Abstract
To assess the effect of multipotent stromal cells (MSC) on monocytes, 3-day cultures were performed of freshly isolated monocytes in MSC-conditioned medium (CM). As a control condition, monocytes were stimulated with low dose macrophage colony-stimulating factor (M-CSF). Monocytes were isolated from peripheral blood mononuclear cell (PBMC) populations by magnetic activated cell sorting (MACS) using CD14 microbeads.
Materials and Reagents
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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)]
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Multipotent stromal cells (MSC) from healthy donors
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Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies, catalog number: 31870-082 )
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Penicillin/streptomycin (5,000 U/ml) (Life Technologies, catalog number: 15070-063 )
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L-glutamin (200 mM) (Life Technologies, catalog number: 25030-024 )
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Fetal calf serum (FCS) (Greiner Bio-One GmbH, catalog number: 758072 )
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Phosphate buffered saline (PBS) (produced by in-house pharmacy)
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M-CSF (Pepro Tech, catalog number: 300-25 )
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CD14 MicroBeads (human) (Miltenyi Biotec, catalog number 130-050-201 )
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Antibodies
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Anti-CD14 PE (BD Biosciences)
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Anti-CD206 APC (BD Biosciences)
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Anti-CD163 PerCP-Cy5.5 (BioLegend)
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Anti-CD80 PE-Cy7 (BioLegend)
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Trypsin/EDTA (0.05%, phenol red) (Life Technologies, InvitrogenTM, catalog number: 25300-054 )
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Culture medium (see Recipes)
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Proliferation medium (see Recipes)
Equipment
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T75 culture flasks
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6-well plates (Sigma-Aldrich, catalog number: CLS3506 )
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37 °C, 5% CO2 cell culture incubator
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Microscope
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Centrifuge
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Hemocytometer (counting chamber)
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MS Columns (Miltenyi Biotec, catalog number: 130-042-201 )
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MiniMACS separator (Miltenyi Biotec, catalog number: 130-042-102 )
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MACS MultiStand (Miltenyi Biotec, catalog number: 130-042-303 )
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24-well plates (Sigma-Aldrich, catalog number: CLS3526 )
Procedure
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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.
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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.
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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.
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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.
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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.
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Plate 2.5 x 106 monocytes in a 24-wells plate in 400 µl culture medium.
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Add 600 µl of cell-free MSC-CM to the monocyte cultures.
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As control condition, add M-CSF to the monocyte cultures at a concentration of 5 ng/ml.
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Place the cultures for 3 days at 37 °C in a 5% CO2 cell culture incubator.
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Day 3: Collect monocytes.
Note: When the monocytes are attached, place the plates for 15 min on ice.
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Spin down monocytes at 350 x g for 10 min.
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Add 120 µl of PBS and count dead/alive cells using a hemocytometer.
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Analyze monocytes with flowcytometry/ isolate mRNA for follow-up analysis.
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Antibodies for flowcytometry to analyze the monocytes:
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Anti-CD14 PE
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Anti-CD206 APC
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Anti-CD163 PerCP-Cy5.5
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Anti-CD80 PE-Cy7
Recipes
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Culture medium
RPMI medium
10% FCS
P/S (100 U/ml)
L-glutamin (100 U/ml)
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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
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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.
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Melief, S. M., Schrama, C. L. M. and Roelofs, H. (2015). Monocyte-MSC Co-cultures.
Bio-protocol 5(2): e1384. DOI:
10.21769/BioProtoc.1384.