transfer the solution into the smaller tube using a glass pipette. Centrifuge the suspension at 110 g for 5 min.
11. Prepare a 15% BSA by adding 500 µl of a 30% BSA solution to 500 µl of F12 medium. Slowly pipette the solution down the wall of a 15 ml tube to create a gradual protein trail. At this stage, it is helpful to hold the tube at a 45° angle and slowly release the BSA using the numbers on the falcon tube as a reference for the forming "track".
12. Aspirate the supernatant from step 2.10 leaving 500 µl at the bottom of the tube and resuspend the cell pellet in the same medium. Slowly pipette the suspension along the protein trail previously prepared in step 2.11 (use the numbers on the tube as a reference) and centrifuge at 500 g for 5 min.
13. Aspirate the supernatant and resuspend the pellet in 1 ml of modified BS medium.
    NOTE: The volume to re-suspend the DRG neurons can be made up to the actual volume required, depending on the final cell concentration desired and the number of samples to be seeded. One animal will provide enough cells for a 24-well plate experiment.
14. Incubate the seeded samples at 37 °C, 5% CO₂ for 2 hr to allow cell attachment and finally add fresh BS medium supplemented with 50 ng/ml of NGF.