Long-term primary culture of highly-pure rat embryonic hippocampal neurons of low-density.

In order to develop a simplified method for long-term primary culture of highly-pure rat embryonic hippocampal neurons of low-density (10(3) cells/cm(2)), we optimized and modified conventional culturing methods. The modifications of our simplified method include: (1) combinational application of two growth substrates, tail collagen and poly-L-lysine, to coat plastic culture dishes and coverslips for a better neuronal attachment; (2) dissociation of hippocampal tissues with combinational use of two milder enzymes (collagenase and dispase) and trypsin of a lower concentration to minimize enzymatic damages to cultured neurons; (3) a cell pre-plating step to preliminarily eliminate the contaminating non-neuronal cells; (4) a modified culture medium as a critical step to promote highly pure neurons of low-density for a long term; and (5) appropriately reduced frequency and volume of refreshment of the culture medium. Using our modified method, the beta-tubulin III-immunostained and Hoechst 33342 counterstained neurons harvested a steady and healthy growth with a longer culture time of over 35 days, and a clear distinction between TAU-1- and MAP2-immunoreactive neurites was apparent at the early culturing period. In addition, the purity of neurons was over 95% at the different time points in comparison with the control culture using conventional serum-free method in which most neurons degenerated and died within 5 days. Thus, our modified method proved to be a simple, feasible as well as time- and resource-saving approach for a long-term survival of pure rat embryonic hippocampal neurons of low-density.