RANKL-Based Osteoclastogenic Assay from Murine Bone Marrow Cells

Abstract

The osteoclast is the unique type of cell that resorbs bone in vivo and it is required for normal skeletal development and postnatal homeostasis. Osteoclast deficiency impairs skeletal development during embryogenesis and results in osteopetrosis and impaired tooth eruption. In contrast, excessive osteoclast formation in adults results in bone loss in a number of conditions, including osteoporosis, rheumatoid arthritis, and metastatic bone disease. Osteoclasts are derived from monocytes/macrophages; they can be generated in vitro by treatment of these precursor cells with macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). This chapter describes procedures for generating osteoclasts from mouse bone marrow cells in vitro using M-CSF and RANKL and assessing their ability to form resorption lacunae on thin bone slices.

1. Introduction

Osteoclasts, the multinucleated cells that resorb bone, are derived from precursors in the monocyte/macrophage lineage and participate in the essential biological processes of bone modeling and remodeling during embryonic development and post-natal growth. Increased osteoclast activity is responsible for pathological bone loss and destruction in many diseases, such as osteoporosis, rheumatoid arthritis, and cancer cell metastasis to bone. Although osteoclasts had been identified in tissue sections and characterized in ex vivo organ cultures [1] and their origin from hematopoietic precursors had been established [2], the first papers describing osteoclast generation from precursors in vitro were published by Allen et al. in 1981 [3, 4]. More efficient methods to generate osteoclasts in vitro were reported later by Ibbotson et al. in 1984 [5] and Roodman et al. in 1985 [5, 6] in Dr. Greg Mundy’s group.

In these early osteoclastogenic assays, primary bone marrow cells were cultured in medium containing 1,25-dihydroxy-vitamin D3 for 1–2 weeks to form multinucleated cells with several osteoclast characteristics, including positive staining for tartrate-resistant acid phosphatase (TRAP) and formation of resorption pits on bone slices. The establishment of an osteoclastogenic assay greatly improved understanding of how osteoclasts formed and were activated in response to cytokines and growth factors as these became available as pure recombinant proteins. However, a major limitation of the assay was consistency of results because the extent of osteoclastogenesis largely depended on the source of the serum, 1,25-dihydroxy-vitamin D3, and the culture medium used. The discovery of receptor activator of nuclear factor kappa-B ligand (RANKL) in 1997 [7, 8] as an essential factor for osteoclast formation along with macrophage-colony stimulating factor (M-CSF) dramatically improved the success and efficiency of osteoclastogenic assays. Unlike 1,25-dihydroxy-vitamin D3 which promotes osteoclastogenesis by stimulating RANKL production by stromal cells, RANKL directly binds to its receptor, RANK, on precursor cells to promote osteoclast differentiation. RANKL can induce osteoclast formation from RANK-expressing cells from multiple sources, including bone marrow, blood, spleen, liver, and lymph nodes.

In this chapter, we will describe detailed protocols for RANKL-mediated osteoclast formation from mouse bone marrow cells, along with TRAP staining and bone resorption assays, two commonly used criteria for osteoclast identification and functional analysis, respectively. There are other osteoclast assays, such as cocultures of osteoclast precursors with various types of cells that produce RANKL, especially osteoblastic cells. Some cell lines also can give rise to osteoclast-like cells in the presence of RANKL, such as the RAW264.7 cell line. However, the RANKL-based osteoclastogenic assay using primary bone marrow cells is the most commonly used assay to generate osteoclasts in vitro. Protocols for other osteoclastogenic assays have been described [9].

The RANKL-based osteoclastogenic assay utilizes soluble forms of RANKL and monocyte colony stimulating factor (M-CSF), both of which are essential for proliferation and survival of monocytes into osteoclast precursors. M-CSF also induces expression of RANK on these precursors and RANKL completes their differentiation into osteoclasts as well as osteoclast activation and survival, which are also supported by M-CSF.

2. Materials

2.1. Instruments

1. Autoclaved scissors.

2. Autoclaved fine forceps.

3. 10 ml disposable syringes

4. 21 gauge needles

5. 70% ethanol.

2.2. Reagents

1. Alpha minimal essential medium (α-MEM).

2. Fetal bovine serum (FBS) (see Note 1).

3. Penicillin/Streptomycin (P/S) solution 10,000 U/ml.

4. Phosphate buffered saline (PBS).

5. RBC lysis buffer.

6. 10% neutral buffered formalin phosphate

7. 0.5% toluidine blue in PBS

8. 96 well culture plate

9. 10 cm petri dish

10. 15 ml tube

11. Recombinant murine RANKL (R&D). Make 10 μg/μl of stock solution and store in aliquots at −80 °C.

12. Recombinant human M-CSF (R&D). Make 30 μg/μl of stock solution and store in aliquots at −80 °C (see Note 2).

13. Bone slices: Individual bone slices are prepared from the cortices of bovine long bones obtained from a local slaughterhouse. Muscles and other soft tissues are scraped from the cortices, which are then cut into longitudinal blocks ~1 × 2 × 4 cm using an EXAKT Pathology Saw. Uncut or cut bone blocks can be stored at −20 °C in a closed container. Blocks are mounted into the vice on a Buehler IsoMet Low Speed Cutting Machine and 300 μm thick slices are cut. Approximately 4.7 mm (3/16 in.) diameter circular discs are punched from these slices using a McGill Hole punch. Bone slices are washed with distilled water 4 times, placed in 70% ethanol for 30 min at room temperature, washed with autoclaved 1× PBS 4 times, dried in a sterilized incubator hood, and stored at −20 °C in a closed container. The discs are placed into the wells of 96-well plates for osteoclast resorption assays.

2.3. Solutions and Media

1. TRAP buffer: Combine 9.2 g sodium acetate anhydrous, 11.4 g of L-(+)tartaric acid, 950 ml of distilled water, and 2.8 ml of glacial acetic acid. Dissolve and adjust pH to 4.7–5.0 with 5 M sodium hydroxide (NaOH) to increase or glacial acetic acid to decrease pH. Bring total volume to 1 l with distilled water.

2. 5 M NaOH (for pH adjustment): Combine 50 g NaOH pellets and 250 ml of distilled water.

3. Naphthol AS-BI Phosphate Substrate (store for 3 weeks at 4 °C) (Solution #2): Combine 5 mg of Naphthol AS-BI Phosphate and 250 μl ethylene glycol monoethyl ether.

4. TRAP Solution (50 ml, freshly made): Combine 30 mg Fast Red Violet LB salt in 50 ml of TRAP buffer and mix well (Solution #1). Add 250 μl of Solution #2. Mix Solution #1 and Solution #2 and keep in the dark at 4 °C.

5. Washing medium (50 ml of α-MEM-2% FBS-P/S): Combine 49 ml of α MEM, 1.0 ml of FBS, and 0.5 ml P/S.

6. Osteoclast culture medium (50 ml of α-MEM-10% FBS-P/S): Combine 45 ml of α-MEM, 5.0 ml of FBS, 0.5 ml of P/S, 0.5 ml of nonessential amino acid (NEAA), and 0.5 ml of L-glutamine (see Note 3).

3. Methods

3.1. Preparation of Bone Marrow Cells

1. Harvest bone marrow cells from long bones of 2–4-month-old mice (although any age of mice older than 1 month can be used). In fact, any tissue containing monocytes/macrophages can be used to culture osteoclasts (see Note 4).

2. Sacrifice mice by carbon dioxide asphyxiation, followed by cervical dislocation to ensure death, according to a protocol approved by the Institutional Animal Care and Use Committee.

3. Sterilize the mouse by immersing it whole in 50 ml of 70% ethanol for 2–5 min.

4. Remove the femora and tibiae of both hind limbs using sterilized dissecting scissors and forceps by cutting through the skin surrounding the hip joint. Tear the skin posteriorly toward the feet to remove it. Disarticulate the hip joint and remove the muscle surrounding the femur and tibia. Place clean bones in a 10 cm petri dish.

5. Cut open both ends of each femur and tibia to expose the marrow cavity. Flush out bone marrow with 10 ml washing medium using a 21 gauge needle. Pass the cells through the needle twice to make single cell suspensions. Collect the cells with 10 ml αMEM with 2% FBS into a 15-ml tube (see Note 5).

6. Centrifuge the tube at 1000 × g for 5 min at room temperature. Discard the supernatant.

7. Resuspend the cell pellet in 2 ml of washing buffer, add 8 ml of RBC lysis buffer and mix thoroughly. Incubate the cells for 10 min at room temperature.

8. Spin down and discard the supernatant.

9. Resuspend the cells with 10 ml wash buffer and repeat step 7.

10. Resuspend the cells in 10 ml of osteoclast culture medium and mix thoroughly.

11. Mix 10 μl of cell suspension with 90 μl of osteoclast culture medium. Count cell numbers in a hemocytometer: Cell number/ml = cell count (# of cells in 4 squares of a hemocytometer) × 10 × 10⁴.

3.2. Cell Culture

1. On day 0, seed bone marrow cells at 4–6 × 10⁴ cells in 200 μl of osteoclast medium per well in a 96-well plate with 5 ng/ml M-CSF for 2 days (see Note 6).

2. On day 2, remove 100 μl medium from the culture and replace with 100 μl of freshly made osteoclast culture medium containing 5 ng/ml M-CSF and 10 ng/ml RANKL for 2 days. An alternative is to add RANKL at day 0 (see Notes 7 and 8).

3. On day 4, observe the cells under an inverted microscope. In general, osteoclasts begin to form after 2 days (or 4 days if RANKL is given on day 0) of RANKL treatment [10]. To assess early stages of osteoclast formation, the culture can be stopped and fixed with 10% neutral formalin. To observe later stages of osteoclast formation, replace half the medium with freshly made osteoclast culture medium containing 5 ng/ml M-CSF and 10 ng/ml RANKL at day 4, stop the culture by discarding the culture medium and fix the cells with 10% neutral formalin on the next day (day 5). Large multinucleated cells can be seen under an inverted microscopy at day 4 to 5 (Fig. 1a) (see Note 9).

4. Remove medium and add 200 μl of 10% formalin for 20 min at room temperature. Remove formalin and wash cells thoroughly with water (see Note 10).

Fig. 1.

Osteoclast culture assays. Bone marrow cells were isolated from C57BL/6 mice and were cultured with M-CSF for 2 days and followed by M-CSF and RANKL for an additional 4–8 days. (a) The cells were cultured on a plastic culture dish and observed under an inverted light microscope. The image shows large multinucleated osteoclasts. The border of an osteoclast is outlined by red arrows. Mononucleated cells are indicated by green arrows. (b) Cells were cultured on a plastic culture dish and subjected to TRAP staining. TRAP+ osteoclasts are indicated by green arrows. (c) Cells were cultured on a bone slice and subjected to toluidine blue staining after removal of cells. Resorption pits are indicated by red arrows

3.3. Staining Cells for TRAP Activity

1. Add 60 μl of TRAP solution for 10–30 min at room temperature. To speed up the staining, the plate can be heated to 37 °C. Check under microscope for large red/purple multinucleated cells to decide when to stop staining. Examples of TRAP+ osteoclasts are demonstrated in Fig. 1b.

2. Wash the cells thoroughly with water.

3. Counter stain the cells with Mayer’s Hematoxylin for 30 s followed by 0.5% Ammonia Water for 30 s.

4. Wash the cells thoroughly with water.

5. Air dry.

3.4. Bone Resorption

1. Place bone slices in the wells of a 96 well dish in 200 μl of α-MEM-10% FBS-P/S for 2 h to overnight in a cell culture incubator at 37 °C.

2. Remove medium, seed 4–6 × 10⁴ cells on top of the bone slices. Follow Subheading 3.3 above to culture osteoclasts with 5 ng/ml M-CSF and 10 ng/ml RANKL.

3. Remove half the culture medium and replace with new M-CSF and RANKL every other day. Examine for osteoclast formation under an inverted microscope every day. After 4–5 days, large multinucleated cells will have formed. Stop the culture and fix the cells on the bone slices 4 days after large osteoclasts are seen under the microscope, as described in Subheading 3.3 above (see Notes 11 and 12).

4. Stain the bone slices in the 96-well plates for TRAP activity, as described in Subheading 3.4, but without Mayer’s Hematoxylin counter staining.

5. Quantify osteoclast numbers.

6. Dip bone slices in 0.5% toluidine blue solution for a few seconds to stain the pits and brush the bone slice with a tooth-brush with water to remove the cells.

7. Dip bone slices in 0.5% toluidine blue solution again for a few seconds and wipe off the staining solution with a piece of paper.

8. Air dry.

9. Examine resorption pits by turning the slices upside down and viewing them under an inverted microscope. Examples of bone resorption pits are demonstrated in Fig. 1c.

4. Notes

1. We typically test different lots of FBS from different suppliers in the osteoclast assay to find the best lot of FBS that supports the maximum number of osteoclasts in standard culture conditions. We then purchase a large amount of the best lot of FBS (2000–3000 ml) and store it at −80 °C.

2. We have found that recombinant human M-CSF induces more osteoclasts than recombinant murine M-CSF. Conditioned medium from M-CSF-expressing cells can be used to replace recombinant M-CSF. We use 1:50 dilution of the M-CSF-conditioned medium [11].

3. We make a small amount of osteoclast culture medium with M-CSF or/and RANKL.

4. We have demonstrated that cells from peripheral blood [12], spleen [11], liver and popliteal lymph nodes from adult C57/B6 mice can differentiate to mature osteoclasts using this protocol. If spleen (some osteopetrotic mice do not have bone marrow) is used to culture osteoclasts, mesh the spleen in a cell strainer [11]. Seed 2 × 10⁵ spleen cells (after lysis of red blood cells) in each well of a 96-well plate.

5. We obtain approximately 5 × 10⁷ total cells per mouse including both legs.

6. We seed different densities of cells to determine the optimal cell numbers at the beginning of each new project, for example when using cells from a new transgenic or knockout mouse.

7. Be careful. We add medium very gently because a high force from addition of medium can disturb cells on the bottom of the well. A vigorous pipetting technique can result in fewer osteoclasts, uneven cell distribution, and huge well-to-well variation.

8. Different amounts of RANKL (1–10 ng/ml) and M-CSF (3–30 ng/ml) can be used to study the responses of cells to osteoclastogenic cytokines, for example to determine if they act synergistically.

9. Typically, osteoclasts are formed 1–2 days after the second RANKL addition.

10. Plates can be stored in −20 °C (discard formalin without washing) for months before TRAP staining.

11. Observe osteoclasts daily under an inverted microscope after 4–5 days of culture to determine if there are osteoclasts present on the plastic around the bone slices. These can be seen as large multinucleated cells (Fig. 1a). If there are osteoclasts, wait for another 2–3 days before stopping the experiment to allow time for resorption pits to form.

12. TRAP staining can be done beforehand.