Survival and growth of hippocampal neurons in defined medium at low density: advantages of a sandwich culture technique or low oxygen

The study of development and plasticity of hippocampal circuitry would greatly benefit from methods which allow the long-term culture of neurons at low density under precisely defined culture conditions. We report that isolated hippocampal neurons from embryonic day 18 rats can be cultured for several weeks at low densities which permits the determination of individual connections. A serum-free medium was modified from the formulation of Romijn to include the biological anti-oxidants vitamin E, glutathione, pyruvate, catalase and superoxide dismutase. Neuronal survival of 80% and neuritogenesis greatly exceeded that seen in serum-based cultures. It appeared that vitamins E, A and linolenic acid promoted neuritogenesis. The beneficial effects of the antioxidants suggested a toxic role of oxygen. To directly test this, cultures were incubated in reduced oxygen (9%) and compared to those in the normal 19.7% oxygen (95% air). After 3 days in culture, neurons with processes in 9% oxygen were more than double those in normal oxygen. Neuronal survival and neurite growth could be improved if the cells were grown on a substrate-coated surface covered with a coverslip. Under this condition, cells show a ring of growth between the center and the edge of the coverslip. In 9% oxygen, this ring was closer to the edge of the coverslip than in normal oxygen. The coverslip did not serve as an additional substrate for attachment since it left the neurons attached to the original substrate. However, removal of the coverslip leads to cell death within 24 h, suggesting that the cells had been exposed to a toxic factor. Variations in glial cell content (less than 10%), pH, and pCO2 were demonstrated to be unlikely explanations of the higher survival. These results suggest that growth in a diffusion-limited space, reduction of oxygen concentration to physiological levels and control of toxic oxidation with physiological antioxidants can greatly improve the survival and neuritogenesis of isolated hippocampal neurons in primary culture.