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.