Objective To establish a low density,high purity and high stability in vitro culture method of primary hippocampal neurons of fetal rats by co-culturing hippocampal and cortical cells,so as to obtain higher purity and better vitality of primary hippocampal neurons disease.Methods The fetal rat hippocampal tissue was isolated from 16-18 days pregnant SD rats,then cut and digested by 0.125%trypsin.The obtained cell suspension was filtered by 200 mesh cell sieve,and then the obtained cell suspensions were then inoculated into the inner layer and outer ring of the culture plate in a surrounding form.They were co-cultured in DMEM/F12 medium containing 10%horse serum.After 4-6 hours of cell adhesion,the culture medium was changed to maintenance medium(Neurobasal+2%B27+0.5 mmol/L glutamine).Then the cell viability was detected with CCK-8 kit and the purity of hippocampal neurons was detected by immunofluorescent staining.Results Hippocampal neurons grew well and formed crisscross neural networks after 5 days.And it could survive for 3 weeks.The purity of hippocampal neurons was up to 98%.Conclusion The method of co-culturing hippocampal and cortical cells can obtain high-purity,high activity,high survival rate,and high stability primary hippocampal neurons from fetal rats,which can provide certain experimental conditions for the study of hippocampal neuron related diseases in the nervous system and is worthy of promotion and application.
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