Growth of Cerebral Organoids

Table of Contents

• Introduction
• Materials
• hES Media
• Neural Induction Media
• Differentiation Media
• Differentiation Media + RA
• Protocol
• References

Introduction

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!

Materials

• H9 ES cells or iPSCs
• Basement membrane extract (generic alternative)
• FGF2
• ROCK inhibitor Y-27632 (HB2297)
• DMEM:F12 medium
• Neurobasal medium
• N2 supplement
• B27 supplement (with and without vitamin A)
• Glutamax or equivalent L-glutamine supplement
• 2-Mercaptoethanol
• MEM-NEAA
• Insulin
• Heparin sodium salt
• Retinoic acid

hES Media

Component	Concentration
FGF2	4 ng ml-1
ROCK inhibitor Y-27632 (HB2297)	50 μM

Neural Induction Media

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

Differentiation Media

Component	Concentration
DMEM:F12 medium & neurobasal containing N2 supplement	1:200
B27 supplement w/o vitamin A	1:100
2-Mercaptoethanol	3.5 μl/L
Insulin	1:4000
Glutamax	1:100
MEM-NEAA

Differentiation Media + RA

Component	Concentration
Differentiation Media (see above)
B27 supplement with vitamin A	1:100
Retinoic Acid

Protocol

1. Dissociate H9 ES cells or iPSCs from their feeder layer of mouse embryonic fibroblasts (MEFs) to generate a suspension of single cells.
2. Seed the single cells into 96-well culture plates utilizing human ES media supplemented with FGF2 and 50 μM ROCK inhibitor Y-27632 (HB2297). 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.
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.
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

• Lancaster MA et al. (2013) Cerebral organoids model human brain development and microcephaly. Nature 501: 373. PMID: 23995685