title: Methods for isolating highly-enriched embryonic spinal cord neurons: a comparison between enzymatic and mechanical dissociations.
abstract: Spinal cord neuronal culture is a useful system to study normal and abnormal functions of the spinal cord. For many bioassays, obtaining large quantities of highly purified spinal cord neurons is required. However, technical difficulties exist in obtaining these cells reliably and consistently. By comparing two dissociation methods, mechanical and enzymatic dissociations, we found that the enzymatic dissociation of embryonic day 14-15 spinal cords resulted in significantly higher cell yield than the mechanical dissociation (25.40 +/- 5.41 x 10(6) versus 3.43 +/- 0.52 x 10(6) cells per 12 embryos; n = 6/group; p < 0.01). Furthermore, cell viability was significantly higher after the enzymatic than the mechanical dissociation (83.40 +/- 3.08% versus 32.81 +/- 3.49%, n = 4/group; p < 0.01). In both methods, highly purified populations of primary neurons were obtained (mechanical: 85.17 +/- 2.84%; enzymatic: 87.67 +/- 2.52%; n = 3/group). Critical measures that affect culture outcomes include, but not limited to, the age of embryo, cell seeding density, dissociation time, and elimination of non-neuronal cells. Thus, the present study has identified the enzymatic dissociation method to be a preferred method for obtaining large quantity of highly-enriched embryonic spinal cord neurons.
authors.affiliation: Department of Neurobiology, Shanghai Jiaotong University School of Medicine, 280 South Chong-Qing Road, Shanghai 200025, PR China.
authors.forename: Xiao-Yan
authors.initials: XY
authors.lastname: Jiang
authors.forename: Sai-Li
authors.initials: SL
authors.lastname: Fu
authors.forename: Bao-Ming
authors.initials: BM
authors.lastname: Nie
authors.forename: Ying
authors.initials: Y
authors.lastname: Li
authors.forename: Lin
authors.initials: L
authors.lastname: Lin
authors.forename: Lan
authors.initials: L
authors.lastname: Yin
authors.forename: Yan-Xia
authors.initials: YX
authors.lastname: Wang
authors.forename: Pei-Hua
authors.initials: PH
authors.lastname: Lu
authors.forename: Xiao-Ming
authors.initials: XM
authors.lastname: Xu
mesh_terms: D000367:Age Factors; D000818:Animals; D002452:Cell Count / Q000379:methods; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured / Q000187:drug effects; D004622:Embryo, Mammalian; D004798:Enzymes / Q000494:pharmacology*; D005260:Female; D005455:Fluorescent Antibody Technique; D019563:Mechanics; D009474:Neurons / Q000502:physiology*; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D013116:Spinal Cord / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D014404:Tubulin / Q000378:metabolism
chemical_list: D004798:Enzymes; C488416:Tubb3 protein, rat; D014404:Tubulin