Methods for isolating highly-enriched embryonic spinal cord neurons: a comparison between enzymatic and mechanical dissociations.
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.
Jiang
Xiao-Yan
XY
Department of Neurobiology, Shanghai Jiaotong University School of Medicine, 280 South Chong-Qing Road, Shanghai 200025, PR China.
Fu
Sai-Li
SL
Nie
Bao-Ming
BM
Li
Ying
Y
Lin
Lin
L
Yin
Lan
L
Wang
Yan-Xia
YX
Lu
Pei-Hua
PH
Xu
Xiao-Ming
XM
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
D004798:Enzymes; C488416:Tubb3 protein, rat; D014404:Tubulin