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

Take a section of a rat's spinal column and divide it to remove the spinal cord.

Detach the dorsal root ganglia, or DRGs, a cluster of neurons surrounded by satellite glial cells, or SGCs.

Place the DRGs in a growth medium. Remove the nerve roots to reduce SGC contamination.

Treat with collagenase to degrade the tissue matrix and wash to remove the enzymes.

Introduce trypsin to disrupt intercellular connections. Add a serum containing proteins that deactivate trypsin.

Add the growth medium and mechanically dissociate the tissue. Filter the suspension to isolate single cells and centrifuge to remove tissue debris.

Resuspend the cells to layer them on a density gradient medium, and centrifuge.

Neurons with a higher density settle at the bottom, facilitating the separation of SGCs.

Resuspend the neurons in a culture medium. Transfer them onto a culture substrate-coated well, allowing cell attachment.

Supplement with nerve growth factors for neuronal survival.

To obtain the DRG neurons after harvesting the spinal cord, begin by removing any dorsal parts and then, use sterile and sharp surgical scissors to divide the spinal column in half along the longitudinal axis, exposing the cord tissue. Cut the spinal column into two smaller segments below the level of the rib cage, and then use fine forceps to gently remove all the cord tissue, taking care not to pull and remove the DRG roots, which appear as white filaments coming directly out of the canals.

Once all of the core tissue has been removed, use very fine forceps to pull the entire DRG root, reaching deep into the vertebral canals and taking care not to damage the roots of the ganglia. Place the DRG into a 60-square-millimeter Petri dish containing 3 to 4 milliliters of Ham's F-12 medium, supplemented with antibiotics. Then, under a dissecting microscope, use sterile forceps and a scalpel to clear away any excess nerve roots surrounding the ganglia to reduce satellite cells' contamination.

Next, transfer the DRG into a 35-square-millimeter Petri dish containing 1.8 milliliters of fresh F-12 medium, and add 200 microliters of collagenase type-4 stock solution. Incubate the DRG for one hour, and then carefully aspirate the medium with a glass pipette, taking care not to aspirate or damage the DRG.

Repeat the collagenase step and wash the DRG with F-12 medium. After the second wash, add 1.8 milliliters of F12 medium and 200 microliters of trypsin to the cells, removing the trypsin and adding 1 milliliter of medium supplemented with 500 microliters of FBS to arrest the enzymatic reaction. Then, aspirate the medium and gently wash the DRG with F-12 medium three times to remove all traces of the serum.

Now, feed the cells with 2 milliliters of fresh F-12 medium and use a glass pipette to carefully transfer the cell suspension into a 15-milliliter tube. Pipette up and down 8 to 10 times to gently dissociate the neurons, and then allow the pellet to accumulate at the bottom of the tube. When the pellet is settled, transfer the supernatant into a new tube, and add 2 milliliters of fresh F12 medium to the pellet, repeating the mechanical dissociation and medium transfer until the suspension becomes homogeneous.

Then, triturate the cell suspension three to four times, followed with filtration of the resulting homogenized suspension through a 100-micron cell strainer into a new 50-milliliter tube. Centrifuge the cells in a 15-milliliter tube. While they are spinning, slowly pipette freshly prepared 15% bovine serum albumin down the inside of a 15-milliliter tube held at a 45-degree angle to create a gradual protein trail using the numbers on the tube as a reference for the forming track.

Then, aspirate the supernatant from the cells, saving the last 500 microliters for resuspending the pellet, and slowly dispense the cells along the protein trail into the new tube. After spinning down the cells again, resuspend the pellet in 1 milliliter of modified BS medium and seed the cells in a small volume of medium in a 24-well plate for two hours. When the cells have attached, add fresh BS medium supplemented with 50 nanograms per milliliter of nerve growth factor.