Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

This protocol was carried out in accordance with The National Council for the Control of Animal Experimentation (Concea) and with the approval of the Ethics and Use of Animals Committee (CEUA). C57BL/6 mice were purchased from Biotério Central of Federal University of Minas Gerais (UFMG), Belo Horizonte, Brazil. Personal protective equipment (PPE) such as laboratory coats, gloves, and eye protection should be used at all steps described in this protocol.

1. Preparation of fibroblast L-929 lineage supernatant as a source of M-CSF

1. Defrost L-929 fibroblast lineage cells (American Type Culture Collection Certified Cell Line-ATCC CCL1) and start the culture process immediately to preserve the viability.
2. In a laminar flow biosafety cabinet, using a 1,000 µL pipette, transfer the L-929 fibroblast lineage cells from the vial to a 50 mL sterile centrifuge tube.
3. Use a serological pipette to add 50 mL of sterile phosphate-buffered saline (PBS), free from calcium and magnesium.
4. Centrifuge at 300 x g, 4 °C for 10 min. Discard the supernatant.
5. Resuspend the pellet in 20 mL of Dulbecco′s modified Eagle′s medium-F12 supplemented with 10% fetal bovine serum (FBS) and 1 mL of penicillin/streptomycin (P/S) (DMEM/F12-10) using a serological pipette.
6. Transfer the resuspended cells to a T75 cell culture flask.
7. Incubate in a 37 °C, 5% CO₂ incubator until full confluence.
   NOTE: While manipulating the flask, be careful to avoid wetting the cover with solution, as this can be a source of contamination. To obtain a convenient amount of supernatant, it is necessary to replicate the cells after they cover the full surface of the cell culture flask. L-929 cells are inhibited by contact. Warm trypsin/ethylenediaminetetraacetic acid (EDTA) solution, DMEM/F12-10, and sterile PBS to 37 °C to start the cell culture expansion, as described in the following steps.
8. Once full confluence is reached, discard the supernatant from the cell culture flask, inside the laminar flow biosafety cabinet.
9. Wash the cell culture flask with 20 mL of sterile PBS using a serological pipette and discard the solution.
10. Add 10 mL of trypsin/EDTA (0.05% solution) to the cell culture flask containing the adhered cells.
11. Transfer the cell culture flask from laminar flow to a 37°C, 5% CO₂ incubator and incubate for 3 min.
12. Shake the cell culture flask to dissociate the cells from the surface of the culture flask and use a microscope to ensure that all the cells have been dissociated.
13. Inactivate the trypsin/EDTA (0.05% solution) with 10 mL of DMEM/F12-10 medium.
14. Transfer the solution using a serological pipette to a 50 mL sterile centrifuge tube.
15. Centrifuge at 300 x g at 4 °C for 10 min. Discard the supernatant.
16. Resuspend the pellet in 10 mL of DMEM/F12-10 medium.
17. Add 1 mL of the cell suspension to a T175 cell culture flask (1:10 culture).
18. Repeat the procedure to obtain ten T175 cell culture flasks.
19. Add 50 mL of DMEM/F12-10 to each cell culture flask.
20. Incubate in a 37 °C, 5% CO₂ incubator until full confluence.
    NOTE: These steps guarantee 500 mL of L-929 cell supernatant. On the day 5, after the cells have covered the T175 cell culture flask surface, the supernatant will be enriched for M-CSF and must be collected¹⁴.
21. Remove the cell culture flask from the incubator on day 5 after contact inhibition.
22. Transfer the medium to a 50 mL sterile centrifuge tube.
23. Centrifuge at 3,200 x g, at 4 °C for 10 min.
24. Collect the supernatant enriched with M-CSF and transfer the solution to a 50 mL sterile centrifuge tube. Store in a freezer at -20 °C.
    ​NOTE: Centrifugation is extremely important to remove cells and debris from the supernatants.
25. L-929 supernatant is a convenient and inexpensive source of macrophage colony-stimulating factor (M-CSF) and typically contains 5 to 10 ng/mL of M-CSF⁸. These supernatants also contain trace amounts of other cytokines and growth factors, but they do not exert a profound influence on macrophage physiology¹⁵. However, purified M-CSF can be purchased and used as an alternative to L-929 supernatants at concentrations of 1 to 2 ng/mL⁸.

2. Removal of the femur and tibia

1. Proceed with 8-week-old, C57BL-6 wild-type male mouse euthanasia by cervical dislocation after carbon dioxide asphyxiation, in accordance with normative resolution number 18 of the National Council for Animal Experimentation (Concea) and with approval of the Ethics and Use of Animals Committee (CEUA).
2. Soak the mouse with 70% ethanol solution and use sterile scissors to make a 1 cm incision along the abdomen.
3. Remove the skin until the muscle of the hind legs is fully exposed.
4. Remove the hind legs at hip height, being careful not to break the femur and tibia.
5. Put the hind legs in a conical centrifuge tube with 70% ethanol solution.
   ​NOTE: Always use pathogen-free mice. Be sure to collect both legs to achieve a greater number of cells. Step 2 is performed in a non-sterile environment (benchtop). Hence, it is important to remove the femur and tibia carefully so they remain intact to avoid bacterial contamination. The remaining steps of this procedure are carried out in a laminar flow biosafety cabinet. The time of exposure of the legs to 70% ethanol solution should be limited to 10 min. If the time is longer, bone marrow progenitors can be affected.

3. Obtain bone marrow progenitors from the lumen of the tibia and femur

1. Remove the legs from the 70% ethanol solution and transfer them to sterile PBS.
2. Using forceps and disinfectant wipes to remove all muscle and fascia. The bone should be clean after this step.
3. Use a sterile surgical scalpel blade to cut the bone epiphysis on both ends to expose the bone marrow.
4. Fill a 20 mL syringe with 10 mL of sterile PBS supplemented with 2% penicillin/streptomycin solution and connect a 26 G needle.
5. Hold the bone using anatomical dissection forceps with one hand, and use the other to insert the needle into the bone cavity. Be careful to not crush the bone with the forceps.
6. Wash out the inside of the bone with the PBS and collect the bone marrow in a sterile conical centrifuge tube. After this step, the bone cavity should appear white.
7. Centrifuge the collected cells for 10 min at 300 x g at 4 °C.
8. Discard the supernatant and resuspend the cell pellet in 1 mL of DMEM/F12-10.
9. Homogenize and add another 9 mL of DMEM/F12-10 medium to bring the cells up to a total of 10 mL.

4. Macrophage culture

1. Add 1 mL of cell progenitors to each of the 100 mm x 20 mm round plastic Petri dishes (total of 10 dishes), spreading the cells across the plates with a pipette to obtain a uniform distribution. Do not use tissue-culture treated dishes.
2. Add 9 mL of DMEM/F12-10 supplemented with 20% of the L-929 cell supernatant to each dish.
3. Incubate in a 37 °C, 5% CO₂ incubator.
4. On the 3^rd day, add 10 mL of DMEM/F12-10 medium supplemented with 20% of the supernatant of L-929 cells to each dish. In this way, the culture dishes now have a total of 20 mL of culture medium, sufficient for the growth of the cells until the end of their maturation.
   NOTES: Tissue culture-treated dishes must not be used because macrophages, as natural adherent cells marked by strong interactions with surfaces, will not easily dissociate from the dish later. Usually, cell progenitors from one mouse (two femurs) can be seeded in 10 culture dishes. Cell progenitors will transform to macrophages, which can be used from incubation day 7 to 10. The maturation confirmation is identified by changes on the morphology of macrophages. Mature macrophages are adherent and show heterogeneous morphology, explained by the emission of pseudopodia in different directions. Examples of these mature cells are shown in the representative results.

5. Harvesting macrophages

1. Discard the supernatants from all the culture dishes.
2. Wash each culture dish with 10 mL of warm (37 °C) sterile Mg²⁺ and Ca²⁺ free PBS.
3. Discard the solution from each dish.
4. Pre-warm non-enzymatic cell dissociation solution to 37 °C and add 3 mL to each dish. Incubate for 10 min in a 37 °C, 5% CO₂ incubator. Use an inverted microscope to visualize if the macrophages are dissociating from the culture dishes.
   NOTE: The use of non-tissue culture treated plastic plates should allow the easy dissociation of cells and avoid the need to scrape the cells off the plate.
5. Wash the dishes using a serological pipette with the non-enzymatic cell dissociation solution over and over, performing circular movements to confirm all the macrophages are dissociated from the dish.
6. Collect the solution of all the culture dishes together to a conical 50 mL centrifuge tube.
7. Add 10 mL of sterile pre-warmed PBS to the empty culture dishes and proceed as in step 5.4. This procedure is a repetition of step 5.4 and intends to guarantee that all the macrophages are collected.
8. Centrifuge at 300 x g, 4 °C for 10 min.
9. Discard the supernatant and resuspend the pellet with 1 mL of DMEM/F12-10. Homogenize slowly and carefully up and down using the pipette to avoid damaging the cells.
10. Prepare an aliquot of 10 µL of macrophage solution diluted with 10 µL of Trypan Blue to count the cells using the hemocytometer.
    NOTE: At day 7, each dish will produce approximately 2 x 10⁶ macrophages. This means that one mouse can produce approximately 2 x 10⁷ primary macrophages when harvested in ten plates. Since M-CSF only drives the maturation of macrophages, the procedure guarantees a population of cells that is essentially just macrophages and free from other contaminating leukocytes¹⁶.