Here we present a protocol for the isolation of BMMs from SD rats, called the secondary adherence method.
With a decrease of bone mineral density, bones are more likely to fracture, thus negatively affecting a patient’s quality of life. The growth and development of bones are mainly regulated by osteoblasts and osteoclasts. It has been widely accepted that osteoclasts are derived from bone marrow monocyte-macrophage cells (BMMs). BMMs and other hematopoietic stem cells are located in the bone marrow cavity. Therefore, isolating single stable BMMs from different and heterogeneous cell populations is a huge challenge. Here we present a protocol for the isolation of BMMs from SD rats, called the secondary adherence method. Adherent cells cultured for 24-48 h in primary culture were collected. Flow cytometric analysis showed that approximately 37.94% of the cells were CD11b/c+ (monocyte-macrophage surface antigen). Tartrate resistant acid phosphatase (TRAP) staining and western blot analysis demonstrated that BMMs could differentiate into osteoclasts in vitro. The above findings suggested that the secondary adherence cells could be considered as a suitable cellular model for osteoclast differentiation research.
It has been reported that monocyte-macrophage lineage cells existing in the bone marrow can differentiate into blood monocytes and tissue macrophages1,2. The above cells, which can differentiate into osteoclasts to balance bone growth and development, are commonly used as a cell model to induce osteoclasts in vivo3,4. Bone marrow is a special tissue containing several different types of cells, which include but are not only limited to bone marrow mesenchymal stem cells, bone marrow monocyte-macrophage cells (BMMs), hematopoietic stem cells, endothelial cells, and immune cells. In fact, several previous studies suggested that adherent cells rushed out of the bone marrow cavity of the long bone could differentiate into osteoblasts, osteoclasts, chondrocytes, or adipocytes5,6,7,8. Although, different isolation and culture methods have been used to produce different homogeneous cell populations, there are still great challenges in isolating and culturing BMMs from a variety of different cell types.
Several methods have been developed to extract bone marrow mononuclear macrophages (BMSCs). However, the majority of these methods are complex9,10,11. For example, density gradient centrifugation requires a specialized kit and the operation is time-consuming and cumbersome. This method is suitable for the isolation of BMMs from high-volume blood samples, but not from bone marrow samples9,12,13. In addition, extracting tissue samples using collagenase digestion is a complex and time-consuming procedure; this method is not recommended for the isolation of BMMs from bone marrow samples14,15. In addition, although flow separation can result in highly purified monocyte/macrophage populations, it requires very large sample sizes and high instrument and equipment requirements10,16. Additionally, the microbead enrichment method is extremely expensive and is not feasible in a general laboratory17.
Therefore, in the current study a convenient, fast, and cheap method was proposed for the isolation of mononuclear macrophages from the bone marrow. Bone marrow cells adhered for different time points were used to isolate BMMs using a secondary adherence method. BMMs extracted with the above method could induce the formation of osteoclasts in vitro, thus providing a simple and convenient cell model for the future study of osteoporosis in vitro.
All experiments in this study were conducted in accordance with the animal experiment guidelines of the Zhejiang Chinese Medical University Laboratory Animal Research Center (Approval No: IACUC-20181029-11).
1. Cell extraction
2. FACS staining of the cell
3. Wright-Giemsa staining
4. TRAP staining
5. Western blot
The secondary adherent cell population was stable and uniform. With the continuous cell proliferation, the majority of cells became larger, with irregular shape and grew into a radial adherent disk (Figure 2C,D). Flow cytometry showed that the percentage of cells expressing CD11b/c, a molecular marker on the surface of monocyte-macrophage lineage cells, was approximately 37.94% (Figure 2A,B). To further verify that the CD11b/c positive cells were monocyte-macrophage lineage cells, the differentiation of secondary adherent cells into osteoclasts was induced following cell treatment with RANKL and M-CSF. The TRAP staining results showed that compared with the control group, the number of intracellular purple-red granules was significantly increased in cells induced with RANKL and M-CSF (Figure 2E,F). Furthermore, the expression levels of the osteoclast-specific proteins TRAP and cathepsin K were significantly increased (Figure 2G,H).
Figure 1: Flow chart of the secondary adherent cell extraction method. Please click here to view a larger version of this figure.
Figure 2: (A–B) After three generations of stable culture of secondary adherent cells, CD11b/c positive cells were detected by flow cytometry. (C–D) The morphology of secondary adherent cells after three generations is shown (C for white light, D for Wright-Giemsa staining). (E–F) TRAP staining was used to detect the effect of RANKL and M-CSF on osteoclast formation (E for control; F for RANKL and M-CSF). (G–H) The cells were treated with RANKL and M-CSF for 7 days and the expression levels of TRAP and cathepsin K were determined by western blot analysis. Data are expressed as the mean ± SD of three independent experiments. ***P < 0.001 vs. the control group. TRAP, tartrate resistant acid phosphatase; RANKL, receptor activator of nuclear factor-κB ligand; M-CSF, macrophage colony-stimulating factor. Please click here to view a larger version of this figure.
Table 1: Method for extracting macrophages Please click here to download this Table.
Osteoclasts are one of the most significant cell types involved in the occurrence and development of bone diseases, as well as one of the primary objects of bone disease research20. Monocyte/macrophages can differentiate into osteoclasts. Since mononuclear macrophages (RAW264.7 cells) are too expensive to buy and are easily activated during culture, it is difficult to perform in vitro differentiation experiments using this cell line. Although several methods have been developed for extracting monocytes/macrophages from bone marrow, including density gradient centrifugation, collagenase digestion, microsphere enrichment and flow cytometry (Table 1), these methods are time-consuming, while they require high sample volumes and costly equipment. Therefore, the current study aimed to propose a simple and rapid method to extract monocytec/macrophages from bone marrow samples.
There are many cell populations in bone marrow, including BMSCs, endothelial cells and immune cells in addition to BMMs. As we all know that BMSCs can differentiate into osteoblasts, and this process will affect the differentiation of BMMs into osteoclasts21. Uniform and stable BMMs are significant factors for inducing osteoclast differentiation in vitro, so it is particularly important to consider these limiting factors when isolating and extracting BMMs. At the same time, the ability of BMMs to differentiate into osteoclasts will also weaken during the continuous passage. Therefore, it is challenging to isolate BMMs from bone marrow and successfully induce them into osteoclasts.
We found that there are differences in the adherence time of adherent cells in the bone marrow. More specifically, BMSCs basically adhered to the culture dish wall during 0-24 h of culture, while BMMs begin to adhere to the culture dish wall after 24 h. Based on the above finding, the secondary adherence method was selected to isolate BMMs. Suckling SD rats (age, 1-10 days old) were selected to extract BMMs, since the BMMs of young rats exhibit a stronger differentiation ability. The bone marrow cells of SD rats were collected, and after culturing for 24 h, the cell suspension was transferred and cultured for another 24 h. The collected secondary adherent cells contained a large number of BMMs. Following culture, the secondary adherent cells were gradually purified and became stable and uniform. The flow cytometry results demonstrated that the percentage of CD11b/c positive cells reached approximately 37.94%. Furthermore, TRAP staining and western blot analysis showed that the extracted BMMs could differentiate into osteoclasts. This finding was consistent with a previous study also suggesting that BMMs extracted by the secondary adherence method could be successfully differentiated into osteoclasts22.
The secondary adherence method can be used to simply and quickly extract mononuclear macrophages from the bone marrow, without requiring high-tech lab equipment. At the same time, this method is suitable for isolating cells from a small bone marrow sample, thus overcoming the cumbersome and time-consuming difficulties observed in the previous methods commonly used to extract mononuclear macrophages. Overall, BMMs extracted by the secondary adherence method could be successfully differentiated into osteoclasts in vitro, thus providing a stable cell model for the in vitro study of osteoclasts.
The authors have nothing to disclose.
This work was supported by the Natural Science Foundation of Zhejiang Province (grant no. LY19H060001) and the Zhejiang Traditional Chinese Medicine Science and Technology Plan Project (no. 2022ZB093).
35 mm2 cell climbing slices | NEST Biotechnology | 80102 | |
Anti-cathepsin K | Abcam | ab19027 | 1:1,000 |
Anti-CD11 isotype control | Abcam | ab172730 | 1 μg/test,1.675 mg/Ml |
Anti-CD11b/c | Absin | abs124232 | 1μg/test, 1 mg/mL |
Anti-TRAP | Abcam | ab191406 | 1:1,000 |
Anti-β-actin | Beyotime | AF5003 | 1:1,000 |
Cell climbing slices | NEST Biotechnology | 80102 | |
Cell culture dish | corning | 430167 | |
Cell culture flask | corning | 430168 | |
Dulbecco's modified eagle medium (DMEM) | Gibco | C11995500BT | |
Fetal bovine serum (FBS) | Gibco | 10099141C | |
Goat anti-rabbit IgG | Abcam | ab150077 | for IF, 1:2,000 |
goat anti-rabbit IgG | Abcam | ab6721 | for WB, 1:2,000 |
M-CSF | Pepro tech | 400-28 | |
PBS | Biosharp | BL302A | |
RANKL | Pepro tech | 400-30 | |
SD rat | Shanghai SLAC Laboratory Animal Co, Ltd | 1-10 days old | |
SDS-PAGE gel preparation kit | Solarbio | P1200 | |
TRAP/ALP Staining Kit | Wako | 294-67001 | |
Trypsin-EDTA solution | Biosharp | BL512A | |
Wright-Giemsa solution | Keygen Biotech | KGA225-1 |