Based on doi:10.3791/65176 (2023) with modifications

1. Once the iPSCs have reached 80%-90% confluency, wash them with DPBS, and add 400 μL of accutase, incubate at 37C until cells de-attach (5-7min).

2. Add 1mL of prewarmed (37°C) DMEM/F12 to stop the reaction, and pipette up and down 5x-10x (not more than 10x) to dissociate the cells from the cell culture dish and to obtain a single cell suspension.

3. Transfer the cell suspension to a 15 mL conical centrifuge tube, and centrifuge the cells at 300 × g for 5 min at room temperature.

4. Aspirate the supernatant, and resuspend the pellet in 2 mL of DMEM/F12 or culture media with 50μM pro-survival compound (PSC).

5. Use 10 μL of the cell suspension to count the cells using a Neubauer chamber.

6. Adjust the cell suspension to a concentration of 9,000 cells per 150 μL (60,000 cells/mL) using iPSC culture medium supplemented with 50μM PSC.

7. To generate EBs, seed 150 μL of the cell suspension into each well of an ultralow attachment 96-well plate. While pipetting, gently shake the tube containing the cell suspension to prevent the cells from sedimenting. When adding cells to the wells of the 96w plate, try to do one slow motion to not create separate drops.

8. Culture the EBs in a humified atmosphere of 5% CO2 and 95% air at 37 °C (0 days post seeding [dps]). Do not disturb the EBs within the first 24h after seeding.

9. After ~48 h (2 dps), change the medium to iPSC culture medium without PSC. Remove 100 μL of medium per well, and add 150 μL of prewarmed (37°C) fresh medium without PSC. Go row by row.

10. Perform further medium changes every other day; remove 150 μL of medium from each well, and add 150 μL of prewarmed (37 °C) fresh medium without PSC per well.

NOTE: After 4-5 dps, the periphery of the EBs should become translucent.

NOTE: In general, good-quality EBs should have smooth contours and translucent borders at this stage. The time points of neural induction slightly differ between primate species and iPSC lines. Neural induction for marmoset EBs usually needs to be started at 4 dps, for rhesus macaque at 5 dps, and for human and chimpanzee EBs at 4-5 dps, depending on the state of the EBs.

1. Remove 150 μL of medium from each well of the first row of the 96-well plate, and add 150 μL of prewarmed (37 °C) neural induction medium (NIM) (table below) per well in the same row.

2. Continue changing the medium as described above row by row for the whole 96-well plate. Perform further neural NIM changes every other day by removing 150 μL of NIM from each well and adding 150 μL of prewarmed (37 °C) fresh NIM.

NOTE: From this point on, the marmoset EBs should be cultured under the same conditions as the other primate EBs (humified atmosphere of 5% CO2 and 95% air at 37 °C).

NOTE: Once the EBs have developed a pronounced, translucent, radially organized neuroepithelium on the surface, structural support needs to be provided for the development of ventricle-like structures. This is achieved by embedding the EBs into a basement membrane matrix. Due to differences in development rates, marmoset and rhesus macaque EBs are ready for embedding already at 7 dps, while human and chimpanzee EBs are usually embedded at 8-9 dps. For simplicity, basement membrane matrix refers only to Matrigel in this protocol.

1. In preparation for embedding, UV-sterilize scissors, forceps, a small rack for 0.2 mL tubes, and three to six squares of parafilm treated with 80% EtOH under the laminar flow hood for 15 min. Let the basement membrane matrix thaw on ice for 30min-1h (~1.5 mL of Matrigel is usually enough for 96 EBs).

NOTE: Always keep the Matrigel on ice.

2. Create a 4 x 4 dimple grid on the parafilm. Place the parafilm grid on the 0.2 mL tube rack so that the paper-enveloped side is facing up, and gently press a gloved finger against each hole of the rack.

3. Remove the paper, and cut the dimple grid out of the parafilm square using scissors to adjust its size to fit into a 60 mm cell culture dish. Place the dimpled parafilm in a 60mm dish.

4. Using a pipette with a cut 200 μL pipette tip, carefully transfer the EBs one after another from the well of the culture dish to the parafilm dimples.

5. After moving 16 EBs to the grid, take a new 200μL pipette tip, and remove the remaining medium from the dimples.

6. Pipette one drop (~15 μL) of basement membrane matrix onto each dimple containing one EB.

7. Take a 10 μL pipette tip or a micro dissection tool and quickly move the EBs into the center of each droplet without disturbing the droplet borders.

8. Check if some droplets are too small or EBs are to close to the borders, add more Matrigel to these drops.

9. Place the dimpled parafilm with the basement membrane matrix drops inside a 60 mm cell culture dish, and incubate for 15-30 min at 37 °C to allow the Matrigel to polymerize.

10. To detach the matrix-embedded EBs from the parafilm, add 5 mL of differentiation medium (DM) without vitamin A (table below) to the dish, and turn the parafilm square upside-down using forceps so that the side with the EBs is facing the bottom of the dish.

11. Carefully shake the dish to make the basement membrane matrix drops containing the EBs detach from the parafilm. If some of them are still attached, take an edge of the parafilm square using forceps, and rapidly roll it up toward the center of the dish multiple times.

12. Culture the cerebral organoids on an orbital shaker at 65 rpm in a humified atmosphere of 5% CO2 and 95% air at 37 °C. Keep them in DM without vitamin A with medium changes every other day. To induce the production of neurons, switch to DM with vitamin A (retinoic acid, RA) after 13 dps for marmoset and rhesus macaque cerebral organoids or 14-15 dps for human and chimpanzee cerebral organoids. From this point on, change the medium every 3-4 days.

NOTE: To support neuronal survival, DM with vitamin A can be supplemented with 20 μg/mL human neurotrophin 3 (NT3), 20 μg/mL brain-derived neurotrophic factor (BDNF), and 1 μL/mL basement membrane matrix from 40 dps on.