Sep 29, 2025
Human iPSC-derived MgBr assembloids
- Maria Jose Perez J.1
- 1Institut Imagine
- Team Deleidi
Protocol Citation: Maria Jose Perez J. 2025. Human iPSC-derived MgBr assembloids. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl46x7ogo5/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: September 25, 2025
Last Modified: September 29, 2025
Protocol Integer ID: 228165
Keywords: derived mgbr assembloids human ipsc, mgbr assembloids human ipsc, human ipsc, ipsc
Funders Acknowledgements:
ASAP
Abstract
Human iPSC-derived MgBr assembloids
Troubleshooting
Seed 9,000 iPSCs into each well of a Nunclon‱ Sphera‱ 96-well microplate (Cat. number 174925; Thermo Fisher Scientific) to generate embryoid bodies (EBs).
Culture EBs from day 0 to day 5 in dual SMAD inhibition medium consisting of mTeSR‱ Plus supplemented daily with 1% NEAAs, 1% GlutaMAX, 100 μM β-mercaptoethanol, 200 nM LDN 193189 hydrochloride (LDN), 10 μM SB, and 50 μM Y-27632 (ROCK inhibitor).
On day 6, switch to cortical induction medium consisting of 1:1 DMEM-Ham’s F-12 and Neurobasal medium supplemented with 1% NEAAs, 1% GlutaMAX, 1% N2, 1% B27 without vitamin A, 100 μM β-mercaptoethanol, 20 ng/mL FGF2, and 20 ng/mL EGF.
On day 7, embed EBs in Matrigel at a 3:2 ratio of Matrigel to medium to form the embedding mixture.
Wash EBs with fresh medium, embed in Matrigel in a 6-well ultralow-attachment plate, and incubate the Matrigel–EB mixture for 30 minutes at 37°C.
Maintain organoids in cortical induction medium until day 14.
From days 14 to 30, maintain organoids in differentiation medium consisting of 100% Neurobasal medium supplemented with 1% NEAAs, 1% GlutaMAX, 1% N2, 1% B27 without vitamin A, 1% PS, 100 μM β-mercaptoethanol, 2.5 μg/mL insulin, 200 μM ascorbic acid, 20 ng/mL BDNF, and 1 mM dcAMP.
On day 20, dissociate Matrigel from organoids and place plates on an orbital shaker to increase oxygenation and improve medium distribution.
On day 30, transfer individual brain organoids into wells of a Nunclon‱ Sphera‱ 96-well microplate to generate iPSC-derived MgBr assembloids.
On day 10 of in vitro differentiation, seed 2 × 10^5 predifferentiated iPSC-derived microglia on top of each organoid in a final volume of 200 μL assembloid maturation medium.
Prepare assembloid maturation medium with 100% Neurobasal medium supplemented with 1% NEAAs, 1% GlutaMAX, 1% N2, 1% B27, 1% PS, 100 μM β-mercaptoethanol, 20 ng/mL BDNF, 1 mM dcAMP, 10 ng/mL GDNF, 100 ng/mL IL-34, 100 ng/mL M-CSF, and 10 ng/mL GM-CSF.
Change maturation medium twice daily for 3 days without removing microglia in suspension.
Transfer assembloids to a 6-well ultralow-attachment plate after 3 days.
After 2 additional days, place MgBr assembloids on an orbital shaker and maintain in maturation medium until day 45 (15 DPI) or day 65 (35 DPI) of differentiation.