Generating Mature Cerebellar Organoids From Induced Pluripotent Stem Cells Using Single-Use Bioreactors

1. Seeding of human iPSCs in the bioreactor

1. Incubate iPSCs grown as monolayers in mTeSR1 supplemented with 10 µM of ROCK inhibitor Y-27632 (ROCKi). Add 1 mL of supplemented medium to each well from a 6-well tissue culture plate and incubate for 1 h at 37 °C, 95% humidity, and 5% CO₂.
   NOTE: ROCKi is used to protect dissociated iPSCs from apoptosis.
2. After 1 h of incubation, aspirate the spent medium from each well and wash 1x with 1 mL of 1× PBS per well.
3. Add 1 mL of the cell detachment medium (see Table of Materials) to each well of a 6-well plate and incubate at 37 °C for 7 min until cells detach easily from the wells with gentle shaking.
4. Pipette the cell detachment medium up and down with a P1000 micropipette until the cells detach and dissociate into single cells. Add 2 mL of complete cell culture medium to each well to inactivate enzymatic digestion and pipette the cells gently into a sterile conical tube.
5. Centrifuge at 210 × g for 3 min and remove the supernatant.
6. Resuspend the cell pellet in culture medium (i.e., mTeSR1 supplemented with 10 µM of ROCKi) and count the iPSCs with a hemocytometer using trypan blue dye.
7. Seed 15 × 10⁶ single cells in the bioreactor (maximum volume of 100 mL) with 60 mL of mTeSR1 supplemented with 10 µM of ROCKi at a final cell density of 250,000 cells/mL.
8. Insert the vessel containing the iPSCs in the universal base unit placed in the incubator at 37 °C, 95% humidity, and 5% CO₂.
   NOTE: The bioreactor stirring is maintained for 24 h by setting the universal base unit control to 27 rpm to promote iPSC aggregation.

2. Differentiation and maturation of human iPSC-derived aggregates in cerebellar organoids

1. Define the day of single cell seeding as day 0.
2. On day 1, collect 1 mL of the iPSC aggregates sample using a serological pipette. Maintain the bioreactor under agitation as before by placing the universal base unit with the bioreactor containing the aggregates in a sterile flow prior to collecting the sample. Plate the cell suspension in an ultra-low attachment 24-well plate. Check that iPSC-derived aggregates are formed.
3. Acquire images with an optical microscope using a total magnification of 40x or 100x to measure aggregate diameter.
4. Measure the area of the aggregates in each image using FIJI software.
   1. Select "Analyze | Set Measurements" from the menu bar and click on "Area" and "OK".
   2. Select "File | Open" from the menu bar to open a stored image file. Select the line selection tool presented in the toolbar and create a straight line over the scale bar presented in the image. Select "Analyze | Set scale" from the menu bar.
   3. In "Known distance," add the expanse of the image's scale bar in µm. Define the "Unit of length" as µm. Click on "Global" to maintain the settings and "OK". Select Oval Selection in the toolbar.
   4. For each aggregate, delineate the area with the oval tool. Select "Analyze | Measure". Calculate their diameter based on measured area, considering that aggregates are approximately spherical using
      
      with A as the area of the aggregate.
5.  When the average diameter of the aggregates is 100 µm, replace 80% of the spent medium with fresh mTeSR1 without ROCKi. When aggregates reach 200–250 μm in diameter, replace all the spent medium with gfCDM (Table 1), letting the organoids settle at the bottom of the bioreactor.
   NOTE: If the average aggregate diameter exceeds 350 μm, do not start the differentiation protocol. Repeat the seeding of single cells. Generally, it takes around 1 day for the aggregate to reach an average diameter of 100 µm.
6. Insert the bioreactor containing the aggregates in the universal base unit placed in the incubator at 37 °C, 95% humidity, and 5% CO₂.
7. Decrease the bioreactor agitation to 25 rpm.
8. On day 2, repeat steps 2.2, 2.3, and 2.4 to evaluate the aggregate diameter. Add 30 μL of FGF2 (final concentration, 50 ng/mL) and 60 μL of SB431542 (final concentration, 10 μM) to 60 mL of gfCDM differentiation medium (Table 1). Replace all spent medium from the bioreactor with the supplemented gfCDM. Repeat step 2.6.
   NOTE: SB431542 is crucial to inhibit mesendodermal differentiation, inducing neural differentiation. FGF2 is used to promote the caudalization of the neuroepithelial tissue.
9. On day 5, repeat steps 2.2, 2.3, 2.4, and 2.8.
   NOTE: Aggregate size should increase during the differentiation protocol. However, the diameter is only critical when the differentiation starts because this parameter could influence the efficacy of differentiation.
10. On day 7, repeat steps 2.2, 2.3, and 2.4. Dilute FGF2 and SB431542 to 2/3: Add 20 μL of FGF2 and 40 μL of SB431542 to 60 mL of gfCDM differentiation medium. Replace all spent medium from the bioreactor with supplemented gfCDM. Repeat step 2.6 and increase bioreactor agitation to 30 rpm.
11. On day 14, repeat steps 2.2, 2.3, and 2.4. Add 60 μL of FGF19 (final concentration, 100 ng/mL) to 60 mL of gfCDM differentiation medium. Replace all spent medium from the bioreactor with gfCDM supplemented with FGF19. Repeat step 2.6.
    NOTE: FGF19 is used to promote polarization of mid-hindbrain structures.
12. On day 18, repeat steps 2.2, 2.3, 2.4, and 2.11.
13. On day 21, repeat steps 2.2, 2.3, and 2.4. Replace all spent medium from the bioreactor with complete neurobasal medium (Table 1). Repeat step 2.6.
    NOTE: Neurobasal medium is a basal medium used to maintain the neuronal cell population within the organoid.
14. On day 28, repeat steps 2.2, 2.3, and 2.4. Add 180 μL of SDF1 (final concentration, 300 ng/mL) to 60 mL of complete neurobasal medium. Replace all spent medium from the bioreactor with complete neurobasal medium supplemented with SDF1. Repeat step 2.6.
    NOTE: SDF1 is used to facilitate the organization of distinct cell layers.
15. On day 35, repeat steps 2.2, 2.3, and 2.4. Replace all spent medium from the bioreactor with complete BrainPhys medium (Table 1). Repeat step 2.6.
    NOTE: BrainPhys is a neuronal medium that supports synaptically active neurons.
16. Replace 1/3 of the total volume every 3 days with complete BrainPhys medium until day 90 of differentiation.

Table 1: Stock solutions and media preparation. Listed are all the components and volumes used to prepare media for the iPSCs maintenance and differentiation protocol, as well as stock solutions of growth factors and small molecules. For stock solutions, all stock concentrations and protocols for reconstitution are listed.

Table 2: Solutions for preparation of organoids for cryosectioning and immunostaining. Listed are all the components and volumes used to prepare the solutions used in the preparation of organoids for cryosectioning and immunostaining.