Cortical organoid differentiation protocol

Cortical Organoid Differentiation Protocol

Day 0: Aggregating iPSC Cells

Clean areas of differentiation from iPSC colonies (typically 2-6 days after passaging) with thin point pipette tip

Aspirate Media, wash cells/debris with 1xPBS

(If starting from colonies on feeders, first use 1-3 minute Accutase treatment to selectively remove feeder cells, and plate 1.2x the cell number to account for remaining feeders)

Dissociate colonies with Accutase by placing 1mL Accutase per well of a 6 well plate Place cells covered in Accutase for 3-5mins at 37°C 

Use cell lifter (Corning) to lift cells, pipette up/down once to capture cells and move to a 15mL conical tube.

Place 9mL of Organoid Media #1 into 15mL conical tube for a total of 10mL (9ml media + 1ml cells in Accutase).

Spin cells down with Organoid #1 media at 300g for 5 mins

While spinning cells, prep Organoid Media #1 including small molecules: Rock inhibitor (Y-27632) at 1:500, SB431542 at (1:1000), IWR1-e (1:2000).

**Always add small molecules fresh to media #1 for the amount needed for a single day of feeding. SB431542 and IWR1-e are always added to Media #1, but Rock inhibitor is only supplemented into media for the first 6 days.

Resuspend 1 million cells in 10ml of Organoid Media#1 containing small molecules. Count cells.

Plate 1 million cells (10,000 cells/well) in 96 well ultra low attachment v-bottom plate (S-bio MS- 9096VZ) in a volume of 10mL media per plate. Using using a p200 multi-channel pipette add 100ul/well. Let cells settle by gravity.

Place cells back in 37C and do not disturb for 2 days.

Day 2: Half-volume media change (Media#1 including Rock inhibitor)

Remove 50uL media/well (so as not to disturb the cell aggregates) and replace with 100ul/well Organoid Media#1 media +Rock inhibitor + SB431542 + IWR1-e using a p200 multi-channel pipette.

Day 4 and 6: Media changes (Media#1 including Rock inhibitor)

Remove and replace 100ul/well Organoid Media#1 media +Rock inhibitor + SB431542 + IWR1-e using a p200 multi-channel pipette

Day 8-18: Media changes (Media#1 without Rock-inhibitor)

Remove and replace Media #1 with SB431542 + IWR1-e every other day, no longer supplementing with Rock-inhibitor (typically M/W/F – it’s ok to skip weekends)

Day 18-35: Move organoids into 6 well low attachment plate in Media#2 and place on orbital shaker

Use widebore p200 pipette tips on multichannel pipette to transfer organoids into 6 well ultra-low attachment plate (Corning 3471)

Place on orbital shaker in 37C and shake cells to increase oxygenation and prevent organoids aggregating together (ORBIShaker CO₂ model BT4001 – 85rpm)

Remove and replace 2mL Media#2 on M/W/F schedule

Day 35-70: Media #3

Remove and replace 3mL Media#3 on M/W/F schedule

Day 70 and beyond: Media #4

Remove and replace 4mL Media#4 on M/W/F schedule
 

CORTICAL ORGANOID DIFFERENTIATION MEDIA RECIPES