May 16, 2025

Public workspaceiPSC-derived organoid generation

DOI
dx.doi.org/10.17504/protocols.io.n92ldnxp7v5b/v1
  • Irene Fernández-Carasa1,2,
  • Veronica Testa1,2,
  • Miquel Vila3
  • 1Department of Pathology and Experimental Therapeutics, Bellvitge University Hospital- IDIBELL, 08908 Hospitalet de Llobregat, Spain;
  • 2Institute of Biomedicine of the University of Barcelona (IBUB), Carrer Baldiri Reixac 15-21, Barcelona 08028, Spain.;
  • 3VHIR-CIBERNED-ASAP
  • Vilalab Public
Miquel Vila
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DOI: dx.doi.org/10.17504/protocols.io.n92ldnxp7v5b/v1
Protocol Citation: Irene Fernández-Carasa, Veronica Testa, Miquel Vila 2025. iPSC-derived organoid generation. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldnxp7v5b/v1
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: May 16, 2025
Last Modified: May 16, 2025
Protocol Integer ID: 218435
Funders Acknowledgements:
ASAP
Grant ID: ASAP-020505
Abstract
iPSC-derived organoid generation
iPSCs are grown until 90% of confluence and 10 μM of RI is added to culture plate 4/6 hours before starting the protocol. Then, cells are dissociated in 1:1 accutase/DPBS, detached and centrifuged at 800xrpm for 5 minutes.
Cell pellet is resuspended in 1 mL of fresh mTeSR‱1, passed through a 40 μm cell strainer (Clear Line‱) and counted. Dissociated iPSCs are plated in ultra-low attachment 6-well plastic plate (Corning‱) at a density of 4 x 106 cells per well, with mTeSR‱1 medium supplemented with 10 μM of RI.
From then on, cells are kept under rotation on CO2 resistant orbital shaker (Leica) at 120xrpm and maintained under normal incubation conditions.
After 2 days, medium is replaced with SRM supplemented with LDN and SB, a dual SMAD inhibition to promote neuronal commitment.
On day 4, and every 3 days, half of SRM medium is replaced with fresh factors and the addition of SAG, Purmorphamine, CHIR and AA.
On day 12, SRM medium is replaced by NBN2B27-vitA, which consisted of Neurobasal‱ supplemented with 1x N2, 2x B27‱-vitA, 1% of P/S and 1% of Glut. N2B27-vitA is changed every other day by the addition of 100 nM of LDN, 1 μM of SAG, 2 μM of Purmorphamine, CHIR and AA, 50 ng/mL of FGF-2, and 50 ng/mL of Endothelial Growth Factor (EGF; R&D Systems), to promote conversion towards a DAn fate.
From day 22 onwards, spheres are matured to vmO in B27-vitA supplemented with BDNF, GDNF, TGF-β3, cAMP and AA. Half of the medium is changed every 2-3 days and vmO are cultured until day 45, before being used for experiments.