May 16, 2025

Public workspaceiPSC derived microglia (hMG) culture

DOI
dx.doi.org/10.17504/protocols.io.81wgbkeyngpk/v1
  • 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.81wgbkeyngpk/v1
Protocol Citation: Lucas Blasco-Agell, Veronica Testa, Miquel Vila 2025. iPSC derived microglia (hMG) culture. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbkeyngpk/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: 218433
Funders Acknowledgements:
ASAP
Grant ID: ASAP-020505
Abstract
iPSC derived microglia (hMG) culture
Single iPSC colonies are manually passaged onto fresh Matrigel-coated 6 cm2 plastic plate.
After 2-4 days, protocol starts by supplementing mTeSRTM1 medium with 80 ng/mL of Bone Morphogenetic Protein (BMP)-4 every day from day 0 to day 4.
From day 5, SP34 medium is employed, which consisted of StemProTM-34 SFM (Gibco‱) with 1% of P/S and 1% of Ultraglutamine (Glut; Lonza). For two days, SP34 medium is supplemented with 80 ng/ml of VEGF, 100 ng/ml of Stem Cell Factor (SCF) and 25 ng/ml of Fibroblast Growth Factor (FGF)-2.
After day 7 until day 14, SP34 is supplemented with 50 ng/ml of Fms-like tyrosine kinase 3-Ligand (Flt3-L), 50 ng/ml of IL-3, 50 ng/ml of SCF, 5 ng/ml of Trombopoietin (TPO) and 50 ng/ml of M-CSF, changing the medium at day 10.
The last step consisted on the addition to SP34 of Flt3-L, M-CSF and 25 ng/ml of GM-CSF from day 14, changing the medium every 3-4 days. At day 35, floating hMG progenitors are collected from the culture’s supernatant and passed through a 70 μm Filcon‱ Syringe-Type nylon mesh (BD Biosciences) to ensure a single-cell suspension and to remove cell clumps.
Cells are counted, centrifuged at 300xg for 10 minutes and cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco‱) with 1% of P/S and 1% of Glut, supplemented by M-CSF and 50 ng/ml IL-34.
Medium is changed with fresh factors every 2-3 days. hMG progenitors are plated onto Matrigel-coated glass coverslips in 24-well plates (25,000 cells/well), on uncoated 96-well plates (10,000 cells/well) or 6-well plates (200,000 cells/well) depending on the experiment.
hMG mature cells are cultured for one week, before being used for experiments. For treatments, 100 ng/mL of LPS or 5 ug/mL of NM are added for a total of 24 hours.