This article describes a method for culturing mouse embryonic neuronal progenitor cells to form three-dimensional neuron balls. The technique utilizes hanging drops to facilitate cell aggregation and growth.
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.
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