Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages

Bone marrow encloses both hematopoietic and mesenchymal stem cell populations that are self-renewable and can be differentiated into various cell lineages. Hematopoietic stem cells in the bone marrow give rise to myeloid and lymphoid lineages¹. Mesenchymal stem cells produce osteoblasts (bone), adipocytes (fat), or chondrocytes (cartilage)². These cells have multiple applications in the field of cell biology and tissue engineering, including gene therapy^3,4. Progenitor cells present in the bone marrow differentiate into specific cell types in the presence of lineage-specific growth factors. Erythropoietin promotes the proliferation of erythroid progenitor cells, granulocyte colony-stimulating factor (G-CSF) stimulates the growth of neutrophil colonies, and thrombopoietin regulates the production of platelets as a few examples of lineage-specific growth factors⁵. Cell surface antigen labeled FACS and magnetic-activated cell sorting (MACS) are well-established methods for isolation and purification of the specific bone marrow-derived cell types⁶.

Though neonatal studies are advancing toward finding the causes of neonatal deaths and addressing the complications during premature births, direct therapeutic development remains an unmet medical need. Smith and Davis stated, "Pediatric patients remain therapeutic orphans"⁷. There are several challenges, such as small samples, lifelong effects of the outcome, and ethical issues in obtaining consent in clinical studies of neonates⁸. Hence, there is a high demand for in vivo and in vitro study models specific to neonates to achieve translational relevance. Because of the similarities between anatomical and tissue levels, short gestational periods, and litter sizes, rodents are the most studied mammalian model system.

Here, we describe a detailed, highly feasible, and reproducible procedure for isolating bone marrow from 7-9-day-old mouse pups and their ability to differentiate into macrophages. However, a variety of cell lineages could be achieved with the use of distinct differentiation signals. We also demonstrate the presence of cell surface markers and the presence of in vitro phagocytic activity expected for bone marrow-derived macrophages (BMDMs).

All procedures were approved by the West Virginia Institutional Animal Care and Use Committees and were performed following the recommendations of the Guide for the Care and Use of Laboratory Animals by the National Research Council. C57BL/6J mouse pups were used for this study. The details of all the reagents and equipment used are listed in the Table of Materials.

1. Media preparation

1. Prepare 3 mL of MEM culture media supplemented with 10% FBS, 2 mM glutamine, 25 mM HEPES, and penicillin (100 U/mL)/streptomycin (100 µg/mL) in a 5 mL centrifuge tube, and keep it on ice.
2. If needed for storing bone marrow progenitors in liquid nitrogen, prepare freezing media containing 10% DMEM, 80% FBS, and 10% DMSO.
3. For complete DMEM, prepare DMEM supplemented with 10% FBS, 2 mM glutamine, 25 mM HEPES, and penicillin (100 U/mL)/streptomycin (100 µg/mL).
4. For macrophage differentiation, prepare DMEM supplemented with 10% FBS, 2 mM glutamine, penicillin (100 U/mL)/streptomycin (100 µg/mL), and 10% L929-cell supernatant^9,10,11.

2. L929-cell supernatant preparation

1. Thaw L929 cells stored in liquid nitrogen and transfer to a 15 mL centrifuge tube containing 5 mL of complete DMEM. Centrifuge at 350 x g for 5 min at room temperature.
2. Resuspend the cells in 5 mL of complete DMEM media and seed them into a T25 tissue culture flask at the density of 2×10⁵ cells. Count the cells with an automated cell counter using 0.4% trypan blue. Incubate at 37 °C with 5% CO₂ until they reach 70% confluency.
3. To detach the cells, first wash them with 5 mL of phosphate-buffered saline (PBS). Aspirate the PBS and replace it with 2.5 mL 0.05% trypsin-EDTA.
4. Incubate at 37 °C with 5% CO₂ for 5 min and add 5 mL of complete DMEM.
5. Collect the cells and centrifuge at 350 x g for 5 min at room temperature.
6. Resuspend the cell pellet in 15 mL of complete DMEM media and transfer it to a T75 flask.
7. Incubate at 37 °C with 5% CO₂ until the cells reach 90% confluency, and then collect the medium and centrifuge at 500 x g for 5 min at 4 °C. Filter the media containing M-CSF through a 0.45 µm filter and freeze at -80 °C in 5 mL aliquots.

3. Animal preparation

1. Under a biosafety cabinet, carefully separate the 7-9-day old C57BL/6J mouse pups (a litter of 6 pups) from the dam and place them in a separate cage.
2. Soak a cotton ball in 2 mL of veterinary-grade isoflurane in a bell jar or other containment chamber.
3. Place a pup in the chamber and close the lid. Monitor the neonate for approximately 90 s to ensure it becomes motionless and unconscious.
4. Quickly remove the pup and decapitate it with sharp scissors (following institutionally approved protocols) before the pup can regain consciousness.
   NOTE: Neonates do not breathe sufficiently deep for euthanasia by isoflurane inhalation alone.
5. Ensure to close the lid of the container after each pup is removed. The same cotton ball can be used for 5-7 pups.

4. Isolation of neonatal bone marrow

1. Sterilize the body of the neonate with 70% ethanol.
2. “Using forceps and fine-tipped surgical scissors, make an incision between the
   abdomen and hind legs. Remove the skin by pulling toward the foot of the hind limbs.
3. Scrape the connective tissue and muscle attached to the bones using sharp-edged forceps.
4. Cut the tibia and femur, as shown in Figure 1, with scissors to detach and remove. Using forceps, place these bones in the media tube on ice. Repeat the process for each pup.
5. Transfer the bones with the help of forceps to a 40 µm strainer with a collection tube. Crush the bones and release the marrow by pressing with a 3 mL syringe plunger against the strainer.
6. Centrifuge the cell suspension at 350 x g for 5 min at 4 °C.
7. Aspirate the supernatant and resuspend the cells by gentle pipetting in 0.2% NaCl, a hypotonic solution for the lysis of erythrocytes. Immediately add an equal volume of 1.6% NaCl to bring the solution to isotonic.
8. Centrifuge at 350 x g for 5 min at 4 °C and remove the lysis solution.
9. Resuspend in complete DMEM for immediate use or freezing media for storage and count the cells using a hemocytometer or automated cell counter.
   NOTE: For the present study, this method yielded 6.55 (± 1.44) × 10⁶ bone marrow cells/pup (Figure 1E).
10. Store the cells at -80 °C in freezing media or use them immediately as described below.
    NOTE: Because of the age, bones are transparent and clear enough to visualize the marrow through them. It is important to be careful while pulling the bones as the application of slight pressure on them may be enough to release the marrow. The marrow in the neonatal bones is in the form of liquid rather than an intact thread found in adult bones. It is difficult to collect the marrow if it escapes during the collection of the bones.

5. Differentiation of neonatal bone marrow-derived macrophages

1. Seed 2 × 10⁷ bone marrow cells per T75 flask in 10 mL of complete DMEM that contains 10% L929-cell supernatant (2.5 mL) as a source of M-CSF. Incubate the culture dishes at 37 °C with 5% CO₂ for 5 days. Add 2 mL of L929-conditioned media on day 3 of differentiation.
2. Observe the cells under the microscope daily using an inverted microscope and imaging system with 20x magnification. Upon differentiation, macrophages should appear adherent, elongated, and heterogenous (Figure 2).
3. To harvest the macrophages for downstream assays, aspirate the differentiation media.
4. Wash the cells with 5 mL of PBS to remove non-adherent cells and serum proteins that will interfere with detachment. Aspirate the PBS.
5. Harvest the cells by adding 5 mL of 0.05% trypsin-EDTA to the flask. Place the culture flask at 37 °C with 5% CO₂ incubator for 5 min and add 5 mL of DMEM.
6. Pipette the cells to detach and centrifuge at 350 x g for 5 min at room temperature.
7. Dissociate the cell pellet in 1 mL of complete DMEM, and count and check the cell viability using trypan blue. BMDMs isolated using this method are 7.05 (± 2.52) ×10⁵ cells/pup (Figure 1E).
   NOTE: Observe the presence of spindle-shaped cells that begin to appear on day two of differentiation (Figure 1B, red arrows). If the color of the media turns yellow, indicating it is spent, add more L929-conditioned media on day 4 of differentiation; complete replacement of media is not recommended.

6. Immunolabeling and flow cytometric analysis

1. Block non-specific antibody labeling of the BMDMs (2x10⁵/label) by treating with 10 µL/10⁷ cells of FcR blocking reagent according to manufacturer recommendations in a volume of 100 μL/label flow cytometry buffer (PBS supplemented with 2 mM EDTA and 0.5% bovine serum albumin). Keep on ice for 10 min.
   NOTE: The cells can be blocked in bulk or in individual wells or tubes. All subsequent steps indicate amounts and volumes per individual cell marker label at a final volume of 100 μL.
2. Wash the cells by adding 200 µL of flow cytometry buffer and centrifuge at 525 x g for 7 min at room temperature.
3. Remove the supernatant and seed the cells in a U-bottom 96 well plate at a density of 5 × 10⁵ cells per well. Stain cells by adding FVS780-Live/Dead cell-counting dye diluted to 3,000-fold and 0.625 µL of BV786-CD11b, PE-F4/80, and AlexaFluor488-CD206 in 100 µL of flow cytometry buffer either individually or in combination^12,13,14,15. Incubate the cells for 45 min on ice covered with foil.
4. Add 100 µL of flow cytometry buffer and wash the cells by centrifuging at 525 x g for 7 min at room temperature.
5. Aspirate the supernatant and vortex the cell pellet gently. Add 100 µL of 0.4% paraformaldehyde to each well and incubate overnight at 4 °C.
6. Wash the cells by adding 100 µL of flow cytometry buffer and pellet by centrifugation at 525 x g for 7 min at room temperature.
7. Resuspend the cells in 400 µL of flow cytometry buffer and analyze using a flow cytometer.

7. In vitro assay to evaluate the phagocytic efficacy of neonatal bone marrow-derived macrophages

1. Resuspend the BMDMs in complete DMEM without phenol red or antibiotics and seed them at a density of 2 × 10⁵/ quadrant in a 35 mm quad dish at a volume of 500 µL.
2. To prepare the bacterial inoculum at a multiplicity of infection (MOI) of 25 or other desired MOI, remove a pre-calculated stock of Escherichia coli O1:K1:H7 stored at -80 °C.
3. Aliquot the desired volume of bacteria into a 1.7 mL microcentrifuge tube. Wash the bacteria by adding PBS to a total volume of 1 mL. Pellet the bacteria by centrifugation at 2,000 x g for 5 min at room temperature. Repeat the washing step.
4. Resuspend the bacterial pellet in 50 µL of PBS and add green fluorescent pH-sensitive dye to a final concentration of 500 µM. This is a pH-sensitive dye with no fluorescent signal at neutral pH and fluoresces brightly in acidic environments, such as acidified phagosomes, during the process of phagocytosis.
5. Incubate the bacterial cells for 20 min in the dark for dye conjugation.
6. Wash the bacteria four times with 1 mL of PBS by centrifugation at 1000 × g for 5 min at room temperature.
7. Resuspend the bacteria in 500 µL of complete DMEM without phenol red and add to the BMDM cultures.
8. Incubate the BMDMs at 37 °C in 5% CO₂ incubator for 4 h and then add 200 ng of a cell-permeable red fluorescent dye that stains the lysosomes.
   NOTE: Phagocytic bacteria can be visualized by fluorescent microscopy.

Using the method outlined in this study, 25 to 37 million bone marrow cells can be successfully isolated from a litter size of five C57BL/6 mouse pups. This method has been validated with litter sizes ranging from 5 to 7 pups. The minimum age for isolation in our experiments has been 7 days old. Depending on the litter size and the number of cells required for the experiment being less than a million, researchers could attempt this protocol for mice younger than 7 days old. In the presence of L929-cell supernatant as a source of M-CSF, bone marrow cells were differentiated into macrophages in 5 days (Figure 2). Formation of spindle-shaped cells was observed on the second day of differentiation (Figure 2B), nearly half of the cells showed a spindle shape on day three (Figure 2C), and most of the cells adhered and formed elongated spindle shapes on day five of differentiation (Figure 2D). The yield of bone marrow-derived macrophages (BMDMs) with this method was 2.5-5 million cells from 5 pups of 7 days of age.

To characterize the differentiated BMDMs phenotypically, cells from 5-day cultures were immunolabeled for CD11b, CD206, and F4/80. Forward/side scatter and single-marker gating schemes can be seen in Supplementary Figure 1. The results from the flow cytometry analysis demonstrated that 76.4% of the BMDMs are positive for both CD11b and F4/80 (Figure 3A). The F480-CD11b⁺ population is consistent with the approximate number of cells positive for CD206 (Figure 3B). The latter is generally considered a marker consistent with M2-like macrophages, and while the abundance of CD206 labeling can range as much as two-fold more, a higher proportion of F4/80 labeling is consistent with the ability of M-CSF to promote differentiation to an M1-like phenotype^16,17(Figure 3).

We further evaluated the capability of the differentiated neonatal BMDMs to phagocytose bacteria and traffic them to acidified compartments as a functional measure. The bacteria labeled with a pH-sensitive dye should only fluoresce green when phagocytosed and trafficked to acidified compartments. Abundant green fluorescent bacteria phagocytosed inside the BMDMs were detected following infection (Figure 4A). The green fluorescence further localizes with red fluorescence indicative of acidified lysosomes (Figure 4B,C). The phagocytosis and appearance of green pH-sensitive dye-positive neonatal BMDMs were also observed throughout the 4 h infection (Figure 4D and Supplemental video). The functional activity displayed here is consistent with M1-like inflammatory macrophage activity.

Figure 1: Isolation of 7-day-old C57BL/6J mouse pups bone marrow. (A) Hind limb bones of 7-day-old pups in a 100 mm dish. (B) Hind limb of a 7-day-old pup after removing the skin and subcutaneous tissue; black dotted lines indicate the place to cut for the bone marrow extraction. (C) The neonatal hind limb processed bones with marrow prior to crushing. (D) The neonatal hind limb processed bones after crushing using a syringe plunger in a strainer. (E) The mean number ± standard error of bone marrow (BM) and bone marrow-derived macrophage cells obtained from three independent experiments. Please click here to view a larger version of this figure.

Figure 2: Differentiation of BMDMs from 7-day-old neonatal bone marrow cells. Bone marrow cells on day 1 (A), day 2 (B), day 3 (C), and day 5 of differentiation (D). Red arrows on panel B indicate spindle-shaped adhered cells on day 2 of differentiation. Scale bars: 200 µm. Please click here to view a larger version of this figure.

Figure 3: Detection of murine macrophage markers using flow cytometric analysis. Differentiated neonatal bone marrow-derived macrophages showing the expression of F4/80 and CD11b (A) or CD206 and CD11b (B) surface antigens. Please click here to view a larger version of this figure.

Figure 4: Phagocytosis of pH-sensitive dye-labeled E. coli by neonatal BMDMs. (A) Bone marrow-derived macrophages showing phagocytosed pH-sensitive dye-labeled (green) E. coli after 4 h of infection. (B) BMDMs labeled with stain for acidified lysosomes (red). (C) Merged image of panels (A) and (B). Scale bars: 20 µm. (D) An overlay of green fluorescent bacteria on macrophages shown in phase contrast. Magnification in (D) is the same as that in (A). Please click here to view a larger version of this figure.

Supplementary video. A 4 h video of live cell imaging of green florescent bacteria by neonatal BMDMs as in Figure 4 panel A. Please click here to download this Video.

Supplementary Figure 1: Flow cytometry analysis of BMDMs. (A) BMDMs were first gated on FSC and SSC to remove debris and doublets. Single staining of F4/80 (B), CD11b (C), and CD206 (D) is shown. Please click here to download this File.

Research involving neonatal mouse models can present a number of challenges. Neonates have a developing immune system that is unique compared to adults⁸. As such, data generated from adult animal models should not be assumed to apply to newborns, and several published works have articulated this idea well^18,19. Therefore, neonatal-specific models and sources of cells are necessary to study the intricacies of the early-life immune response. However, due to the size, sensitivity, and delicate nature of neonatal mice, the quantity of biological samples can be limited, and the collection process can be time-consuming. This protocol establishes a simple and time-efficient procedure for extracting bone marrow from neonatal mice.

Adult mouse bones are large enough to insert a needle and easily flush out the marrow. Conversely, accessing neonatal bone marrow with a needle is challenging due to its size and fragility. Some studies have employed enzymatic digestion with collagenase type II and dispase²⁰. Here, due to the soft and tiny nature of neonatal bones, we adopted a crushing method to release the bone marrow. After purification, depending on the research application of interest, these cells can be differentiated into specific cell types.

Neonates exhibit distinct phenotypic macrophage populations²¹. This study elucidated the differentiation of BMDMs from day 7-9 aged pups' bone marrow cells without using enzymes. Critical steps involved in the methodology include the overall delicate nature of neonatal mouse pups, the collection of the small bones in a manner that does not permit the release of bone marrow, and locating the proper place in the tibia and femur to make scissor cuts. We observed that neonatal BMDMs are more sensitive than adult BMDMs in culturing, and a longer duration of isolation of bone marrow from neonates prohibited their differentiation capability. Hence, extraction from more than 7 pups at a time is not suggested. Additionally, the addition of BMDM differentiation media on day two of differentiation also resulted in poor outcomes such as poor differentiation and lower viability.

This protocol used L929 cell supernatant as the source of M-CSF for the differentiation of bone marrow cells into macrophages. L929 cells are murine fibroblasts known to produce high amounts of M-CSF¹⁰. Macrophages are expected to be exposed to other substances secreted in the L929 supernatant, along with M-CSF. Commercial purified M-CSF can also be used for macrophage differentiation; however, we have not directly compared it with the use of L929 culture supernatant. It is also important to note that L de Brito Monteiro et al. observed distinct metabolic profiles between macrophages generated using L929 and commercial M-CSF²².

This method established a time-saving, economical approach without the use of any digestive enzymes. The technique yielded reliable isolation of neonatal bone marrow that resulted in clear differentiation of neonatal BMDMs. Isolated neonatal BMDMs can be further used to study neonatal macrophage dynamics during various infections and the regulation of inflammation by these cells. Limitations of the method include a learning curve for the initial establishment and adaptation to the pulling process of the small bones, which eventually improves with experience and minimizes the duration of the process with increased viability of the cells. The litter sizes of pups are an additional consideration for the number of macrophages that can be differentiated depending on experimental need.