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

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.