Cryopreservation of transfected primary dorsal root ganglia neurons.
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.
Seggio
Angela M
AM
Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, United States. seggia@rpi.edu
Ellison
Karen S
KS
Hynd
Matthew R
MR
Shain
William
W
Thompson
Deanna M
DM
D000818:Animals; D000831:Animals, Newborn; D048429:Cell Size; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D003952:Diagnostic Imaging; D005727:Ganglia, Spinal / Q000166:cytology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D016501:Neurites / Q000502:physiology; D009474:Neurons / Q000145:classification / Q000166:cytology / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection / Q000379:methods; D014404:Tubulin / Q000378:metabolism
C488416:Tubb3 protein, rat; D014404:Tubulin; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins