Generating 3D Co-culture Spheres of Astrocytes and Neurons to Induce Synapse Formation

Take a multiwell plate coated with a surfactant to reduce the surface tension of the well.

Add the desired ratio of astrocytes and neurons in a suitable culture medium.

Centrifuge to settle the cells and then incubate.

The low surface tension prevents cells from sticking to the well surface, causing their aggregation.

The cells adhere to each other through various membrane interactions, including adhesion molecules, forming a 3D coculture sphere.

Collect the spheres and transfer them to a tube.

Allow the spheres to settle. Remove the supernatant and add fresh culture medium.

Transfer the spheres to a spinner flask containing a suitable growth medium and incubate with constant spinning.

In the sphere, the neurons extend projections towards each other, forming a junction called a synapse, while the astrocyte extends towards the synapse, establishing a close association with the neurons.