1. Seeding of human iPSCs in the bioreactor
- 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.
- After 1 h of incubation, aspirate the spent medium from each well and wash 1x with 1 mL of 1× PBS per well.
- 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.
- 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.
- Centrifuge at 210 × g for 3 min and remove the supernatant.
- 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.
- 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.
- 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
- Define the day of single cell seeding as day 0.
- 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.
- Acquire images with an optical microscope using a total magnification of 40x or 100x to measure aggregate diameter.
- Measure the area of the aggregates in each image using FIJI software.
- Select "Analyze | Set Measurements" from the menu bar and click on "Area" and "OK".
- 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.
- 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.
- 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.
- 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.
- Insert the bioreactor containing the aggregates in the universal base unit placed in the incubator at 37 °C, 95% humidity, and 5% CO2.
- Decrease the bioreactor agitation to 25 rpm.
- 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.
- 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.
- 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.
- 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.
- On day 18, repeat steps 2.2, 2.3, 2.4, and 2.11.
- 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.
- 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.
- 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.
- 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.
| Media preparation | mTeSR1
Final volume: 500 mL | 1. Thaw mTeSR1 5× supplement at room temperature (RT) or at 4 °C overnight and mix with basal medium
2. Store complete mTeSR1 medium at 4 °C for up to 2 weeks or prepare 40 mL aliquots and store at -20 °C
3. Pre-warm complete mTeSR1 at RT before use |
gfCDM (growth factor-free chemically defined medium)
Final volume: 60 mL | 30 mL Ham's F12 30 mL IMDM 600 µL chemically defined lipid concentrate (1 % v/v) 2.4 µL monothioglycerol (450 μM) 30 µL apo-transferrin (stock solution at 30 mg/mL in water, final concentration: 15 μg/mL) 300 mg crystallization-purified BSA (5 mg/mL) 42 µL insulin (stock concentration at 10 mg/mL, final concentration: 7 µg/mL) 300 µL P/S (0.5% v/v, 50 U/ml penicillin/50 μg/ml streptomycin) |
Neurobasal
Final volume: 60 mL | 60 mL of Neurobasal medium 600 µL N2 supplement 600 µL Glutamax I 300 µL P/S (0.5 % v/v). |
Complete BrainPhys
Final volume: 60 mL | 60mL of BrainPhys 1.2 mL NeuroCult SM1 Neuronal Supplement 600 µL N2 Supplement 12 µL BDNF (final concentration: 20 ng/mL) 12 µL GDNF (final concentration: 20 ng/mL) 300 µL Dibutyryl-cAMP (stock concentration: 100 mg/mL in water, final concentration: 1 mM) 42 µL ascorbic acid (stock concentration: 50 µg/mL in water, final concentration: 200 nM) |
| Stock solutions of growth factors and small molecules | Basic fibroblast growth factor (bFGF/FGF2)
Stock concentration: 100 µg/mL | 1. Reconstitute in 5 mM Tris, pH 7.6, at a concentration of 10 mg/mL
2. Dilute with 0.1 % BSA in PBS (v/v) to a final stock concentration of 100 µg/mL |
Stromal cell-derived factor 1 (SDF1)
Stock concentration: 100 µg/mL | 1. Reconstitute in water at a concentration of 10 mg/mL
2. Dilute with 0.1 % BSA (v/ v) in PBS to a final stock concentration of 100 µg/mL. |
Brain-derived neurotrophic factor (BDNF)
Stock concentration: 100 µg/mL< |
Glial cell-derived neurotrophic factor (GDNF)
Stock concentration: 100 µg/mL |
Fibroblast growth factor 19 (FGF19)
Stock concentration: 100 µg/mL | 1. Reconstitute in 5 mM sodium phosphate, pH 7.4, at a concentration of 10 mg/mL
2. Dilute with 0.1 % BSA in PBS (v/v) to a final stock concentration of 100 µg/mL |
ROCK inhibitor Y-27632
Stock concentration: 10mM | Reconstitute in DMSO at a concentration of 10 mM. |
SB431542
Stock concentration: 10mM |
Insulin
Stock concentration: 10 mg/mL | 1. Reconstitute 10 mg of insulin in 300 µL of 10 mM NaOH
2. Carefully add 1 M NaOH until the solution becomes clear-transparent
3.Fill to 1 mL with sterile water |
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.
Gelatin/Sucrose
Final concentration: 7.5%/15% w/w | 1. Weigh 15 g of sucrose and 7.5 g of gelatin in a sterile Schott Glass Bottle and mix well
2. Pre-warm the PBS 1× at 65 °C
3. Add pre-warmed PBS 1× to a final weight of 100 g and mix well
4. Place the Schott Glass Bottle in a heating plate at 65 °C and shake until the gelatin melts
5. Incubate at 37 °C until the solution stabilizes |
