Purification and culture of adult rat dorsal root ganglia neurons

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Abstract
To study the trophic requirements of adult rat dorsal root ganglia neurons (DRG) in vitro, we developed a purification procedure that yields highly enriched neuronal cultures. Forty to fifty ganglia are dissected from the spinal column of an adult rat. After enzymatic and mechanical dissociation of the ganglia, myelin debris are eliminated by centrifugation on a Percoll gradient. The resulting cell suspension is layered onto a nylon mesh with a pore size of 10 μm. Most of the neurons, the diameter of which ranged from 17 μm to > 100 μm, are retained on the upper surface of the sieve; most of the non‐neuronal cells with a caliber of < 10 μm after trypsinization go through it. Recovery of neurons is achieved by reversing the mesh onto a Petri dish containing culture medium. Neurons to non‐neurons ratio is 1 to 10 in the initial cell suspension and 1 to 1 after separation. When these purified neurons are seeded at a density of 3, 000 neurons/cm2 in 6 mm polyornithine–‐laminin (PORN‐LAM) coated wells, neuronal survival (assessed by the ability to extend neurites), measured after 48 hr of culture, is very low (from 0 to 16%). Addition of nerve growth factor (NGF) does not improve neuronal survival. However, when neurons are cultured in the presence of medium conditioned (CM) by astrocytes or Schwann cells, 60–80% of the seeded, dye‐excluding neurons survive. So, purified adult DRG neurons require for their short‐term survival and regeneration in culture, a trophic support that is present in conditioned medium from PNS or CNS glia. As NGF has no survival‐promoting effect on these purified adult neurons, the trophic effect of CMs is likely to be mediated by non‐NGF molecule(s).

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