Jan 29, 2025
Large-Scale Production of Human iPSC-Derived Macrophages
- Lilia Rodriguez1,
- Zaya Mohamad1,
- Janelle Drouin-Ouellet1
- 1Faculté de pharmacie, Université de Montréal, Montréal, Canada
Protocol Citation: Lilia Rodriguez, Zaya Mohamad, Janelle Drouin-Ouellet 2025. Large-Scale Production of Human iPSC-Derived Macrophages. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo9w59v4o/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: January 28, 2025
Last Modified: January 29, 2025
Protocol Integer ID: 119288
Keywords: Culturing of iPSC, Generation of embroid bodies, Harvesting EBs , Differentiation to monocytes and macrophages
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP 000525
Disclaimer
This protocol needs prior approval by the users' institutional review board (IRB) or equivalent ethics committee(s).
Abstract
To differentiate iPSC-derived macrophages, we adopted and modified a previously published protocol. Starting from the pluripotent state, we induced mesoderm and subsequent hemogenic endothelium differentiation in preformed and size-controlled embryoid bodies (EBs) for four days by supplementing the culture medium with recombinant human bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), and stem cell factor (SCF). On day 5, floating EBs are reseeded for attachment to culture vessels in “factory” medium containing human macrophage colony-stimulating factor (M-CSF) and interleukin 3 (IL-3). A key modification to some previously published protocols is the reseeding of the EBs on growth factor-reduced (GFR) Matrigel-coated culture vessels.
Materials
Reagents
| A | B | C | |
| Reagents | Supplier | Cat# | |
| BMP4 | Peprotech | 120-05ET | |
| SCF | Peprotech | 300-07 | |
| VEGF | Peprotech | 100-20 | |
| X-VIVO 15 | Lonza | BE02-053Q | |
| M-CSF | Peprotech | 300-25 | |
| IL-3 | Peprotech | 200-03 | |
| Y-27632 dihydrochloride | Miltenyi Biotech | 130-106-538 | |
| mTeSRTM Plus | STEMCELL Technologies | 100-0276 | |
| GlutaMAX | Life Technologies | 35050061 | |
| 2-mercaptoethanol | Life Technologies | 31350-010 | |
| Matrigel® hESC-Qualified Matrix | Fisher Scientific | 08-774-552 | |
| Matrigel® Growth factor reduced | Fisher Scientific | CB-40230 | |
| Aggrewell 800 well (24 wells) | STEMCELL Technologies | 34811 | |
| Anti-Adherence Rinsing Solution | STEMCELL Technologies | 7010 | |
| Nunc™ EasYFlask™ Cell Culture Flasks | Thermo Fisher | 156499 | |
| Suspension Flat Bottom 6-well Cell Culture Plate | Sarstedt | 83.3920.500 | |
| Suspension Flat Bottom Tissue culture dish, 100 x 20 mm | Sarstedt | 83.3902.500 | |
| Strainers, 40 mm | Corning | 352340 | |
| Strainers, 70 mm | Fisher Scientific | 22-363-548 | |
| Gentle Cell Dissociation Reagent | STEMCELL Technologies | 100-0485 | |
| DPBS (Without Mg2+ or Ca2+) | Gibco | 14190144 | |
| 0.5 M EDTA | Invitrogen | 15575-038 | |
| DMEM/F12, HEPES | Gibco | 11330032 |
Complete Media: mTeSR+ with BMP4 (50 ng ml−1), VEGF (50 ng ml−1), SCF (20 ng ml−1)
Factory Media: X-VIVO 15 Supplemented with 100 ng/mL M-CSF, 25 ng/mL IL-3, 2 mM GlutaMAX, 0.050 mM b-mercaptoethanol
Macrophage media: X-VIVO 15 supplemented with 100 ng/mL M-CSF, 2 mM GlutaMAX and 0.050 mM b-mercaptoethanol
EDTA solution: 0.05 mM EDTA in DPBS (Without Mg2+ or Ca2+)
Human BMP-4 Recombinant Protein, PeproTech®Thermo Fisher ScientificCatalog #120-05ET-10UG
Recombinant Human SCFpeprotechCatalog #300-07
Recombinant Human VEGFpeprotechCatalog #100-20
X-Vivo 15 with Recomb Transf Gent L-Gln Phen Red 1 LLonzaCatalog #BE02-053Q
Recombinant Human M-CSFpeprotechCatalog #300-25
Recombinant Human IL-3peprotechCatalog #200-03
StemMACS™ Y27632Miltenyi BiotecCatalog #130-106-538
mTeSR plus media kitSTEMCELL Technologies Inc.Catalog #100-0276
GlutaMAX™ SupplementThermo Fisher ScientificCatalog #35050061
2-Mercaptoethanol (50 mM)Gibco - Thermo FischerCatalog #31350010
Corning™ Matrigel™ hESC-Qualified MatrixFisher ScientificCatalog #08-774-552
Corning™ Matrigel™ GFR Basement Membrane MatrixThermo Fisher ScientificCatalog #CB-40230
AggreWell™800 24-well Plate, 1 pack 1 Plate
STEMCELL Technologies Inc.Catalog #34811
Anti-Adherence Rinsing SolutionSTEMCELL Technologies Inc.Catalog #07010
Nunc™ EasYFlask™ Cell Culture Flasks, T75, filterThermo FisherCatalog #156499
Cell culture plate 6 well surface: Suspension flat baseSarstedtCatalog #83.3920.500
10 cm Petri Dishes SureGrip Sycamore Life SciencesCatalog #Sarstedt 83.3902.500
Falcon 40 µm Cell StrainerCorningCatalog #352340
Fisherbrand™ Sterile Cell StrainersFisher ScientificCatalog #22-363-548
Gentle Cell Dissociation ReagentSTEMCELL Technologies Inc.Catalog #100-0485
DPBS, no calcium, no magnesiumThermo Fisher ScientificCatalog #14190144
UltraPure 0.5M EDTA, pH 8.0Thermo Fisher ScientificCatalog #15575-038
Gibco™ DMEM/F-12 HEPESThermo Fisher ScientificCatalog #11330032
Culturing of iPSC
Culturing of iPSC
Maintain pluripotent hiPSC on Matrigel hESC in mTeSR+ medium.
Passage the cells in aggregates every 4 days with Gentle Cell Dissociation Reagent, followed by re-seeding with ROCK inhibitor (10 micromolar (µM) Y-27632) included in the medium for the first 24:00:00 after plating.
Authenticate cells by STR profiling upon receipt and check monthly for mycoplasma contamination by PCR.
Differentiation protocol: Preparation of Aggrewell 800 plates (Day 1)
Differentiation protocol: Preparation of Aggrewell 800 plates (Day 1)
8m
8m
Warm basal media (DMEM/F12) and complete media.
Open AggreWell plates in a biosafety cabinet.
Note
NOTE: Do not expose AggreWell plates to organic solvents, including ethanol or isopropanol.
Pre-treat wells with Anti-Adherence Rinsing Solution by adding 500 µL for a well of 24-well plate.
Centrifuge plate at 1300 x g, 00:03:00 in a swinging bucket rotor fitted with plate holders.
Note
NOTE: Plates must be well balanced. Prepare a balance plate using a standard plate filled with water to match the weight and position of the AggreWell plate.
3m
Observe the plate under a microscope to ensure that bubbles have been removed from microwells. If bubbles remain trapped in any microwells, centrifuge at 1300 x g, 00:05:00 . Aspirate Anti-Adherence Rinsing Solution from the wells.
5m
Rinse each well with 2 mL of warm basal medium. Aspirate medium from the well.
Add 1 mL of warm complete medium with ROCK inhibitor (10 μM Y-27632) to each well to be used for a 24-well plate.
Differentiation protocol: Generation of Embryoid Bodies (EBs)
Differentiation protocol: Generation of Embryoid Bodies (EBs)
1d 0h 5m
1d 0h 5m
Prepare a single-cell suspension in the desired medium (mTeSR+).
To achieve EBs size of 10,000 cells per EB, plate 4.0 x 106 in each well and add complete medium with ROCK inhibitor (10 micromolar (µM) Y-27632) to each well to achieve a final volume of 2 mL/well.
Prepare a centrifuge balance plate using a standard plate filled with water to match the weight and position of the AggreWell plate.
Pipette cells up and down gently several times to ensure even distribution of cells throughout the well. Be careful not to introduce bubbles into the microwells.
Immediately centrifuge the AggreWell plate at 100 x g, 00:05:00 to capture cells in the microwells, using the balance plate prepared.
5m
Observe the plate under a microscope to verify that cells are evenly distributed among the microwells.
Incubate the plate at 37°C with 5% CO2 and 95% humidity for 24:00:00 .
1d
Differentiation protocol: Changing medium in Aggrewell plate (Day 1-4)
Differentiation protocol: Changing medium in Aggrewell plate (Day 1-4)
Warm complete medium (no need for ROCK inhibitor at this point).
Perform a 50 - 75% medium change by removing slowly 1 - 1.5 mL of medium from each well.
Replace with 1 - 1.5 mL of the fresh complete medium by slowly pipetting down the wall of the well. Slowly dispensing the medium helps to prevent displacement of EBs/spheroids from the microwells.
Differentiation protocol: Harvesting EBs from Aggrewell plates (Day 4)
Differentiation protocol: Harvesting EBs from Aggrewell plates (Day 4)
Warm basal and completed medium.
Using a serological pipette:
Note
Alternative option: This step can be achieved by using P1000 cut tips.
Remove approximately half of the culture medium from the well.
Dispense the medium firmly back onto the surface of the plate to dislodge the EBs/spheroids from the microwells. Repeat if necessary.
Use the 40 µm strainer and a 50 ml conical tube for separation of EBs/spheroids from single cells.
Place strainer on top of the tube with the arrow pointed upward. Add 1 mL of PBS to wet the surface of the strainer.
Gently aspirate the dislodged EBs/spheroids. Pass the EB/spheroid suspension through the strainer.
Note
NOTE: The aggregates will remain on the filter; any unincorporated single cells will flow through.
Using a serological pipette, dispense 1 mL (24-well plate) of the warm basal medium across the entire surface of the well to dislodge any remaining EBs/spheroids.
Collect wash and pass over the strainer.
Repeat this wash step 3 times. Wash can be repeated until you do not see more EBs under the microscope in the AggreWell.
Invert the strainer, and place over a new conical tube of the same size. Collect the EBs/spheroids by washing with 2 – 5 mL of complete medium per well harvested.
Transfer correct number of EBs to Matrigel-GFR coated plates/flasks. Swirl the plate/flask gently to distribute the EBs around the whole surface.
Note
NOTE: EBs are differentiated in either 6-well plates (15 EBs/well), T75 (75 EBs), or T175 flasks (175 EBs).
Differentiation protocol: Differentiation to Monocytes and Macrophages: (Day 4 onwards)
Differentiation protocol: Differentiation to Monocytes and Macrophages: (Day 4 onwards)
Differentiate EBs in X-VIVO 15 Supplemented with 100 µL M-CSF, 25 µL IL-3, 2 millimolar (mM) GlutaMAX, 0.050 millimolar (mM) b-mercaptoethanol with fresh medium added weekly.
Note
Cells can be maintained in this medium for up to 6 months.
After approximately 4 weeks, collect weekly the monocytes emerging into the supernatant and replenish differentiation cultures with fresh medium. Strain (70 um) harvested cells. Use it either directly or plate onto ultra-low attachment tissue culture plates.
Differentiate cells in suspension (monocytes) into macrophages by culturing in X-VIVO 15 supplemented with 100 µL M-CSF, 2 millimolar (mM) GlutaMAX and 0.050 millimolar (mM) b-mercaptoethanol for 7 days until cells are adherent and elongated.
Note
NOTE 1: We usually plate 4.0 x 106 monocytes per 10 cm dish. Scale up or down accordingly.
NOTE 2: On day 3 after plating do a half media change of macrophage media.
Detach macrophages by washing in 1X PBS -Ca, -Mg and then incubating the plate at 37 °C for 10-15 min in 0.05 millimolar (mM) EDTA solution followed by gently scraping.
Count cells and stain them for flow cytometry or plate them for experiments in ultra-low attachment plates.
Quality control for iPSC-derived macrophages
Quality control for iPSC-derived macrophages
Flow cytometry for CD14+, CD11b+, CD45+.
Protocol references
1. Adapted from Gutbier, Cowley, Patsch et al. Int. J. Mol. Sci. 2020, 21, 4808; doi:10.3390/ijms21134808.