Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.
Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity.
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to naïve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.
Dendritic cells (DCs) are the most important specialized antigen-presenting cells of our immune system. Immature DCs (iDCs) reside in the skin or in mucosal tissues and are therefore among the first immune cells to interact with invading pathogens. DCs represent the bridge between the innate and the adaptive immune system1, since they can activate T- and B-cell responses following pathogen detection. Furthermore, they contribute to pro-inflammatory immune responses because of the secretion of high amounts of cytokines, such as IL-1β, IL-6, and IL-12. DCs also activate NK cells and attract other immune cells to the site of infection by chemotaxis.
DCs can be divided into immature dendritic cells (iDCs) and mature dendritic cells (mDCs)2 based on their morphology and function. After the recognition of foreign antigens by one of the many pattern recognition receptors (e.g., toll-like receptors, C-type lectins, or complement receptors) abundantly expressed on the cell surface, iDCs undergo major changes and start to mature. During this maturation process, receptors for antigen capture are down-regulated, whereas molecules essential for antigen presentation are up-regulated3. Mature DCs up-regulate the major histocompatibility complexes I and II (MHC I and II), co-stimulatory molecules like CD80 and CD86, which are essential for antigen presentation and activation of T-lymphocytes. Additionally, the expression of chemokine receptor CCR7 on the cell surface is induced, which enables the migration of DCs from peripheral tissues to the lymph nodes. The migration is facilitated by the "rolling" of DCs along a chemokine ligand 19 (CCL19/MIP-3b) and chemokine ligand 21 (CCL21/SLC) gradient to the lymph nodes4-6.
Following migration, mDCs present the processed antigen to naïve CD4+ and CD8+ T cells, thus initiating an adaptive immune response against the invading pathogen7. This interaction with T cells in the lymph nodes is also associated with the spread of the virus8. Other in vitro studies revealed that DCs efficiently capture and transfer HIV to T cells and that this transmission results in a vigorous infection9-12. These experiments highlight that in vivo HIV exploits DCs as shuttles from the periphery to the lymph nodes. During antigen presentation, DCs secrete key interleukins that shape the differentiation of effector T helper cells, and therefore, the outcome of the entire immune response against the microbe is determined at this very interaction. Apart from type 1 (Th1) and type 2 (Th2) effector T cells, other subsets of CD4+ T helper cells (e.g., type 17 (Th17) and type 22 (Th22) T cells) have been described, and their induction and function have been investigated thoroughly. DCs are furthermore involved in the generation of regulatory T cells (Tregs)13,14. These cells are immunosuppressive and can stop or down-regulate induction or proliferation of effector T cells and are thus crucial for developing immunity and tolerance.
Human conventional DCs (cDCs) comprise several subsets of cells with a myeloid origin (i.e., Langerhans Cells (LCs) and dermal and interstitial DCs) or a lymphoid origin (i.e., plasmacytoid DCs (pDCs)). For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used as a model for dermal DCs. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by the addition of interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) to monocytes isolated from healthy donors12,15-18. Dendritic cells can also be directly isolated from dermal or mucosal biopsies, or can even be developed from CD34+ hematopoietic progenitor cells isolated from umbilical cord blood samples obtained ex utero. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. After incubation for 5 days, human monocytes under specific conditions are differentiated into iDCs and are ready for experimental procedures in a non-clinical setting.
Ethics statement: Written informed consent was obtained from all participating blood donors by the Central Institute for Blood Transfusion & Immunological Department, Innsbruck, Austria. The use of anonymized leftover specimens for scientific purposes was approved by the Ethics Committee of the Medical University of Innsbruck.
1. Enrichment of Peripheral Blood Mononuclear Cells (PBMCs)
2. Isolation of Monocytes by Anti-human CD14 Magnetic Particles
3. Stimulation of Isolated Monocytes with IL-4 and GM-CSF
After centrifugation of anti-coagulated blood using a sucrose cushion, peripheral blood mononuclear cells (PBMCs) are enriched in an interphase on top of the density gradient medium (Figure 1). After the PBMCs are drawn off, FACS analysis is performed to characterize the different cell populations within the PBMCs using lineage markers (e.g., CD3 for T lymphocytes, CD14 for monocytes, and CD19 for B lymphocytes). Figure 2A shows the results of a representative FACS analysis of PBMCs collected and stained after density gradient centrifugation. Out of the gated population (Figure 2A, upper plot), we detected 4.03% B lymphocytes, 28.20% monocytes, and 67.62% other cells (Figure 2A, middle plot), of which 43.62% were T lymphocytes (Figure 2A, lower plot).
PBMCs are then incubated with CD14 magnetic particles and, following incubation on a magnet, the negative fraction containing the peripheral blood lymphocytes (PBLs) is analyzed by FACS using the selection of abovementioned lineage markers. Compared to PBMCs, we measured similar percentages of B lymphocytes (4.65%, Figure 2B, middle plot), higher percentages of other cells (88.61%, Figure 2B, middle plot), and strongly reduced percentages of monocytes (6.59%, Figure 2B, middle plot). Characterization of the positive fraction (Figure 3) demonstrated a high separation efficiency, since the purity of the isolated cells is over 97% (Figure 3, left plot), of which 99.56% (Figure 3, right plot) expressed high levels of CD14 on the cell surface.
IL4 and GM-CSF stimulation of monocytes for 5 days results in differentiation into monocyte-derived dendritic cells, which are comparable to dermal dendritic cells in morphology, behavior, and receptor expression19. FACS analysis of monocyte-derived dendritic cells on day 5 shows a homologous population (Figure 4, left plot) expressing high levels of CD11b, CD11c (not shown), and the C-type lectin DC-SIGN. No expression of the DC maturation marker CD83 is detected (Figure 4, right plot), and cells also lose the monocyte marker CD14 (not shown).
Figure 1: Separation of blood components by density gradient centrifugation. PBMCs are enriched in an interphase on top of the separation medium by density gradient centrifugation. Layers before (left) and after (right) centrifugation are shown. Please click here to view a larger version of this figure.
Figure 2: Flow cytometric analyses of PBMCs (A) and PBLs (B). (A) PBMCs are harvested, stained for lineage markers (mouse-anti-human CD3, CD14, and CD19 antibodies), and analyzed by flow cytometry. Dot plots of a representative result are shown. (B) The negative fraction containing PBLs is characterized by lineage markers (mouse-anti-human CD3, CD14, and CD19 antibodies) and analyzed by flow cytometry. Dot plots of a representative result are shown. Please click here to view a larger version of this figure.
Figure 3: Flow cytometric analyses of monocytes. The isolated monocytes (positive fraction) are characterized using lineage markers (mouse-anti-human CD3, CD14, and CD19 antibodies) and analyzed by flow cytometry. Dot plots of a representative result are shown.
Figure 4: Flow cytometric analyses of monocyte-derived dendritic cells. Monocytes are stimulated for 5 days with IL4 and GM-CSF and stained for characteristic DC markers, like CD11b, CD11c (not shown), the C-type lectin DC-SIGN, and the maturation marker CD83. Dot plots of a representative result are shown.
This protocol describes the generation of monocyte-derived dendritic cells (MDDCs) through the isolation of human monocytes from anti-coagulated blood using a magnetic nanoparticle-based assay. In this protocol, the centrifugation steps are performed upstream the cell isolation procedure, which leads to an enrichment of the PBMC fraction. Although cells are lost during centrifugation, to overlay the content of a whole blood pack on density gradient medium would require 200-300 ml of the density gradient medium and therefore cannot be considered cost efficient. Additionally, the enrichment of PBMCs by centrifugation results in a cleaner cell population, which positively influences the attachment of the anti-human CD14 magnetic particles during the isolation process. Magnetic-nanoparticle isolation of monocytes results in a viable monocyte population, which can be further used for in vitro differentiation to various DC subsets or macrophages.
Advantages of this technique compared to other protocols used in the past, like adherence-mediated purification on tissue culture or gelatin-coated plastic dishes, are the high purity and homogeneity of the isolated cell population, as well as the convenience and speed. In general, it is important to work quickly but thoroughly when working with cells in order to avoid prolonged exposure to non-physiological conditions. The anti-human CD14 magnetic particles used in this protocol are optimized for the positive selection or depletion of CD14-bearing leukocytes; therefore, the magnetic nanoparticles bind directly to the cells of interest and are not removed after the procedure. This could be an issue that researchers must address before certain experiments or analyses are performed, since results could be influenced or altered by cell-bound magnetic nanoparticles. Alternatively, negative selection assays are available, which are designed to deplete unwanted cells and to keep the cells of interest untouched. For the generation of MDDCs, the anti-human CD14 magnetic nanoparticles used to isolate monocytes present no complication because, during the differentiation into MDDCs, cytokine-stimulated monocytes down-regulate the CD14 receptor. Thus, the magnetic nanoparticles are removed without further elution or detaching procedures, and the MDDCs are tested negative for CD14 receptor expression by flow cytometry. Since culture medium containing FCS is used during the differentiation in this protocol, MDDCs obtained here are for research use only. For MDDCs in clinical settings, special media without the need of serum have been developed. In addition, autologous plasma can be collected after the first centrifugation step (step 1.1.4), heat-inactivated, and used in the culture medium. Besides autologous plasma, commercially-available human plasma can also be added to the culture medium to avoid possible background reactivity in long-term assays due to xenogeneic proteins in FCS.
Alternatively, dendritic cells can be directly isolated from dermal or mucosal biopsies or can be developed from CD34+ hematopoietic progenitor cells isolated from umbilical cord blood samples obtained ex utero.
Nonetheless, MDDCs are the most widely-used model for dendritic cells, since the direct isolation of DCs from biopsies is more complex to perform. It can also lead to inefficient cell numbers that often vary due to differences in the donor quality. Similar problems can occur when DCs are generated from CD34+ stem cells isolated from cord blood.
For future applications, inducible pluripotent stem cells (iPSCs) could be used instead of CD14-magnetic bead isolation of monocytes from blood. The advantages of using iPSCs are that not only DC subsets, but all hematopoietic cells of interest, can be generated from one donor (T cells, B cells, and NK cells) and that patient-specific iPSCs can be used to characterize the interactions of autologous immune cells in a personalized manner.
The most critical step during the isolation of monocytes is the density gradient centrifugation step, since accurate separation of the red and white blood cells is only achieved when the brake of the centrifuge is turned off. This determines the outcome of the down-stream isolation efficiency.
In conclusion, this protocol is a very fast, efficient, and cost-effective way to generate a viable and homogenous monocyte-derived dendritic cell population through the isolation of human monocytes from anti-coagulated blood.
The authors have nothing to disclose.
We would like to thank our technician Karolin Thurnes, Divison of Hygiene and Medical Microbiology, and Dr. Annelies Mühlbacher and Dr. Paul Hörtnagl, Central Institute for Blood Transfusion and Immunological Department, for their valuable help and support regarding this manuscript. We thank the Austrian Science Fund for supporting this work (P24598 to DW, P25389 to WP).
APC Mouse Anti-Human CD19 Clone HIB19 | BD Biosciences | 555415 | |
APC Mouse Anti-Human CD83 Clone HB15e | BD Biosciences | 551073 | |
BD Imag Anti-Human CD14 Magnetic Particles | BD Biosciences | 557769 | |
BD Imagnet | BD Biosciences | 552311 | |
BSA (Albumin Fraction V) | Carl Roth | EG-Nr 2923225 | |
Costar 6 Well Clear TC-Treated Multiple Well Plates | Costar | 3506 | |
Density gradient media: Ficoll-Paque Premium | GE Healthcare Bio-Sciences | 17-5442-03 | |
Dulbecco’s Phosphate Buffered Saline (D-PBS) | Sigma-Aldrich | D8537 | |
Falcon 10mL Serological Pipet | Corning | 357551 | |
Falcon 25mL Serological Pipet | Corning | 357525 | |
Falcon 50mL High Clarity PP Centrifuge Tube | Corning | 352070 | |
Falcon Round-Bottom Tubes | Corning | 352054 | |
FITC Mouse Anti-Human CD3 Clone HIT3a | BD Biosciences | 555339 | |
Ghost Dye Violet 510 (Cell Viability Reagent) | Tonbo biosciences | 13-0870 | |
GM-CSF | MACS Miltenyi Biotec | 130-093-862 | |
Heat Inactivated FBS (Fetal Bovine Serum), EU Approved Origin (South America) | Gibco | 10500-064 | |
Hettich Rotanta 460R | Hettich | — | |
IL-4 CC | PromoKine | C-61401 | |
Isolation buffer: BD IMag Buffer (10X) | BD Biosciences | 552362 | |
L-Glutamine solution | Sigma-Aldrich | G7513 | |
Microcentrifuge tubes, 1,5 ml, SuperSpin | VWR | 211-0015 | |
PE Mouse Anti-Human CD14 Clone M5E2 | BD Biosciences | 555398 | |
PE Mouse Anti-Human CD209 Clone DCN46 | BD Biosciences | 551265 | |
RPMI-1640 medium | Sigma-Aldrich | R0883 | |
UltraPure 0.5M EDTA, pH 8.0 | Invitrogen | 15575020 |