In vitro culture of adult mouse DRG neurons

This protocol is a detailed description of the method used in the research article Multimodal control of dendritic cell functions by nociceptors, published in Science, 2023. 379, eabm5658

Materials and reagents

Poly-D-Lysine

Laminin

PBS

Leibowitz 15 medium

Percoll

Digestion solution 1 (filter-sterilized):

            60U papain

            0.5 mM EDTA

            1.5 mM CaCl₂

            in HEPES-buffered HBSS

Digestion solution 2 (filter-sterilized):

            4mg/ml Collagenase type-2

            5mg/ml Dispase 

            in HEPES-buffered HBSS

Complete Neurobasal Medium

            B27 supplement

            L-Glutamine

            Penicillin/Streptomycin

            5μM Cytosine β-D-arabinofuranoside 

            25 ng/ml recombinant mouse NGF

Equipment

Surgical tools (fine tweezers, angled microscissors, surgical scissors)

Stereotactic microscope

Water bath

Refrigerated centrifuge

Tissue culture hood

Tissue culture incubator

Preparing the plate:

• Coat wells of a flat-bottom 96-well plate with 50 μl of 100 μg/ml Poly-D-Lysine overnight at 37°C. Coat 8 wells per mouse if using multiple animals, 6 wells if using a single mouse.
• Aspirate Poly-D-Lysine, wash twice with 200 μl of sterile dH₂O, and let air-dry in a tissue culture hood
• Coat with 50 μl of 10 μg/ml Laminin at RT for 1 hour
• Aspirate Laminin and let air-dry in a tissue culture hood 

Harvesting dorsal root ganglia:

• Euthanize the mouse in accordance with institutional guidelines
• Place the animal in a prone position and spray with 70% ethanol
• Remove the skin on the back to expose the spine
• Use surgical scissors to dissect the spine. Cut behind the skull and at the base of the tail and through the connective tissues, ribs, and pelvis.
• Place the dissected spine in ice-cold PBS in a 10 cm dish and use scissors to remove as much connective tissue as possible.
• Cut the spine into 3 pieces roughly corresponding to the cervical, thoracic, and lumbar/sacral segments. 
• Insert one blade of angled microscissors into the spinal canal and cut each segment longitudinally. 
• Remove the spinal cord and dura mater to reveal the dorsal root ganglia. Note that some ganglia may remain attached to the dura when it is peeled off.
• Use fine tweezers to pull out individual ganglia.
• Critical: Make sure to completely remove the axons from each ganglion and all remnants of the dura
• Keep the harvested ganglia in 1ml PBS in a 1.5 ml tube on ice until all ganglia have been harvested.
• Proceed directly to digestion.

Digestion & plating the cells:

• Transfer the harvested ganglia into a 15 ml conical tube with 3 ml (per mouse) of pre-warmed digestion solution 1 and incubate for 10 minutes at 37°C, mixing every 3 minutes.
• Spin down (400 x g, 4 min, 4°C), and carefully remove the supernatant. The pellet is very loose and viscous. If using an aspirator, tilt the tube, and do not try to aspirate liquid all the way to the pellet.
• Resuspend and incubate for 30 minutes at 37°C in pre-warmed digestion solution 2, 3ml per mouse, mixing every 3-5 minutes.
• Spin down (400 x g, 4 min, 4°C) and very carefully remove the supernatant. If using an aspirator, be extremely careful that it doesn’t get close to the pellet. Tilt the tube, and use a pipette to remove leftover liquid if necessary. 
• Resuspend the pellet in 200 μl of L15 medium supplemented with 10% FBS and triturate with a 200 μl pipette tip to ensure complete dissociation. Add 800 μl of L15 + 10% FBS.
• Carefully overlay cell suspension onto 5 ml of well-mixed 20% Percoll in L15 medium (4 ml L15 + 1 ml Percoll)
• Spin at 400 x g, 9 min, 4°C with no break
• Remove both phases of the supernatant as well as the interface by aspirating liquid from the top. Make sure that the material present in the interface is removed completely. Resuspend the pellet in 4 ml B27-supplemented Neurobasal medium.
• Spin down (400 x g, 4 min, 4°C), remove supernatant, resuspend pellet in 1 ml B27-supplemented Neurobasal medium
• Spin down (400 x g, 4 min, 4°C) and remove supernatant.
• Take up the pellet in Complete Neurobasal Medium (+ B27, 5 μM Cytosine β-D-arabinofuranoside, 25 ng/ml NGF), 1 ml per mouse. 
• Plate in the previously-coated 96-well plate, 6 wells if using a single mouse, 8 wells per mouse if using 2 or more mice.

Culturing the neurons 

• Carefully add 75 μl of fresh complete Neurobasal medium into all wells on day 4 after plating.
• If keeping the culture for an extended period of time, very carefully replace half of the culture medium every 5 – 7 days.
• Visually inspect the culture under a microscope before starting any experiments - do not use the culture if cells appear to be dead/dying or clumped, axons are straight and no longer adherent, or if the culture is contaminated with an excessive amount of dead glia/Schwann cells.