We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.
Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.
Cells from the monocytic lineage and their terminally differentiated derivative - macrophages - exhibit a striking plasticity in regard to their biological function, leading to their involvement in such diverse processes as development, tissue repair, and immunity1. The latter is due to their phagocytic and antigen-presenting ability which places macrophages at the crossroads between the innate and adaptive immune response2. However, their capability to secrete cytokines, chemokines, growth factors, and other signaling molecules1 not only augments their immune-modulatory function but also serves as a basis for their additional functions. Attempts to mirror these diverse activation steps in the context of non-microbial mediated conditions have resulted in the M1 and M2 categories3. While this classification is not complete, it allows for a basic understanding of macrophage biology.
Due to these multifaceted capabilities it comes as no surprise that macrophages are associated with many conditions that in some way involve tissue remodeling or inflammation. Next to their fundamental role in the recognition and clearance of invading pathogens4-6, macrophages have increasingly come into focus in atherosclerosis, fibrosis, obesity, and cancer7-10. A reproducible method for the generation of human macrophages is therefore crucial for an understanding of these pathologies. Here we present a method based on the isolation of human monocytes from the peripheral blood of healthy donors by a double density gradient centrifugation technique as described previously11. In order to facilitate differentiation towards macrophages, the isolated monocytic cells are incubated in the presence of low concentrations of M-CSF and normal human serum12. To ease further handling and cell harvest, differentiation is carried out in gas permeable FEP Teflon-coated cell culture bags with a hydrophobic surface12-15. The resulting resting macrophages can be subjected to a wide range of assays as they are still capable of responding in either an M1 or M2-like fashion. Alternative methods of monocyte isolation and subsequent differentiation such as magnetic activated cell sorting (MACS) or counterflow centrifugal elutriation (CCE) have some limitations regarding the yield, cost, and time required. The protocol described herein offers the advantage that it can be carried out with standard laboratory equipment without the need for special reagents (e.g., MACS magnetic beads) or devices (e.g., CCE apparatus) and allows for the processing of large quantities of cells.
Macrophages are important effector cells of the innate immune system and display important functions in immunomodulation, antigen presentation and tissue homeostasis. Due to their remarkable plasticity, they are able to respond to different stimuli with changes of their phenotype. However, so far a lot of data regarding macrophage polarization are obtained in the murine system, although there are reports showing that only around 50% of macrophage polarization markers can be directly translated from mouse to human16. Therefore, we present here a method to obtain primary human macrophages in sufficient number and purity without the need for expensive materials, e.g., MACS magnetic beads or a counterflow centrifugal elutriation device.
Our method is based on the isolation of monocytes from PBMCs and their subsequent differentiation to macrophages in FEP Teflon-coated cell culture bags in the presence of low concentrations of M-CSF11-13. While monocytes make up less than 5 to 10% of peripheral blood leukocytes in humans, upon stimulation they are recruited to peripheral sites where they differentiate to resident tissue macrophages or dendritic cells17. The cytokine M-CSF is important for monocyte survival and drives their differentiation to macrophages18,19. So far, the M-CSF concentrations which were chosen for monocyte differentiation ranged up to 100 ng/ml, however, in our protocol we are able to obtain sufficient numbers of mature macrophages with a M-CSF concentration of only 2.5 ng/ml12,20. In addition, cells are cultured in FEP Teflon-coated cell culture bags which facilitate the detachment of the macrophages and their subsequent seeding in defined cell numbers. Since the bags can be reused several times, this further decreases the costs for the isolation process.
The macrophages obtained by this procedure are highly positive for CD45, CD14, CD11b, CD11c and show expression of the mannose receptor CD206 which argues for a population of pure, mature macrophages21,22. Especially high CD14 expression is typical for macrophages differentiated in the presence of M-CSF23. After seeding of the cells, they display a fast adherence to plastic surfaces with some cells showing a typical spindle-like morphology, while others exhibit a fried egg phenotype. This is in accordance with the observations from other authors18,22,24.
It was reported that monocyte differentiation in the presence of M-CSF leads to M2-polarized macrophages16,25. However, the macrophages isolated by our protocol are still able to respond to a wide range of stimuli including exposure to tumor cell-derived microvesicles and co-culture with tumor cells26,27 or exposure to LPS to which they react with induction of pro-inflammatory genes such as IL-1β, TNFα, Wnt5a, or various matrix metalloproteinases which are considered typical for M1-polarized macrophages3,5,28.
In conclusion, the isolation of monocytes by double density gradient centrifugation and subsequent differentiation towards macrophages in FEP Teflon-coated cell culture bags results in high numbers of macrophages without the need for technically difficult or expensive procedures. The obtained macrophages can be utilized for subsequent analysis ranging from classical activation through LPS to the co-culture with tumor cells.
The authors have nothing to disclose.
The authors would like to thank Mrs. Meike Schaffrinski for her always excellent technical assistance during the last few years.
This work was funded through the German Research Council (DFG) within the joint research group 942 (FOR942) and by the Research Program of the Faculty of Medicine, Georg-August-University Göttingen.
antibodies for immunophenotyping | Beckman Coulter | for example: CD11c-PE (IM1760), CD45-FITC (7782), IgG1-PE isotype control (A07796), IgG1-FITC isotype control (A07795) | |
BioLegends | for example: CD14-FITC (325603), CD206-PE (321105) | ||
Axiovert 200M microscope | Zeiss | ||
calcein-AM | AnaSpec | 89201 | |
combi-stopper closing cones | Braun | 4495101 | |
1x PBS, w/o Ca and Mg | Pan biotech | P04-36500 | for PBS-EDTA (1 mM) add 1 ml 0,5 M EDTA per 500ml PBS |
10x PBS, w/o Ca and Mg | Invitrogen | 14200-067 | |
EDTA (Titriplex III) | Merck | 1084211000 | prepare a 0,5 M solution in H2O, use a sterile filter |
cell dissociation buffer (enzyme-free, PBS-based) | Gibco | 13151-014 | |
Fetal calf serum (FCS) | Invitrogen | 10091148 | heat-inactivated |
Ficoll (density 1.077g/ml) | Biochrom AG | L6115 | |
FACSCanto II | BD Biosciences | ||
goat anti-mouse FITC | santa cruz | sc-2010 | |
LPS from E.coli | Sigma | L8274 | final conc: 100 ng/ml |
Multifuge 3 L-R | Heraeus | ||
Penicillin/streptomycin | Biochrom AG | A2213 | |
Percoll (density 1,131g/ml) | GE Healthcare | 17-0891-02 | |
Perfusor syringe 50ml | Braun | 8728844F | |
Phalloidin-TRITC | Sigma | P1951 | resuspend in methanol (c = 0,1 mg/ml) |
Plastic Disposable Pasteur Pipettes | LVL technologies | 2655181 | |
rh M-CSF | ImmunoTools | 11343117 | Resuspend in 500µl sterile H2O (c = 100 ng/µl), aliquot |
RPMI-1640 with Phenol Red | Gibco | 21875-034 | |
RPMI-1640 without Phenol Red | Gibco | 11835-063 | |
sterilization paper | VP group | 3KFKFS230116 | |
Trypan Blue stain (0,4% w/v) | Sigma | T8154 | |
FEP Teflon-coated cell culture bag, small | CellGenix | 72-C | |
FEP Teflon-coated cell culture bag, large | CellGenix | 197-C |