An improved serum-free method for culturing primary hippocampal neurons from SD neonatal rats

  Objective  To explore the cell survival of the modified serum-free method for culturing primary hippocampal neurons from SD neonatal rats.

  Methods  From February to December in 2018, the hippocampal tissues of 72 SD neonatal rats within 24 hours of birth were dissected and isolated, and the neuron cells were cultured with Neurobasal + B27 modified serum-free medium. Invitroge automatic cell counter was used to count the number of viable and dead cells. The morphological changes of nerve cells were observed using a laser confocal microscope.

  Results  In average, (8.40±2.86)×10⁵ nerve cells were isolated from two hippocampi of each neonatal rat, among which the number of viable cells was (5.48±1.73)×10⁵ and the number of dead cells was (3.08±1.30)×10⁵. The cell survival rate was 69%. Axons was observed to grow on the third day after the cells adhered to the wall, dendrites grew on the sixth day, and axons and dendrites of peripheral nerve cells could be observed to form a neural network on the ninth and twelfth days. On the third day after adherence, the nerve cell body gradually grew larger and fuller, and finally became oblate. All cells could survive for up to 4 weeks.

  Conclusion  Our improved serum-free method is suitable for culturing primary hippocampal neurons. Accurate and rapid dissection and gentle and effective pipetting of hippocampal tissue are the key points of culturing neuronal cells with higher survival and viability.