Apr 28, 2026
Generation of Neural Stem Cells derived Organoids (NSCO)
- 1Core Unit pluripotent Stem Cells and Organoids - Berlin Institute of Health @ Charite, Berlin, Germany
Protocol Citation: Valeria Fernandez Vallone, Judit Kuechler, Harald Stachelscheid 2026. Generation of Neural Stem Cells derived Organoids (NSCO) . protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4j6kzlo5/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: March 09, 2023
Last Modified: April 28, 2026
Protocol Integer ID: 78417
Keywords: organoid, pluripotent stem cell, brain, neural stem cells, neural stem cell organoid, generation of neural stem cell, neural stem cell, differentiating neural stem cell, preserving organoid size, developing cell type, such as astrocyte, astrocyte, nsco growth beyond day, organoids in motion, organoid, organoid size, nsco growth, intended application of the nsco model, cell, cellular composition, dynamic culture on an orbital shaker, nsco model, individual nsco, cell type
Funders Acknowledgements:
European Union’s Horizon 2020 research and innovation program
Grant ID: 825161
Bundesministerium für Bildung und Forschung, Germany
Grant ID: 16GW0191
Abstract
This protocol describes two methods for differentiating neural stem cells (NSCs) into neural stem cell organoids (NSCOs). Cells are first seeded and cultured under the same conditions until day 15. From day 15 onwards, the protocol diverges into either static culture or dynamic culture on an orbital shaker.
The choice between static and dynamic culture depends on the intended application of the NSCO model. The static protocol is suitable for medium- to high-throughput applications, in which individual NSCOs are maintained in multi-well plates and each organoid can be treated as a technical replicate. Under static conditions, NSCOs can be maintained for up to approximately 30 days while preserving organoid size and cellular composition.
The dynamic protocol supports NSCO growth beyond day 30 by maintaining the organoids in motion, which provides additional space for expansion and improves nutrient distribution up to a certain size. Depending on the application, optimal endpoints are typically between 70 and 90 days. Longer differentiation periods allow the emergence of later-developing cell types, such as astrocytes. However, NSCOs cultured under dynamic conditions are grown in bulk, making this format less suitable for high-throughput applications.
Materials
LABORATORY EQUIPMENT AND CONSUMABLES
Use sterile material
- 1/5/10 mL pipettes
- 15/50 mL conical tubes
- 10/200/1000µL tips and micropipettes
- 200 µL bore tips
- 8 channel pipette (30 - 300 µL)
- 96-Well round bottom clear ultra low attachment microplates e.g. Corning, Catalog #7007
- Non-culture treated 6-well plates
- 5/10 mL Combi tips and multi-step dispenser
- Microscope
- Centrifuge
- Class II Biosafety Cabinet
- Cell counting equipment
- Aspirator pump with disposable pipette
- Orbital shaker at 37°C or biorreactor solutions for dynamic culture e.g. ClinoStar (CelVivo)
MEDIA AND REAGENT
- Neurobasal™ MediumThermo Fisher ScientificCatalog #21103049
- PSC Neural Induction MediumThermo FisherCatalog #A1647801 includes Neural Induction Supplement (Thermo Fisher, A16477-01) (see appendix) and Neurobasal Medium
- Advanced DMEM/F-12Thermo Fisher ScientificCatalog #12634-010
- Penicillin/StreptomycinInvitrogen - Thermo FisherCatalog #15140-122
- GlutaMAX™ SupplementThermo Fisher ScientificCatalog #35050061
- ROCK inhibitor (Ri) Y-27632STEMCELL Technologies Inc.Catalog #72308
- B27 supplement without retinoic acid (50x)Gibco - Thermo Fisher ScientificCatalog #17504044 (see appendix)
- Human EGF, Animal-Free Recombinant ProteinpeprotechCatalog #AF-100-15 (see appendix)
- Human FGF-basic (FGF-2/bFGF) (154 aa) Recombinant ProteinpeprotechCatalog #100-18B (see appendix)
- Recombinant Human NT-3peprotechCatalog #450-03 (see appendix)
- Human/Mouse/Rat BDNF Recombinant ProteinpeprotechCatalog #450-02 (see appendix)
- DPBS, no calcium, no magnesiumThermo FisherCatalog #14190144
- BSAMerck MilliporeCatalog #A9543-5G
- Trypan blueThermo Fisher ScientificCatalog #T10282
SUPPORTING PROTOCOLS
1 Neural Stem Cell (NSC) sphere formation and expansion
4w 5d 1h 56m
Experimental timeline
Figure 1. NSCO generation timeline. At day 15 NSCO can continue growing in static conditions (endpoint day 30) or be switched to dynamic conditions (endpoint between day 70-90 as desired).
Day 0
Prepare Neural Expansion Medium (NEM) as describes in Table 1.
Table 1. Preparation of Neural Expansion Medium (NEM)
| A | B | C | D | E | |
| Component | Final concentration | 500 mL | 100 mL | 50 mL | |
| Advanced DMEM/F12 | 0.5X | 245 mL | 49 mL | 24.5 mL | |
| Neurobasal Medium | 0.5X | 245 mL | 49 mL | 24.5 mL | |
| Neural Induction Supplement | 50X | 10 mL | 2 mL | 1 mL | |
| Pen/Strep | 100X | 5 mL | 1 mL | 0.5 mL |
Store medium at +4ºC. Use within one week from the date of preparation.
15m
Thaw NSCs from quality controlled bank as described in hiPSC derived Neural Stem Cells (NSCs) generation, banking and quality control.
15m
Resuspend NSCs in 2 mL NEM supplemented with 10 micromolar (µM) ROCK inhibitor and perform a cell count using Trypan Blue for viability assessment.
Note
NSCs viability upon thawing should be > 70-80% for good sphere formation.
5m
Seed NSCs at a concentration of 7500 cells per well of a 96 round bottom well clear ultra low attachment microplate in 100 µL Neural Expansion Medium (NEM), containing 10 micromolar (µM) ROCK inhibitor per well.
Note
- Preparing a NSCs suspension of 75,000 cells in 10 mL NEM is sufficient for 1 plate seeding.
- NSCs batches and hiPSC of origin may influence cell survival and sphere formation. Increasing ROCK inhibitor concentration to 50µM or alternatively using survival cocktail CEPT are options for improving sphere formation and decreasing cell death.
15m
Centrifuge the plate with NSCs at 300 x g, 00:01:00 to induce faster cell sedimentation
1m
Incubate the plate in a 37 °C , 5% CO2 incubator for 24:00:00 .
1d
Day 1
Prepare Neural Medium according to Table 2.
Table 2. Preparation of Neural Medium (NM)
| A | B | C | D | E | |
| Component | Stock concentration | Final concentration | 100 mL | 50 mL | |
| Neurobasal Medium | NA | NA | 95.6 mL | 47.8 | |
| Glutamax | 100X | 1X | 1 mL | 0.5 mL | |
| B27 (50X) | 50X | 1X | 2 mL | 1 mL | |
| human EGF | 10 µg/mL | 20 ng/mL | 200 µL | 100 µL | |
| human FGF-basic | 10 µg/mL | 20 ng/mL | 200 µL | 100 µL | |
| Pen/Strep | 100X | 1X | 1 mL | 0.5 mL |
Store medium at +4ºC. Use within one week from the date of preparation.
See Appendix for reagent / stock solutions preparation.
15m
Observe sphere formation under the microscope.
Note
- If no compact sphere is formed, discard the plate and start again.
- If some satellite spheres are formed around the main one, leave the plate additional 24:00:00 without perturbation. Spheres may fuse within this time and differentiation can proceed, counting as day 1 when the media is changed. If spheres do not fuse, discard the plate and start again.
5m
It is recommended to follow up NSCO formation and growth taking pictures and assessing size increase which should be exponential until day 15.Change to NM without ROCK inhibitor. For this, carefully aspirate 50 µL media and add 100 µL of fresh NM media per well.
Note
- It is recommended to perform medium removal using a 8 channel pipette very carefully tilting the plate and fill the medium with multi-step dispenser with medium speed.
- Verify upon each aspiration that spheres were not carried with the medium to avoid loosing many NSCO by the end of the process.
- It is recommended to follow up NSCO formation and growth taking pictures and assessing size increase which should be exponential until day 15.
15m
Day 2 and 4
Perform media changes by aspirating 100 µL and adding 100 µL of fresh NM media.
3d
Day 6
Prepare Neural Differentiation Medium (NDM) supplemented with BDNF and NT3 according to Table 3.
Table 3. Preparation of Supplemented Neural Differentiation Medium (NDM)
| A | B | C | D | E | |
| Component | Stock concentration | Final concentration | 100 mL | 50 mL | |
| Neurobasal Medium | 95.8 mL | 47.9 | |||
| Glutamax | 100X | 1X | 1 mL | 0.5 mL | |
| B27 (50X) | 50X | 1X | 2 mL | 1 mL | |
| BDNF | 20 µg/mL | 20 ng/mL | 100 µL | 50 µL | |
| NT3 | 20 µg/mL | 20 ng/mL | 100 µL | 50 µL | |
| Pen/Strep | 100X | 1X | 1 mL | 0.5 mL |
Store medium at +4ºC. Use within one week from the date of preparation.
See Appendix for reagent / stock solutions preparation.
15m
Perform a media change by aspirating 100 µL and adding 150 µL fresh supplemented NDM.
15m
Day 6 to day 15
Perform media change every other day by aspirating 150 µL medium from each well and adding 150 µL per well of fresh supplemented NDM.
Note
Optional: a media change adding 200-250 µL final volume can be performed on fridays for a weekend-free intervention. For next media change (i.e. Monday) aspirate the corresponding volume (200 µL - 250 µL ).
2w
Day 15
Decision point to proceed NSCO culture in static or dynamic conditions. Choose one and follow the correspondent step case.
Static culture4 steps
From Day 15 onwards, continue performing media changes every other day by aspirating 150 µL of medium from each well and adding 150 µL per well of fresh Supplemented NDM. Keep the culture under static conditions (no movement) in an incubator at 37 °C and 5% CO2.
Note
Optional: a media change adding 200-250 µL final volume can be performed on fridays for a weekend-free intervention. For next media change (i.e. Monday) aspirate the corresponding volume (200 µL - 250 µL ).
2w 1d
It is recommended to use NSCO under static conditions until day 30 of culture.
If extension is needed from day 45 onwards, medium change can be performed as described in step 13, using NDM supplemented or not with NT3 and BDNF. Incubate NSCO at 37 °C and 5% CO2.
Expected result
Figure 2. Example image of NSCO culture in static conditions at day 20. NSCO derived from BIHi250-A hiPSC line.
Appendix
1h 30m
REAGENTS PREPARATION
Neural Induction Supplement (10 mL )
Thaw Neural Induction Supplement overnight at 4 °C .
Aliquot in working volumes (e.g. 1 mL ) and store at -20 °C . Do not freeze-thaw more than once.
B27 supplement (50X)
Thaw B27 supplement Overnight at 4 °C .
Make aliquots in working volumes (e.g. 1 mL ) and store at -20 °C . Do not freeze-thaw more than twice.
Human EGF
To prepare a 10 µg/mL stock solution from 50 µg Human EGF, dissolve 50 µg EGF in 5 mL PBS containing 0.1% BSA. Make aliquots in working volumes (e.g. 100 µL ) and store at -80 °C . Do not freeze-thaw more than twice.
Human FGF-basic
To prepare a 10 µg/mL stock solution from 50 µg Human FGF-basic, dissolve 50 µg FGF-basic in 5 mL PBS containing 0.1% BSA. Make aliquots in working volumes (e.g. 100 µL ) and store at -80 °C . Do not freeze-thaw more than twice.
NT3
To prepare 20 µg/mL stock from 100 µg NT3, dissolve 100 µg NT3 in 5 mL PBS containing 0.1% BSA. Make aliquots in working volumes (e.g. 100 µL ) and store at -80 °C . Do not freeze-thaw more than twice.
BDNF
To prepare 20 µg/mL stock from 100 µg BDNF, dissolve 100 µg BDNF in 5 mL PBS containing 0.1% BSA. Make aliquots in working volumes (e.g. 100 µL ) and store at -80 °C . Do not freeze-thaw more than twice.
1h 30m