Feb 03, 2025
Generation of human iPSC-derived tricultures
- 1Institut Imagine
- Team Deleidi
Protocol Citation: Maria Jose Perez J. 2025. Generation of human iPSC-derived tricultures. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzknz8vx1/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: November 06, 2024
Last Modified: February 03, 2025
Protocol Integer ID: 111674
Funders Acknowledgements:
ASAP
Abstract
Generation of human iPSC-derived tricultures
Materials
Final neuronal maturation medium: 1:1 DMEM-Ham’s
F-12 and neurobasal medium, 0.5% N2, 1% B27 without vitamin A, 1% P/S, and 1%
GlutaMAX, supplemented with 200 μM AA, 1 μM purmorphamine, and 100 ng/mL FGF8
(PeproTech) + with 20 ng/mL BDNF (PeproTech) and 1
mM dibutyryl-cyclic AMP (dcAMP, Sigma‒Aldrich).
Generation of human iPSC-derived tricultures
Generation of human iPSC-derived tricultures
Differentiate iPSCs into cortical neurons as per the established protocol ("Generation of human iPSC-derived cortical neurons, astrocytes, and microglia") until they reach 14 days in vitro (DIV).
At 14 DIV, dissociate neurons using Accutase for 5' at 37C
Plate dissociated neurons onto glass coverslips pre-coated with Matrigel 1:30 in DMEM 1X for 30' at 37C
Maintain plated neurons in neuronal maturation medium supplemented with 20 ng/mL BDNF and 1 mM dcAMP
After four days in culture, seed iPSC-derived astrocytes onto the neurons at a 3:1 ratio (neurons:astrocytes)
Maintain neuron-astrocyte cocultures in the final neuronal maturation medium
Three days after adding astrocytes, add iPSC-derived microglial precursors to the coculture at a 3:1:1 ratio (neurons:microglia:astrocytes)
Maintain the tricultures in the final neuronal maturation medium for an additional seven days in final neuronal maturation medium supplemented with 100 ng/mL IL-34, 100 ng/mL M-CSF, and 10 ng/mL GM-CSF