Developing Neuron Balls Using a Hanging Drop Culture Technique

Begin with a single-cell suspension of mouse embryonic neuronal progenitor cells in a neurobasal medium.

The medium contains essential nutrients for maintaining cell viability.

Dispense small drops of this cell suspension on the lid of a non-adhesive culture dish, ensuring they are spaced apart.

Invert the lid, resulting in the formation of hanging drops.

Position the lid above the dish containing a buffer to establish a humid environment that prevents media evaporation and incubate.

Within the hanging drops, the surface tension of the liquid maintains the drop's shape.

This shape confinement restricts the cells to a limited space without spreading.

Additionally, gravitational forces within the drops direct the cells toward the lower region.

As neuronal progenitor cells come into closer contact, they spontaneously aggregate to form a three-dimensional structure known as a neuron ball.

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.