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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).
|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|