What are neuron balls?

Neuron balls are three-dimensional aggregates formed by neuronal progenitor cells during culture.

Why use hanging drops for cell culture?

Hanging drops create a microenvironment that promotes cell aggregation and minimizes evaporation.

What is the optimal cell density for forming neuron balls?

A cell density of 1 million cells per milliliter is recommended for optimal aggregation.

How long should the cells be incubated?

Cells should be incubated for three days at 37 degrees Celsius with 5% CO2.

What medium is used for culturing the cells?

Neurobasal medium supplemented with essential nutrients is used for cell culture.

What is the significance of using mouse embryonic neuronal progenitor cells?

These cells are vital for studying neuronal development and function in a controlled environment.

Developing Neuron Balls Using a Hanging Drop Culture Technique

JoVE Encyclopedia of Experiments

Neuroscience

0 views • 1:46 min • July 8th, 2025

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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.