Developing Neuron Balls Using a Hanging Drop Culture Technique

For preparing neuron balls, adjust the cell density in this cell suspension to 1 million cells per milliliter using NGB medium. Add 7 milliliters of PBS to the bottom part of the culture dishes. Culture the cortical neurons as 10-microliter hanging drops containing 10,000 cells per drop inside the upper lids of 10-centimeter culture dishes. Keep the dishes in an incubator for three days at 37 degrees Celsius with 5% CO₂ under humidified conditions to allow for neuron ball formation.