Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

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 cells^1,2,3. 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 therapies^1,4. 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.

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

1. 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.
2. 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.
3. Cut all the muscles around the femur.
4. 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.
5. Separate the lower limbs from the body using scissors along the inside of the femoral head.
6. Cut the hind legs from the end of the knee joint to obtain a free and complete femur.
7. 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.
8. Immerse the femur in 75% alcohol for 2-5 min.

2. Induction culture of BMDC

1. 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.
2. 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.
3. 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.
4. 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.
5. 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 expression^6,7.

3. Flow cytometric detection of the expression of CD11c, CD80, and MHC II

1. 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.
2. 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.
3. 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.
4. Perform flow cytometry.

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 colonie...

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 DC...

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