Summary
Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.
Abstract
The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (>10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells (DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.
Introduction
Dendritic cells (DCs) are the most powerful antigen-presenting cells (APCs) for activating naïve T cells and inducing specific cytotoxic T lymphocyte (CTL) responses against infectious diseases, allergy diseases, and tumor cells1,2,3. DCs are the primary link between innate immunity and adaptive immunity and play an essential role in immunological defense and the maintenance of immune tolerance. In the last 40 years, many researchers have sought to define the subsets of DCs and their functions in inflammation and immunity. As per those studies, DCs develop along the myeloid and lymphoid lineages from bone marrow cells. Tumor vaccines have gained significant milestones in recent years and have a promising future. Mechanically, tumor vaccines modulate the immune response and prevent tumor growth by activating cytotoxic T lymphocytes using tumor antigens. The vaccine based on DCs plays an important role in tumor immunotherapy and has been identified as one of the most promising anti-tumor therapies1,4. In addition, DCs have been widely used in the testing of new molecular-targeted drugs and immune checkpoint inhibitors5.
Researchers urgently need a high number of high-purity DCs to further study the role of DCs. However, DCs are rare in various tissues and blood, accounting for only 1% of blood cells in humans and animals. In vitro culture of bone marrow dendritic cells (BMDC) is an important method for obtaining large amounts of DC cells. Meanwhile, The Lutz protocol for generating DCs from bone marrow has been widely used by researchers6. Although the protocol is effective in obtaining DC cells, it is complex and expensive, involving the addition of high concentrations of cytokines and the lysis of red blood cells.
In this study, we report a method for isolating almost all bone marrow cells from mouse bone marrow (BM) and inducing differentiation into BMDC after 7-9 days of incubation in vitro, with a lower concentration of GM-CSF and IL-4. This procedure only takes 10 min to harvest almost all bone marrow cells and to suspend them in a complete medium. In brief, we provide an efficient and cost-effective culturing method for BMDC in this research.
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Protocol
All procedures were approved by the Nanjing Medical University Animal Care and Use Committee.
1. Isolation of bone marrow and preparation of BM cells
- Sacrifice C57BL/6 mice (18-22 g, 6-8 weeks old) via CO2 asphyxiation. Fix the mouse on the mouse operating table. Disinfect the surfaces with 70% ethanol.
- Cut the skin of the leg to expose the muscles and femoral artery. Clamp and tear off the femoral artery using two forceps, then pull the proximal end toward the abdomen.
NOTE: Do not cut the femoral artery directly. Otherwise, it will cause excessive bleeding and contaminate the field of vision. - Cut all the muscles around the femur.
- Slowly stretch the lower limbs of the mouse outward until the sound of hip joint dislocation is heard and the femoral head prolapse is visible.
- Separate the lower limbs from the body using scissors along the inside of the femoral head.
- Cut the hind legs from the end of the knee joint to obtain a free and complete femur.
- Remove the muscles attached to the femur using gauze.
NOTE: Do not tear the muscles directly. The femur of mice is delicate, its integrity must be maintained. - Immerse the femur in 75% alcohol for 2-5 min.
2. Induction culture of BMDC
- Rinse the residual alcohol with PBS. Use hemostatic forceps to clamp the middle and bottom part of the femur and another set to clamp the lower end of the femur. The hemostatic forceps are applied laterally to the femur, and the femur is separated from the epiphyseal line.
NOTE: The epiphyseal line is not easily visible until the femur fractures. This and the next steps are all performed in a sterile environment. - Use a 1 mL syringe (needle: 0.6 mm x 25 mm) to penetrate the bone marrow cavity from the epiphyseal line break and rotate the needle to penetrate the femoral head and through the bone marrow cavity. Pulse and flush the bone marrow cavity using 1 mL of the complete medium containing GM-CSF/IL-4 until the bone becomes white.
NOTE: Complete culture medium: 10% FBS, 1% penicillin-streptomycin, 55 µM β-Mercaptoethanol, 10 ng/mL GM-CSF, and 10 ng/mL IL-4. - Resuspend all the cells in 24 mL of complete culture medium (~5 x 105 cells/mL). After mixing, all the medium was seeded on a 6-well plate with 4 mL/well, then incubated at 37 °C with 5% CO2 for 2 days.
NOTE: It is not necessary to lyse erythrocytes. - Replace all the medium after 2 days with medium containing GM-CSF/IL-47. On the fourth, sixth, and eighth days, replace half of the medium with the complete medium containing 10 ng/mL GM-CSF/IL-4.
NOTE: In this step, suspended cells such as erythrocytes and lymphocytes are removed. - Take pictures every day to record cell growth. Starting from day six, harvest one well of cells per day to count the cell number and detect CD11c, CD80, and MHC II expression6,7.
3. Flow cytometric detection of the expression of CD11c, CD80, and MHC II
- After washing the DCs with PBS, resuspend 1 x 106 cells in 100 µL of FACS buffer, add 0.5 µg of anti-mouse CD16/32 antibody, and incubate for 10 min on ice to block non-specific antigen sites.
- Add 1 µg of Percp/cy5.5-CD11c, 0.5 µg of PE-CD80, and 0.04 µg of APC-MHC II antibodies, and incubate on ice for 30 min.
- Centrifuge at 1,000 x g for 3 min at 4 °C, remove the supernatant, add 1 mL of 4% paraformaldehyde, fix for 30 min at 4 °C, and re-suspend in 300 µL of FACS buffer.
- Perform flow cytometry.
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Representative Results
The 1 x 107-1.7 x 107 cells were extracted from two femurs and were re-suspended in 24 mL of medium before being planted in a 6-well plate (Figure 1A). After 2 days, non-adherent cells were removed by completely changing the culture medium. Before changing the medium, a significant number of suspended cells were observed (Figure 1B). After 3 days of culture, small cell colonies began to form. On the sixth day, the size and number of colonies increased significantly. On the seventh day, the number of cells peaked (22 x 106-27 x 106) and then dropped gradually (Figure 2A). The surface of the DC cells became rough with longer pseudopodia, exhibiting typical mature DC cell morphology (Figure 2B). Flow cytometric analysis showed that the ratio of CD11c positive to total cells was 71% on the sixth day, whereas the ratio increased to 96.1% on the tenth day (Figure 2C,2D).
To evaluate the expression of co-stimulatory molecules, CD11c, CD80, and MHC II were co-stained8. As shown as Figure 3A, the expression of CD11c, CD80, and MHC II gradually increased with increasing cultivation time from day 6 to day 10. Additionally, flow cytometric analysis showed a gradual increase in the proportions of CD11c and CD80 double-positive, CD11c and MHCII double-positive (Figure 3B,3D), and triple-positive cells (Figure 3C,3D).
Figure 1: Representative images of DCs at different culture times. (A) Flow-chart of culturing BMDC. (B) Representative images of DCs at different culture times. Bone marrow cells were suspended in 24 mL of culture medium with 10 ng/mL GM-CSF and IL-4 and seeded in a 6-well plate. BF, before changing culture medium; AF, after changing culture medium. Red arrow, DC colony. Please click here to view a larger version of this figure.
Figure 2: The number and purity of DCs at different culture times. (A) Total number of cells in all culture medium. (B) Representative images of DCs. (C,D) Flow cytometric and graphical representation of the analysis of the proportion of DCs. Positive cells were sorted by CD11c. Please click here to view a larger version of this figure.
Figure 3: The expression of co-stimulatory molecules in DCs. (A) The expression of CD11c, CD80, and MHC ΙΙ in DCs. (B) Flow cytometric analysis of the proportion of CD11c and CD80 double-positive, CD11c and MHC ΙΙ double-positive, and triple-positive cells. (C, D) Flow cytometric analysis from Day 6- Day 10 and graphical representation to show the statistical analysis. Please click here to view a larger version of this figure.
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Discussion
Humans and mice have different DC subsets, including classical DCs (cDCs, including cDC1s and cDC2s) plasmacytoid DCs (pDCs), and monocyte-derived DCs (MoDCs)9,10,11. It is generally accepted that cDC1s regulate cytotoxic T lymphocyte (CTL) responses to intracellular pathogens and cancer, and cDC2s regulate immune responses to extracellular pathogens, parasites, and allergens12. A significant number of DCs can be produced in vitro from BM progenitor cells in mice in the presence of GM-CSF and IL-46,13,14. The purity and quantity of DCs play key roles in successful DC immunotherapy. Researchers have shown that two doses of 1 x 105 to 1 x 106 DCs could elicit efficient cytotoxic T lymphocyte (CTL) responses in a xenograft model15,16,17,18. This implies that a high number of high-purity DCs need to be cultivated to further investigate the role of DCs in tumor immunotherapy.
The traditional method of obtaining bone marrow cells is to cut the two ends of the femur and rinse with the medium6,7. In this study, we innovatively used physiological anatomical structures to sever the femur. Hemostatic forceps were used to clamp the lower end of the femur and move it laterally.The epiphyseal line is physiologically weak, making them susceptible to fractures when stressed19. After the femur is separated from the epiphyseal line, a small quantity of bone remains in the marrow cavity. The needle can easily penetrate the bone into the bone marrow cavity. The method is simple to operate, protects precious bone marrow cells, and provides a cellular basis for the culture of a high number of DCs. We cultured the DCs using a combination of 10 ng/mL GM-CSF and 10 ng/mL IL-4, which contributes more to the maturation of DCs than GM-CSF alone. Labeur and Son reported that the combined use of GM-CSF and IL-4 in the medium of DCs induced higher expression of maturation markers, such as IFN-ɣ, TNF-α, and IL-6, and enhanced antigen-presenting ability7,20,21. Son et al. also proposed that the combination of GM-CSF and IL-4 promotes the maturation of DCs7,21.
In this study, we directly cultured the collected bone marrow cells without lysing the erythrocytes and separated them by gradient centrifugation, which may result in cell loss and, ultimately, to a reduction in harvested DCs. Before the medium was changed, there were several suspending cells in the culture system, including erythrocytes, T cells, B cells, and granulocytes. During co-culture, these cells may produce cytokines to promote the maturation of DCs.
In terms of limitations, this protocol only used two femurs of the mouse, excluding the smaller tibias, resulting in the loss of some mesenchymal stem cells. In short, we developed a cost-effective and efficient protocol for the isolation and generation of bone-marrow-derived dendritic cells from mice, which takes only 10 min to separate bone marrow cells. A large number of high-purity DCs were harvested after 6 days to 7 days of incubation with 10 ng/mL of GM-CSF and IL-4.
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Disclosures
All authors declare they have no conflicts of interest and that they have nothing to disclose.
Acknowledgments
This work was supported by Program of Tianjin Science and Technology Plan (20JCQNJC00550), Tianjin Health Science and Technology Project (TJWJ202021QN033 and TJWJ202021QN034).
Materials
| Name | Company | Catalog Number | Comments |
| β-Mercaptoethanol | Solarbio | M8211 | |
| 6-well plate | Corning | 3516 | |
| APC-MHC II | Biolegend | 116417 | |
| FBS | Gibco | 10100 | |
| PE-CD80 | Biolegend | 104707 | |
| Penicillin-Streptomycin | Solarbio | P1400 | |
| Percp/cy5.5-CD11c | Biolegend | 117327 | |
| PRMI-1640 | Thermo | 11875093 | |
| Recombinant Mouse GM-CSF | Solarbio | P00184 | |
| Recombinant Mouse IL-4 | Solarbio | P00196 | |
| TruStain Fc PLUS (anti-mouse CD16/32) Antibody | Biolegend | 156603 |
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