Cultivating Cerebral Organoids

This protocol is intended as a guide only, for full experimental details please read the reference provided.

In Brief

Lancaster et al. describe a protocol to generate cerebral organoids from H9 ES cells.

On Day 0 ES cells or iPSCs were dissociated from mouse embryonic stem cells (MEFs) and separated to make single cells. Cells were then plated in 96-well plates in human ES media containing 50 μM Y-27632. Embryoid bodies were fed every other day for 6 days.

On Day 6 cells are transferred to 24 well plates and grown in induction media. They are fed every other day for 5 further days.

On Day 11 tissue is transferred to droplet Matrigels containing differentiation media. After 4 days of stationary growth the tissue droplets are moved to a spinning bioreactor, and grown in differentiation media containing retinoic acid (RA).

Graphic Protocol Overview of Cerebral Organoid Genesis

Cocktails

hES Media		Neural Induction Media		Differentiation Media		Differentiation Media + RA
FGF (233-FB)	4 ng ml^-1	DMEM:F12 medium		DMEM:F12 medium and neurobasal containing N2 supplement (1:200)		Same as differentiation +
Y-27632 (Cat.No. 1254)	50 μM	N2 Supplement	1:100	B 27 supplement w/o vitamin A (equivalent to N21-MAX Vitamin A Free (AR009))	1:100	B 27 supplement with vitamin A (equivalent to N21-MAX with Vitamin A (AR008))	1:100
		Glutamax (equivalent to GlutaminePlus (B90210)		2 Mercapotoethanol	3.5 μl/L	Retinoic Acid (Cat.No. 0695)
		MEM-NEAA		Insulin (Cat.No. 3435)	1:4000
		Heparin sodium salt (Cat.No. 2812)	1 mg/ml	Glutamax	1:100
				MEM-NEAA