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Growth of Cerebral Organoids

 

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Welcome to this guide on generating cerebral organoids starting from H9 embryonic stem (ES) cells, adapted from the methods established by Lancaster and colleagues. In this approach, ES cells or induced pluripotent stem cells (iPSCs) are first separated into single cells from their feeder layers. They are then expanded in 96-well plates to form embryoid bodies, before moving through a series of media changes to induce neural fate and eventually promote 3D differentiation in a spinning bioreactor. Let's dive into the details to help you get your 3D brain models up and running!

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TCB-32 (Small Molecule FGF2 Replacement)

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TCB-541 (Small Molecule FGF2 Replacement)

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TCB-621 (Small Molecule FGF2 Replacement)

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ROCK inhibitor Y-27632

Selective ROCK inhibitor frequently used for the production of organoids

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Materials

  • B27 supplement (with and without vitamin A)
  • Glutamax or equivalent L-glutamine supplement
  • 2-Mercaptoethanol
  • MEM-NEAA
  • Insulin
  • Heparin sodium salt

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Media Recipes

Aseptically combine components and filter the media before use.

hES Media

Component Concentration
ROCK inhibitor Y-27632 (HB2297) 50 μM

One of:

 FGF2

4 ng/ml 
TCB-32 (HB12632) 400nM
TCB-541 (HB11050) 200nM
TCB-621 (HB17550) 100nM

Please note: FGF2 can be replaced with stable, cost-effective small molecule FGFR1 agonists TCB-32, TCB-541, or TCB-621 to enable weekend-free feeding and significantly reduce media costs. TCB compounds have not been optimised yet for this assay therefore concentrations may need adjusting. TCB concentrations are based upon the original 5x lower FGF2 concentration used by Lancaster et al..

Neural Induction Media

Component Concentration
DMEM:F12 medium 100%
N2 Supplement 1:100
Glutamax 1:100
MEM-NEAA 1:100
Heparin sodium salt 1 μg/ml

Differentiation Media

Component Concentration
DMEM:F12 medium 50%
Neurobasal medium 50%
B27 supplement without vitamin A 1:100
N2 supplement 1:200
2-Mercaptoethanol 3.5 μl/L
Insulin 1:4000
Glutamax 1:100
MEM-NEAA 1:200

Differentiation Media + Vitamin A

Component Concentration
DMEM:F12 medium 50%
Neurobasal medium 50%
B27 supplement with vitamin A 1:100
N2 supplement 1:200
2-Mercaptoethanol 3.5 μl/L
Insulin 1:4000
Glutamax 1:100
MEM-NEAA 1:200

Protocol

1
Dissociate H9 ES cells or iPSCs from their feeder layer of mouse embryonic fibroblasts (MEFs) using dispase treatment and gravity separation to generate a suspension of single cells.
2
Seed the single cells into 96-well culture plates (4500 cells / well) utilizing human ES media supplemented with FGF2 and 50 μM ROCK inhibitor Y-27632 (HB2297). Please note: FGF2 can be replaced with stable, cost-effective small molecule FGFR1 agonists TCB-32, TCB-541, or TCB-621 to enable weekend-free feeding and significantly reduce media costs.
3
Maintain the resulting embryoid bodies by replacing the media every other day for a total of 6 days (these changes can be reduced when using TCB compounds instead of FGF2).
4
On day 6, transfer the cellular aggregates to 24-well plates and switch the culture to Neural Induction Media.
5
Continue to feed the cultures with fresh Neural Induction Media every other day for an additional 5 days.
6
On day 11, carefully transfer the developing tissues into droplets of a basement membrane extract that contain Differentiation Media. Drop the tissues into droplets of cold basement extract then allow to gel at 37°C before transferring to differentiation media.
7
Allow the tissue droplets to grow in stationary culture for 4 days.
8
Following this stationary period, relocate the droplets into a spinning bioreactor vessel and maintain them in Differentiation Media + RA (containing retinoic acid) to promote further development.

References

1
Lancaster MA et al. (2013). Cerebral organoids model human brain development and microcephaly.Nature. PMID: 23995685