Mar 18, 2025

Public workspaceCortical Organoid Differentiation Protocol in 96-well Plates

This protocol is a draft, published without a DOI.
  • 1University College London;
  • 2UCL, Queen Square Institute of Neurology
Adriana Cunha
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Protocol Citation: Gustavo MorroneParfitt, Adriana Cunha, Charlie Arber, Selina Wray 2025. Cortical Organoid Differentiation Protocol in 96-well Plates. protocols.io https://protocols.io/view/cortical-organoid-differentiation-protocol-in-96-w-dyjt7unn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 27, 2025
Last Modified: March 18, 2025
Protocol Integer ID: 119123
Keywords: iPSC, Organoids, Brain Organoids
Funders Acknowledgements:
TransNAT
Grant ID: MR/X008029/1
Abstract
Protocol for cortical organoid generation in 96-well plates. Based on Pasca et al. 2015 (https://doi.org/10.1038/nmeth.3415) with some modifications.
Materials
iPSC media
StemFlex medium

Media for EB generation
StemFlex medium + 10 μM Y-27632.

Media for organoid differentiation
Base media: DMEM/F12 + Neurobasal + N2 + B27 (- Retinoic Acid) + Glutamax + NEAA + Beta-mercaptoethanol.
Days 0-8: Base media + 2 μM XAV939 + 10 μM SB431542 + 100 nM LDN193189.
Days 9-24: Base media + 20 ng/ml FGFb + 20 ng/ml EGF.
Days 25-50: Base media + 20 ng/ml BDNF + Antibiotic-Antimycotic (1X).
Days 50+: Base media.
Days 70+: Base media (B27 + vitA)



Generation of Embryoid Bodies (Day -2)
Generation of Embryoid Bodies (Day -2)
2d 0h 8m
2d 0h 8m
Only start the differentiation into organoids after passaging the iPSCs at least 3 times since thawing. The day you create the EBs is considered Day -2.
Make up media: Amount50 mL of StemFlex + Concentration10 micromolar (µM) of Rock inhibitor (Y-27632).
Aspirate media from 2 wells of iPSCs.
Wash with Amount1.5 mL PBS per well.
Add Amount1 mL of accutase to each well.
Incubate at Temperature37 °C and 5% CO2 for Duration00:05:00
5m
Add Amount4 mL of media (as above) to a Falcon tube.
Take cells in accurate and add them to the Falcon tube with media.
Centrifuge at Centrifigation300 x g for Duration00:03:00
3m
Aspirate supernatant and resuspend in Amount1 mL of media.
Count cells and add Amount100 µL of cell suspension in media to each well of a 96-well V-bottom ultra-low attachment plate to achieve 15,000 cells per well (150,000 per plate).
Incubate for Duration48:00:00 .
2d
Day 0
Day 0
Make up Day 0-8 media (according to Materials section).
Remove as much spent media as possible without disrupting the EBs, wash with PBS and add Amount70 µL Day 0-8 media per well.
Days 1-8
Days 1-8
Feed the EBs daily with the Day 0-8 media until Day 8 by removing and adding Amount70 µL Day 0-8 media per well.
Days 9-24
Days 9-24
Make up Day 9-24 media (according to Materials section).
Feed EBs/organoids once every two days with the Day 9-24 media.
Days 25-50
Days 25-50
Make up Day 25-50 media (according to Materials section).
Transfer organoids to ultra-low attachment 24-well plates and incubate them on a shaker. Alternatively, transfer to T75 flasks treated with ultra-low attachment solution.
Feed organoids twice a week with Day 25-50 media.
Days 50+
Days 50+
Make up Day 50+ media (according to Materials section).
Feed organoids twice a week with Day 50+ media.
Days 70+
Days 70+
Make up Day 70+ media (according to Materials section).

Feed organoids twice a week with Day 70+ media.