Cryopreservation of transfected primary dorsal root ganglia neurons

Authors:
Seggio AM, Ellison KS, Hynd MR, Shain W, Thompson DM
In:
Source: J Neurosci Methods
Publication Date: (2008)
Issue: 173(1): 67-73
Research Area:
Neurobiology
Cells used in publication:
Dorsal root gang. (DRG), rat
Species: rat
Tissue Origin: brain
Platform:
Nucleofector® I/II/2b
Experiment
Two million rat DRGs were nucleofected with 3 µg eGFP plasmid (Rat Neuron Kit, program G-013). Cells were then either immediately plated or cryopreserved. Transfection rates raged from 46-56 % for fresh and cryopreserved neurons; approximately 21 % of the non-transfected and 32 % of the transfected neurons survived cryopreservation, as determined. There was no significant difference in neurite outgrowth between fresh and cryopreserved neurons.
Abstract
Primary dorsal root ganglia (DRG) neurons are often used to investigate the relative strength of various guidance cues to promote re-growth in vitro. Current methods of neuron isolation are laborious and disposal of excess dissected cells is inefficient. Traditional immunostaining techniques are inadequate to visualize real-time neurite outgrowth in co-culture. Cryopreservation, in combination with transfection techniques, may provide a viable solution to both under-utilized tissue and insufficient methods of visualization. This study aims to qualitatively and quantitatively demonstrate successful cryopreservation of primary transfected and non-transfected DRG neurons. Fluorescent micrographs were used to assess morphology after 24h in culture and suggest similarities between freshly isolated neurons and neurons which have been transfected and/or cryopreserved. Quantitative measurements of neuron outgrowth (specifically, primary neurites, branch points and total neurite length) indicate that neuron outgrowth is not altered by cryopreservation. Transfected neurons have stunted outgrowth at 24h.