Cryopreserved rat primary dorsal root ganglion cells: simply thaw and culture

Dissociation and production of rat primary dorsal root ganglion (DRG) cells for culture is labour intensive and offers low yield.

In the past, DRG cells have been considered unsuitable for use in 96-well format assays. Now commercial availability of rat DRG cells provides a solution.

We now offer DRG cells (non-purified), isolated and dissociated at P1, then cryopreserved. The frozen, cryopreserved DRG cells can be thawed and plated at 5000 cells per well on poly-D-lysine coated 96-well plates and grown in neurobasal medium with B27. No further supplementation is required for survival to 28+ days in vitro. This represents a significant step for multi-well DRG cell testing and an opportunity for automated screening³. Moreover, the extended survival time in culture shows that this is an ideal in vitro system for long term testing and toxicity studies^{6, 7}.

The DRG neurons were examined using immunohistochemistry for the growth associated protein GAP43. This protein has a role in signal transduction related to growth cone guidance, calcitonin gene-related peptide (CGRP) (a peptide which is abundantly expressed by nociceptive DRG neurons), and neuron-specific β-tubulin (Figures 1-3). There was an abundance of long, loosely bundled axons within the cultures that followed the pattern of the underlying Schwann cells. Over the first few days, neuronal cell bodies formed ganglionated clusters and fasiculated axon bundles, but with the addition of mitotic inhibitors, DRG neurons and their axons remained dispersed. These cultures were superior for most types of studies. Conversely, cultures not treated with mitotic inhibitors became useless at around day 12 due to detachment of ganglionated clusters from the substrate.

Within 2-3 days in culture, the cryopreserved DRG neurons display extensive neurite outgrowth. On day 10 they show the same size and neurochemical type distribution as freshly dissociated rat DRG neurons. This is consistent with the generally accepted view that after 9 days in culture, DRG neurons are physiologically mature.

DRG neurons are generally classified into two populations on the basis of cell size, morphology, and electro-physiology. However, there is overlap in the size distribution for these populations^{4, 5}. The large neurons represent the mechanoreceptive and thermoreceptive A-type sensory cells. The small neurons are the polymodal nociceptors. This distribution of neurons was evident in the cultured, cryopreserved DRG neurons (Figures 1, 4).

The quality of DRG cell axon outgrowth and development was followed with simple plasmid transfections of eGFP where the outgrowth of fluorescent processes was readily imaged (Figure 4). Using recurrent digital imaging, the cell soma, proximal and distal axon, growth cone, and fine filopodia of transfected neurons were monitored over time (Figure 5). These DRG cells were pseudounipolar neurons with branched neurites (Figures 4, 5).

Growth cone dynamics can be studied easily in vitro using cryopreserved rat DRG cell cultures. Researchers can use these cells in culture to assess environmental cues important in controlling growth cone guidance^{1, 2}. Using TIRF microscopy, the best optical technology for examining adherent processes like growth cones, we examined the growth cones of cryopreserved neurons cultured in 96-well plates for 3 days. Growth cones with typical lamellipodia, flattened at the end where there is adhesive contact with the plate surface, are clearly seen (Figure 6). Each growth cone displayed multiple lamellipodia, all with normal length and morphology.

Taken together, these data show the applicability of cryopreserved DRG cells to in vitro study of axonal outgrowth, path finding, neuropathy, nerve regrowth, sensory receptor physiology, and drug testing.

References

1. Adams, D N, Kao, E Y, Hypolite, C L, Distefano, M D, Hu, W S, and Letourneau, P C (2005) Growth cones turn and migrate up an immobilised gradient of the laminin IKVAV peptide. J Neurobiol. 62:134-47.

2. Hari, A, Djohar, B, Skutella, T, and Montazeri, S (2004) Neurotrophins and extracellular matrix molecules modulate sensory axon outgrowth. Int J Dev Neurosci 22:113-7.

3. Keswani, S C, Rosenberg, B, and Hoke, A (2004) The use of GAP-43 mRNA quantification in high throughput screening of putative neuroprotective agents in dorsal root ganglion cultures. J Neurosci Methods 136:193-5.

4. Banker, G, and Goslin, K (1991) Culturing Nerve Cells, p337-377, MIT Press.

5. Landon, D N (1976) The Peripheral Nerve, p.178-188, Chapman and Hall, London.

6. Price, S A, Hounsom, L, Purves-Tyson, T D, Fernyhough, P, and Tomlinson, D R (2003) Activation of JNK in sensory neurons protects against sensory neuron cell death in diabetes and on exposure to glucose/oxidative stress in vitro. N Y Acad Sci 1010:95-9.

7. Tjiattas, L, Ortiz, D O, Dhivant, S, Mitton, K, Rogers, E, and Shea, T B (2004). Folate deficiency and homocysteine induce toxicity in cultured dorsal root ganglion neurons via cytosolic calcium accumulation. Aging Cell 3:71-6.

By Anthony Krantis, PhD, QBM Cell Science