Cultivating Cerebral Organoids

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

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).

https://resources.tocris.com/images/protocols/cerebral-organoid-protocol-workflow.jpg

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      
https://resources.tocris.com/images/protocols/cerebral-organoids-protocol-timeline.jpg

Reference

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

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