A Technique for Harvesting and Dissociating Dorsal Root Ganglia for Neuronal Cultures

Abstract

Source: de Luca, A. C., et al., Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration. J. Vis. Exp. (2015).

This video demonstrates a method for harvesting neurons from the dorsal root ganglia (DRG). DRG from a rat's spinal column is isolated, treated with enzymes, and then mechanically dissociated to obtain a single-cell suspension. The neurons are separated from glial cells using density gradient centrifugation and cultured in a growth medium.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Experimental Set-up

1. Prior to starting tissue and cell harvest, check that all the tools are sterile. If necessary, autoclave a pair of sharp surgical scissors, a pair of very fine forceps, and a pair of fine standard forceps. Also, as appropriate, sterilize each substrate before cell seeding using UV sterilization, ethanol exposure, or steam sterilization.
2. Preparation of media and stock solutions for neuron harvest and dissociation
   1. Prepare Bottenstein and Sato's (BS) medi....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100 µm cell strainer	BD Biosciences	352360	70 μm strainers (ref. 352350) can be used as alternative
15 mL plastic tubes	Sarstedt	62.554.002
50 mL plastic tubes	Sarstedt	62.547.004
75 cm2 flasks	Corning	BC301
Bovine Serum Albumin (BSA)	Sigma	A9205
Collagenase type IV	Worthington Biochemical	LS004188	Note: this collagenase is only used to dissociate DRG explants
Foetal Bovine Serum (FBS)	Biosera	FB-1001
Glass pipettes	Fisher Scientific	FB50253	Sharp material to be disposed accordingly
Nutrient Mix F12 HAM	Sigma	N6658	Warm at 37 °C in a water bath unless specified
Hank's Balanced Salt Solution (HBSS)	Sigma	H9394	Warm at 37 °C in a water bath unless specified
N-2 supplement (100x)	Invitrogen	17502
Nerve Growth Factor 2.5s Protein, Mouse Submaxillary Glands (NGF)	Millipore	NC011
Penicillin-Streptomycin (PS)	Sigma	P0781
Petri dishes	Corning	430165
Trypsin (2x bovine pancreatic)	Worthington Biochemical	LS003703	This is used for DRG dissociation