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

1. Preparation and Maintenance of 3D Sphere Cocultures

1. Dissociate hAstros from 3D aggregates in suspension.​
   1. Gently dissociate human astrocyte (hAstro) aggregates with detachment solution when dark centers appear and remove spheres that spontaneously attach. Break spheres gently once a week to maintain sphere health and to avoid necrotic cores.
      ​NOTE: Do not exceed 5 min of treatment with detachment solution.
2. Dissociate hNeurons or iNeurons from a 2D monolayer.
   1. Remove media from the 6-well plate and add 500 µL of detachment solution to each well-containing monolayer culture of hNeuron (human neuron) or iNeurons (inducible neurons). Incubate at 37 °C for 5 min. Gently add 2-3 mL DMEM/F12 basal medium to remove attached cells.
   2. Collect cells and media in a 15 mL conical tube and centrifuge at 300 x g for 1 min. Aspirate the supernatant, add 1 mL of fresh media, and pipette up and down gently with a 1,000 µL micropipette to achieve a single-cell suspension.
3. Form 3D spheres using microwell culture plates.
   1. Prepare the plate by adding 0.5 mL of anti-adherence rinsing solution to each well of the microwell plate. Centrifuge plate at 2,000 x g for 5 min. Aspirate rinsing solution, add 1 mL of DMEM/F12 basal medium to each well to wash, and aspirate again.
      NOTE: Always ensure that the centrifuge is balanced during use.
   2. Count each cell type using a hemocytometer or automated cell counter. Add desired ratio of dissociated hAstros and hNeurons or iNeurons to each well in a total volume of 2 mL of neural medium (NM) + Y (include Dox if iNeurons are utilized). Centrifuge plate at 100 x g for 3 min and return to the incubator.
      NOTE: 24-well microwell plates (see the Table of Materials) contain 300 microwells per well. Add a minimum of 6 x 10⁵ cells (for spheres of 2 x 10³ cells each) and a maximum of 6 x 10⁶ cells (for spheres of 2 x 10⁴ cells each) in 2 mL of media per well. For viable spheres of a specific density within this range, use a defined quantity of cells and divide by 300 to calculate the number of cells per sphere. Alternative sphere-forming methods and tools may also be utilized if desired. Follow the manufacturer's instructions for acceptable ranges of cell densities.
   3. Allow cells to self-assemble into densely packed spheres within microwell plates over the next 2 days (Figure 1A). Replace 50% of media if the culture is yellowing.
      NOTE: Exchange only half media and pipette gently when removing and adding media in order not to disturb spheres. Spheres may lift up from microwells and fuse together with higher force.
   4. After 2 days, gently remove spheres from microwells with a 1,000 µL micropipette (Figure 2D). Lightly apply force to the bottom of microwells with media to remove any additional adhered spheres. Let spheres settle in a 15 mL conical tube, aspirate the old media, and add fresh media.
4. Set up the spinner flask bioreactor system for forming 3D spheres.​
   1. Clean the surface of a magnetic stir plate with 70% ethanol, and place it into a cell culture incubator. Autoclave individual spinner flasks to sterilize.
   2. Add spheres with a minimum of 50–60 mL media to each spinner flask (Figure 2D, inset). Place the flask on the magnetic stir plate set at 60 rpm. Culture spheres in spinner flasks until they are ready for data collection.
      NOTE: A minimum of 3 weeks in culture is needed for synapse formation.