I used SD rats (E19) for cortical neuron extraction. Due to the need for subsequent special experiments, I cultured the neurons in a Corning PDL 384-well plate. The culture medium is Neurobasal + 1% B27 + 0.5% P/S. There are two plates, each well containing 10,000 and 15,000 cells respectively, with 25 µL of culture medium. The medium is changed every three days, with half of the volume replaced each time (12.5 µL).

For the first five days of the experiment, there were no issues. This includes after the medium change on the third day, where the neurons were growing well, with significant axon growth and synapse formation. However, after the medium change on the sixth day, I observed on the seventh day that the neurons in both plates were in very poor condition. The cell bodies were no longer smooth at the edges, and many dense, dot-like particles, resembling debris from broken cells, were present in the medium.

Others in the lab have encountered similar issues, but we have not been able to solve them. For example, neurons might look good on Day 7 but then deteriorate suddenly by Day 11. Figure 1 shows the neurons in a good state, and Figure 2 shows them in a poor state. The pictures are not very clear; please excuse that. However, this issue is very important, so I am seeking advice from everyone.