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.

Add the desired ratio of dissociated hAstros and hNeurons or iNeurons in a total volume of two milliliters to each well of a microplate. Centrifuge the plate at 100 times g for three minutes, and return to the incubator for two days.

After one day, densely packed microspheres should have formed in the plate. Use a 1,000-microliter micropipette to gently remove spheres from the microwells. Lightly apply force to the bottom of microwells with medium to remove any additional adhered spheres.

After collecting all spheres in a 15-milliliter conical tube, allow it to settle. Then, wash the spheres by first aspirating the old medium, and then adding at least two milliliters of fresh medium. Add the spheres to a spinner flask containing 50 to 60 milliliters of medium. Place the flask in the incubator on a magnetic stir plate set at 60 RPM. A minimum of three weeks in culture is needed for synapse formation.