Oct 01, 2025
Cortical spheroid differentiation
- Annika Martin1,
- Hanqin Li1,2,
- Dirk Hockemeyer1,2
- 1University of California, Berkeley, Berkeley, CA 94720, USA;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 2081
Protocol Citation: Annika Martin, Hanqin Li, Dirk Hockemeyer 2025. Cortical spheroid differentiation. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8po57g2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 11, 2024
Last Modified: October 01, 2025
Protocol Integer ID: 93404
Keywords: ASAPCRN, cortical spheroid differentiation this protocol, cortical spheroid differentiation, human cortical spheroid, maintenance of human cortical spheroid, early cortical spheroid, procedure, published protocol, formulation
Funders Acknowledgements:
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-000486
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-024409
Abstract
This protocol describes the procedure of differentiating hPSCs into early cortical spheroids following an adaptation of a published protocol (Yoon et al 2019, Paşca et al. 2015).
Protocol overview
A. Media Formulations
B. Growth and Maintenance of Human Cortical Spheroids
Initial notes
A list of reagents and relevant vendor information can be found in the table listed under the materials tab.
Attachments
Materials
Reagent Table:
| Item | Vendor | Catalog Number | |
| DMEM-F12 | Thermo Fisher | 11320082 | |
| DMEM With L-Glutamine and 4.5g/L Glucose; Without Sodium Pyruvate | Thermo Fisher | MT10017CV | |
| Knockout Serum Replacement | Gibco Life Technologies | 10828028 | |
| Newborn Calf Serum,USA origin, Heat Inactivated, sterile-filtered, suitable for cell culture | Sigma-Aldrich | N4762-500ML | |
| Penicillin-Streptomycin (10,000 U/mL) | Gibco Life Technologies | 15140163 | |
| MEM-NEAA (100x) | Gibco Life Technologies | 11140050 | |
| Gibco‱ GlutaMAX‱ Supplement | Gibco Life Technologies | 35050061 | |
| HEPEs buffer | Sigma-Aldrich | H0887-100ML | |
| Neurobasal‱ Medium | Gibco Life Technologies | 21103049 | |
| B-27‱ Supplement (50X), minus vitamin A | Gibco Life Technologies | 12587010 | |
| Dorsomorphin, ≥98% (HPLC) | Sigma-Aldrich | P5499-5MG | |
| SB431542 | Selleck Chemicals | S1067 | |
| Y-27632 – ROCK Inhibitor | Chemdea | CD0141 | |
| Recombinant Human BDNF Protein, CF | R&D Systems | 11166-BD-050 | |
| NT-3 | Sigma | SRP3128 | |
| Heat Stable Recombinant Human bFGF | Thermo Fisher Scientific | PHG0367 | |
| Recombinant Human EGF Protein, CF | R&D Systems | 236-EG-01M | |
| Dimethyl sulfoxide (DMSO) | Thermo Fisher Scientific | BP231-100 | |
| Accutase | Thermo Fisher Scientific | SCR005 | |
| DPBS w/o calcium andmagnesium | Corning | MT21031CV | |
| CountessTM Cell CountingChamber Slides | Thermo Fischer Scientific | C10228 | |
| AggreWell‱800 (6-well plate) | StemCell Technologies | 34821 | |
| Costar‱ 6-well Clear Flat Bottom Ultra-Low Attachment 6-well plate | Corning | 3471 |
Troubleshooting
Media Formulations
hESC KSR Media (500 ml)
400 ml DMEM/F12
100 ml KSR
5 ml Pen-strep
5 ml NEAA
5 ml Glutamax
5 mL HEPES buffer
hESC Wash Media (500 ml)
500 ml DMEM
25mL ml Calf Serum
5 ml Pen-strep
hCS media (500 ml)
500 ml Neurobasal media
10 ml B27 (minus vitamin A)
5 ml pen-strep
5 ml Glutamax
5 mL HEPES buffer
Neural precursor expansion media
50 ml hCS media
40 µl 25 ug/ml FGF (final concentration, 20 ng/ml)
25 µl 40 µg/ml EGF (final concentration, 20 ng/ml)
Neural induction media
50 ml hESC KSR Media
50 µl 10 mM SB431542 (final concentration, 10 µM)
25 µl 10 mM dorsomorphin (final concentration, 5 µM)
Cortical differentiation media
50 ml hCS media
50 µl 20 µg/ml BDNF (final concentration, 20 ng/ml)
50 µl 20 µg/ml NT-3 (final concentration, 20 ng/ml)
Resuspend Dorsomorphin in DMSO at a concentration of 10 mM (1:2000)
Resuspend SB431542 in DMSO at a concentration of 10 mM (1:1000)
Resuspend Rock Inhibitor in DMSO at a concentration of 10 mM (1:1000)
Resuspend BDNF in water at a concentration of 20 µg/ml (1:1000)
Resuspend NT3 in water a concentration of 20 µg/ml (1:1000)
Resuspend FGF at a concentration of 25 µg/ml (1:1250)
Resuspend EGF at a concentration of 40 µg/ml (1:2000)
Growth and Maintenance of Human Cortical Spheroids
35m
Grow stem cells feeder-free on 6-well plates as described in (dx.doi.org/10.17504/protocols.io.b4mcqu2w).
On day -1 of differentiation remove hESC Media and feed with hESC KSR Media + 10uM Rock Inhibitor (1:1000).
Pre-coat an aggrewell plate according to manufacturer instructions.
On day 0 of differentiation, aspirate media and add 1 mL of PBS- onto each well.
Immediately Remove PBS- and add 1 mL of Accutase (1:3 Diluted with PBS-) onto each well of the 6 well plate.
Return plate to incubator for ~00:30:00 or until cells are dissociated into single cell suspension.
30m
Remove plate from incubator. Pool the cells into a 15mL conical tube per plate (6mL total volume).
Using 6 mL hESC Wash media, rinse the wells and dilute the cell-Accutase suspension.
Spin down cells for 00:05:00 at 200 x g to 300 x g .
5m
Remove the supernatant from the falcon tube using a sterile glass pipette. Be careful not to aspirate any of the cells.
Resuspend the cells in 5 mL of hESC KSR Media + 10uM Rock Inhibitor (1:1000).
Using a 5mL serological pipette, filter cells through a 40 µm cell strainer into a new 50 ml conical tube.
Take two sets of 10 µL of cell suspension. Mix each set with 10 μl trypan blue dye, which comes with the Countess Cell Counting Chamber Slides.
Count cells with Countess automated cell counter or hemocytometer, averaging the counts from the two sets.
Resuspend the cells to a concentration of 18 million cells per 5 mL of media (3.6 million cells per mL) in hESC KSR Media + 10µM Rock Inhibitor (1:1000).
If needed, combine tubes from multiple plates and re-concentrate by spinning down at 200 x g to 300 x g and resuspending in an appropriate volume of hESC Media + Rock Inhibitor 1:1000.
Transfer 5 mL of suspension to one well of a pre-coated 6-well Aggrewell 800 plate. Return plate to the incubator.
The next day (Day 1), prepare neural induction media.
Remove the aggrewell plate from the incubator and use a serological pipette to transfer the newly formed Embryoid Bodies (EBs) to a 15 ml conical tube.
Allow EBs to settle (1-2 mins, but check before aspirating to ensure EB retention) and aspirate off media, taking special care to not suck up the EBs.
Resuspend in 5 ml of neural induction media and deposit into one well of a 6-well ultra-low attachment plate.
Repeat steps 17-20 to change media each day until Day 6.
On Day 6, remove cell suspension from the plate and deposit into a 15 ml falcon tube
Allow spheroids to settle to the bottom of the tube (~1-2 mins).
While settling, make neural precursor expansion media.
Remove the supernatant from the settled spheroid suspension.
Resuspend spheroids in neural precursor expansion media.
Return to the Ultra-Low Attachment plate and return the plate to the incubator.
Repeat steps 22-27 to feed cells every day until Day 16 and then every other day until Day 25
On day 25, prepare Cortical differentiation media.
Remove cell suspension from the plate and deposit into a 15 ml falcon tube.
Allow spheroids to settle to the bottom of the tube (~1-2 mins).
Remove the supernatant from the settled cell suspension.
Resuspend cells in Cortical differentiation media.
Return to the Ultra-Low Attachment plate and return the plate to the incubator.
Repeat steps 30-34 every four days (d29, 33, 37, 41) day until Day 43
On day 43, change the media as above, but replace with only hCS media with no additives.
Change media by sedimenting the spheroids, removing the supernatant, resuspending in hCS Media, and returning to the Ultra-Low Attachment plate every four days until the termination of the experiment.