Dec 03, 2025
Version 2
Cortical Organoid Differentiation Protocol in 96-well Plates V.2
- Gustavo Morrone Parfitt1,
- Adriana Cunha2,
- Charlie Arber2,
- Selina Wray2
- 1University College London;
- 2UCL, Queen Square Institute of Neurology
Protocol Citation: Gustavo Morrone Parfitt, Adriana Cunha, Charlie Arber, Selina Wray 2025. Cortical Organoid Differentiation Protocol in 96-well Plates. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly62regx9/v2Version created by Adriana Cunha
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: April 28, 2025
Last Modified: December 03, 2025
Protocol Integer ID: 189060
Keywords: iPSC, Organoids, Brain Organoids, well plates protocol for cortical organoid generation, cortical organoid differentiation protocol, cortical organoid generation, well plates protocol, well plate
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 medium
StemFlex medium
Medium for EB generation (Day -2)
StemFlex medium + 10 μM Y-27632 (Rock inhibitor).
Medium for organoid differentiation
Base medium: DMEM/F12 + Neurobasal + N2 + B27 (without Vitamin A) + GlutaMAX + NEAA + 2-mercaptoethanol.
Days 0-8: Base medium + 2 μM XAV939 + 10 μM SB431542 + 100 nM LDN193189.
Days 9-24: Base medium + 20 ng/ml FGFb + 20 ng/ml EGF.
Days 25-50: Base medium + 20 ng/ml BDNF + Antibiotic-Antimycotic (1X).
Days 50+: Base medium + Antibiotic-Antimycotic (1X).
Days 70+: Base medium (B27 containing Vitamin A) + Antibiotic-Antimycotic (1X).
Before start
Passage the iPSCs at least 2-3 times since thawing.
Generation of Embryoid Bodies (Day -2)
2d 0h 8m
The day you generate the embryoid bodies (EBs) is considered Day -2.
Make up Day -2 medium (according to Materials section).
Aspirate the spent medium from 2 wells of iPSCs.
Wash with 1.5 mL PBS per well.
Add 1 mL of accutase to each well.
Incubate at 37 °C and 5% CO2 for 00:05:00
5m
Add 4 mL of Day -2 medium to a Falcon tube.
Take cells in accutase and add them to the Falcon tube with medium.
Centrifuge at 300 x g for 00:03:00
3m
Aspirate supernatant and resuspend cells in 1 mL of Day -2 medium.
Count cells and seed 15,000 cells in 100 µL of Day -2 medium per well (i.e. 150,000 cells/ml) of a 96-well V-bottom ultra-low attachment plate (S-BIO).
Leave in incubator at 37 °C and 5% CO2 for 48:00:00 .
2d
Day 0
Make up Day 0-8 medium (according to Materials section).
Remove as much spent medium as possible without disrupting the EBs, wash once with PBS and add 70 µL of Day 0-8 medium per well.
Days 1-8
Feed the EBs daily with the Day 0-8 medium until Day 8 by removing and adding 70 µL Day 0-8 medium per well.
Days 9-24
Make up Day 9-24 medium (according to Materials section).
Feed EBs/organoids once every two days with the Day 9-24 medium.
Days 25-50
Make up Day 25-50 medium (according to Materials section).
If desired, transfer organoids to ultra-low attachment 24-well plates and incubate them on a shaker.
Feed organoids twice a week with Day 25-50 medium. If kept in 96-well plates, continue feeding organoids every two days.
Days 50+
Make up Day 50+ medium (according to Materials section).
Feed organoids twice a week with Day 50+ medium. If still in 96-well plates, continue feeding organoids every two days.
Days 70+
Make up Day 70+ medium (according to Materials section).
Feed organoids twice a week with Day 70+ medium. If still in 96-well plates, continue feeding organoids every two days.