Materials and Reagents

Peripheral blood mononuclear cells (PBMC) [isolated from a buffy coat from a healthy donor using Ficoll-Paque (own pharmacy) density gradient (1.077 g/cm³)]
Multipotent stromal cells (MSC) from healthy donors
Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies, catalog number: 31870-082 )
Penicillin/streptomycin (5,000 U/ml) (Life Technologies, catalog number: 15070-063 )
L-glutamin (200 mM) (Life Technologies, catalog number: 25030-024 )
Fetal calf serum (FCS) (Greiner Bio-One GmbH, catalog number: 758072 )
Phosphate buffered saline (PBS) (produced by in-house pharmacy)
M-CSF (Pepro Tech, catalog number: 300-25 )
CD14 MicroBeads (human) (Miltenyi Biotec, catalog number 130-050-201 )
Antibodies
1. Anti-CD14 PE (BD Biosciences)
2. Anti-CD206 APC (BD Biosciences)
3. Anti-CD163 PerCP-Cy5.5 (BioLegend)
4. Anti-CD80 PE-Cy7 (BioLegend)
Trypsin/EDTA (0.05%, phenol red) (Life Technologies, Invitrogen^TM, catalog number: 25300-054 )
Culture medium (see Recipes)
Proliferation medium (see Recipes)

Equipment

T75 culture flasks
6-well plates (Sigma-Aldrich, catalog number: CLS3506 )
37 °C, 5% CO₂ cell culture incubator
Microscope
Centrifuge
Hemocytometer (counting chamber)
MS Columns (Miltenyi Biotec, catalog number: 130-042-201 )
MiniMACS separator (Miltenyi Biotec, catalog number: 130-042-102 )
MACS MultiStand (Miltenyi Biotec, catalog number: 130-042-303 )
24-well plates (Sigma-Aldrich, catalog number: CLS3526 )

Procedure

MSC cultures are generated from aspirated bone marrow: bone marrow-derived mononuclear cells are isolated using Ficoll-Paque density gradient (1.077 g/cm³) and plated at 1.3 x 10⁵ cells/cm² in proliferation medium.
Cultures were incubated at 37 °C and 5% CO₂. After 3-4 days non-adherent cells were removed, and medium was refreshed every 3-4 days until confluence was reached. The MSC monolayer was detached using trypsin/EDTA, and cells were reseeded at 4,000 cells per cm² for further expansion.
MSC (passage 2-5) are cultured at confluency in a T75 culture flasks in 10 ml culture medium for at least 3 days without refreshing the medium.
The medium is aspirated from the cultures (= MSC-CM). Spin down MSC-CM at 350 x g for 10 min to obtain cell-free MSC-CM.
Day 0: Isolate monocytes from freshly obtained PBMC from a buffy coat by MACS using CD14 microbeads and MS Columns according to the manufacturer’s instructions.
Plate 2.5 x 10⁶ monocytes in a 24-wells plate in 400 µl culture medium.
Add 600 µl of cell-free MSC-CM to the monocyte cultures.
As control condition, add M-CSF to the monocyte cultures at a concentration of 5 ng/ml.
Place the cultures for 3 days at 37 °C in a 5% CO₂ cell culture incubator.
Day 3: Collect monocytes.
Note: When the monocytes are attached, place the plates for 15 min on ice.
Spin down monocytes at 350 x g for 10 min.
Add 120 µl of PBS and count dead/alive cells using a hemocytometer.
Analyze monocytes with flowcytometry/ isolate mRNA for follow-up analysis.
1. Antibodies for flowcytometry to analyze the monocytes:
  1. Anti-CD14 PE
  2. Anti-CD206 APC
  3. Anti-CD163 PerCP-Cy5.5
  4. Anti-CD80 PE-Cy7

Recipes

Culture medium
RPMI medium
10% FCS
P/S (100 U/ml)
L-glutamin (100 U/ml)
Proliferation medium
Dulbecco’s modified Eagle’s medium-low glucose (DMEM-LG)
10% FCS
P/S (100 U/ml)
L-glutamin (100 U/ml)

References

Melief, S. M., Schrama, E., Brugman, M. H., Tiemessen, M. M., Hoogduijn, M. J., Fibbe, W. E. and Roelofs, H. (2013). Multipotent stromal cells induce human regulatory T cells through a novel pathway involving skewing of monocytes toward anti-inflammatory macrophages. Stem Cells 31(9): 1980-1991.