Name: a rankl-based osteoclast culture assay of mouse bone marrow to investigate the role of mtorc1 in osteoclast formation
Uploaded: 2018-03-15T12:30:00.000+00:00
Duration: 9 min 37 s
Description: Watch this Scientific Journal Video about A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation at JoVE.com

The authors thank Dr. Minghan Tong and S. Kato for kindly providing reagents and mice. We thank the members of the Zou lab for useful discussions. This work was supported in part by grants from 973 Program from the Chinese Ministry of Science and Technology (MOST) [2014CB964704 and 2015CB964503], Clinical Research Program of 9th People&#39;s Hospital, Shanghai Jiao Tong University School of Medicine. Thanks for the help of Core Facility for Cell Biology and Core Facility for Chemical Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.

All procedures relating to the animals were performed according to the protocol approved by the Stanford Administrative Panel on Laboratory Animal Care (APLAC) and were approved by the Animal Care and Use Committee of the Shanghai Institute of Biochemistry and Cell Biology.
1. Preparation
<ol>
	<li>Generate osteoclast specific Raptor deletion mice (Raptorfl/fl; Ctsk-cre, hereafter RapCtsk) by mating Raptorfl/fl mice with Ctsk-cre mice. Use the Raptorfl/fl mice as the WT control in this study.</li>
	<li>Have a container with ice to keep isolated bone.</li>
	<li>Prepare culture medium.
	<ol>
		<li>Prepare &#945;-MEM culture medium by supplementing minimum essential medium alpha (&#945;-MEM) with 1x glutamine, penicillin-streptomycin, and 10% fetal calf serum.</li>
		<li>Prepare bone marrow macrophage induction medium consisting of 50 mL of &#945;-MEM medium and M-CSF at 20 ng/mL.</li>
		<li>Prepare osteoclast induction medium consisting of M-CSF and RANKL at 20 ng/mL, respectively, in 50 mL of &#945;-MEM medium.</li>
	</ol>
	</li>
	<li>Prepare the following buffers before TRAP staining.
	<ol>
		<li>Prepare fix solution consisting of 6.5 mL of acetone, 2.5 mL of citrate solution and 0.8 mL of 37% (vol/vol) formaldehyde solution.</li>
		<li>Prepare TRAP staining solution.
		<ol>
			<li>Prewarm deionized water to 37 &#176;C.</li>
			<li>Add 100 &#181;L of fast garnet GBC base solution and 100 &#181;L of sodium nitrite solution into a 1.5-mL microtube and mix by gentle inversion for 30 s. Leave the mixture to stand for 2 min.</li>
			<li>Prewarm 9 mL of deionized water to 37 &#176;C. Add 200 &#181;L of diazotized fast garnet GBC base solution from step 1.4.2.2, 100 &#181;L of naphthol AS-BI phosphate solution, 400 &#181;L of acetate solution, and 200 &#181;L of tartrate solution.</li>
			<li>Keep the TRAP staining mixture solution in a water bath at 37 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Prepare the following buffer before TRAP activity quantification of culture medium.
	<ol>
		<li>Prepare tartrate buffer (50 mL): 2 mL of 0.33 M L-tartaric acid, 48 mL of 0.33 M sodium tartrate dibasic dehydrate. Adjust the pH value to 4.9.</li>
		<li>Prepare 4-Nitrophenyl phosphate disodium (pNPP) buffer (200 mL): 1.502 g of glycine, 41 mg of MgCl2, 27.2 mg of ZnCl2, and 180 mL of deionized water. Adjust the pH value to 10.4 with 3 M NaOH and the volume to 200 mL with deionized water.</li>
		<li>Prepare pNPP buffer with phosphatase substrate (for one 96-well plate): 49.37 mg of phosphatase substrate and 175 &#181;L of pNPP buffer of step 1.5.2.</li>
		<li>Prepare substrate buffer (9 mL/96-well plate): 6.24 mL of deionized water, 360 &#956;L of acetate solution, and 2.4 mL of tartrate buffer. Mix by vortex and preheat at 37 &#176;C before use.</li>
		<li>Add 90 &#956;L of pNPP buffer with phosphatase substrate(1.5.3) to 9ml of preheated substrate buffer(1.5.4) to make a substrate mixture and vortex for 10 s.</li>
	</ol>
	</li>
</ol>
2. Dissection (Day -1)
<ol>
	<li>Euthanize 3 one-month-old female WT and RapCtsk mice by CO2 separately. Perform CO2 inhalation with appropriate equipment by trained personnel.</li>
	<li>Immerse 6 mice in a beaker with 100 mL of 75% ethanol (vol/vol) for 5 min to prevent bacterial contamination and then place on a dissection board in a supine position. Make an incision at the distal of the tibia vertically and dissect the skin along the hind limb with ophthalmic scissors.</li>
	<li>Cut the articular ligament of the hip joint with scissors and dislocate the hind limbs from the trunk.</li>
	<li>Cut the articular ligament of the knee joint and carefully disassociate the tibia and femur from the knee joint. Gently remove the soft tissue with scissors.</li>
	<li>Place all bones of one genotype in of one mouse in each well of a six-well-plate with 2 mL of &#945;-MEM on ice. 
	NOTE: To preserve cell viability, the bone should be kept in these conditions for less than 1 h.</li>
</ol>
3. Isolation (Day -1)
<ol>
	<li>Fill one well of a six-well plate with 75% ethanol (3 mL) and the other 5 wells with &#945;-MEM medium (3 mL).</li>
	<li>Put all bones of one genotype in the ethanol wash for 15 s and wash bones 5 times with &#945;-MEM medium for 10 s each time.</li>
	<li>Cut off the epiphyses with the scissor and insert a 0.45-mm syringe needle into the bone cavity and flush marrow out with &#945;-MEM medium into a 50-mL centrifuge tube.</li>
	<li>Flush the bone cavity using the same method from the other end of the bone. Repeat at least twice to wash the bone cavity thoroughly until the bone is pale.</li>
	<li>Centrifuge the bone marrow to obtain a cell pellet at 800 x g and 4 &#176;C for 5 min. Aspirate the supernatant.</li>
	<li>Add 3 mL of red blood cell lysis buffer into the centrifuge tube and pipette gently to resuspend the bone marrow. Keep the centrifuge tube on ice for 8 min to lyse red blood cells.</li>
	<li>Add 6 mL of &#945;-MEM medium into the centrifuge tube to stop cell lysis. Centrifuge at 500 x g and 4 &#176;C for 10 min and aspirate the supernatant.</li>
	<li>Resuspend in 3 mL of &#945;-MEM culture medium and place the cells into a six-well plate (generally one mouse per well).</li>
	<li>Incubate at 37 &#176;C in a 5% CO2 incubator overnight.</li>
</ol>
4. Plating (Day 0)
<ol>
	<li>Transfer the supernatant to a 15 mL centrifuge tube to collect the unattached cells the next morning.</li>
	<li>Centrifuge at 800 x g and 4 &#176;C for 5 min and aspirate the supernatant.</li>
	<li>Resuspend in 4 mL of &#945;-MEM culture medium.</li>
	<li>Take 20 &#181;L of the cell solution and mix with 20 &#181;L of Trypan Blue. Add 10 &#181;L of the mixture to the counting chamber and obtain the cell count per mL of solution.</li>
	<li>Add an appropriate volume of &#945;-MEM culture medium to obtain a cell solution of 500,000 cells/mL.</li>
	<li>Add 500 &#181;L and 50 &#181;L of bone marrow macrophage induction medium into each well of a 24-well plate and 96-well plate, respectively.</li>
	<li>Add 500 &#181;L and 50 &#181;L of cell solution into each well of a 24-well plate and 96-well plate, respectively.</li>
	<li>Incubate at 37 &#176;C in a 5% CO2 incubator for 3 days.</li>
</ol>
5. Differentiation (Days 3-7)
<ol>
	<li>Three days after plating, collect 50 &#181;L medium from each well of the 96-well-plate and freeze at -20 &#176;C, and then aspirate the residual medium.</li>
	<li>Add 1 mL and 100 &#181;L osteoclast induction medium into each well of a 24-well plate and a 96-well plate, respectively.</li>
	<li>Incubate at 37 &#176;C for 2 days.</li>
	<li>Collect 50 &#181;L of medium from each well and aspirate the residual medium.</li>
	<li>Add osteoclast induction medium into each well.</li>
	<li>Incubate at 37 &#176;C for 1 day. 
	NOTE: At this time point, large, multinucleated osteoclasts should be seen under an inverted microscope (Figure 2A).</li>
	<li>If osteoclasts are not seen, change the osteoclast induction medium and observe daily until osteoclasts are seen.</li>
	<li>Collect 50 &#181;L of medium from each well of the 96-well plate after osteoclasts have formed.</li>
</ol>
6. Tartrate Resistant Acid Phosphatase (TRAP) Staining
NOTE: Mature osteoclasts may be present after 6-7 days of in vitro culture (step 4).
<ol>
	<li>Aspirate medium and wash gently 3 times with 1x PBS.</li>
	<li>Fix the cells in each well of the 96-well plate with 100 &#181;L of fix solution for 30 s at room temperature.</li>
	<li>After fixation, aspirate the fix solution and wash gently 3 times with deionized water prewarmed to 37 &#176;C. Aspirate deionized water.</li>
	<li>Add 100 &#181;L of TRAP stain into each well of the 96-well plate and place the plate in an incubator at 37 &#176;C for 30 min and shield from light.</li>
	<li>After staining, aspirate the TRAP stain and gently wash 3 times with deionized water.</li>
	<li>Image osteoclasts using an inverted microscope at 40X magnification.</li>
</ol>
7. TRAP Activity Quantification of Culture Medium
<ol>
	<li>Collect 30 &#181;L culture supernatant from each well of the 96-well plate from step 5.1, 5.4 and 5.8; Use 30 &#181;L of non-cultured osteoclast induction medium as the negative control. Place the samples in a new 96-well plate.</li>
	<li>Add 90 &#956;L of substrate mixture(1.5.5) into each well of the 96-well plate.</li>
	<li>Place the plate in an incubator at 37 &#176;C for 1 h and shield from light.</li>
	<li>Stop the reaction by adding 50 &#181;L of 3 M NaOH into each well of the 96-well plate.</li>
	<li>Measure theabsorbance at the wavelength of 405 nm.</li>
</ol>
8. Western Blotting
NOTE: After 6-7 days of in vitro culture in the 24-well plate, mature osteoclasts should be visible.
<ol>
	<li>Aspirate the medium.</li>
	<li>Wash gently with pre-cooled 1x PBS 3 times.</li>
	<li>Aspirate PBS.</li>
	<li>Add 100 &#181;L of 1x SDS lysis buffer containing protease inhibitor cocktail.</li>
	<li>Heat lysates at 105 &#176;C for 10 min and centrifuge at 12,000 &#215; g and 4 &#176;C for 10 min. Collect the supernatants containing proteins.</li>
	<li>Separate the lysates containing 30 &#181;g of protein by 10% SDS-PAGE11 and then transfer to a polyvinylidene fluoride (PVDF) membrane.</li>
	<li>Block the membranes with 5% nonfat milk for 30 min.</li>
	<li>Incubate the membranes with anti-Raptor, anti-P-S6, anti-S6, anti-&#946;-actin at 1:1,000 dilution in 5% nonfat milk at 4 &#176;C overnight.</li>
	<li>Wash the membranes with Tris Buffered Saline with Tween-20 (TBST) for 10 min at room temperature.</li>
	<li>Repeat step 8.9 twice.</li>
	<li>Incubate the membranes with horseradish peroxidase (HRP)-conjugated anti-mouse or rabbit IgG antibodies at 1:5,000 dilution in 5% nonfat milk for 1 h at room temperature.</li>
	<li>Wash the membranes 3 times with TBST for 10 min at room temperature.</li>
	<li>For chemiluminescent detection, add western chemiluminescent HRP substrate on the membranes and incubate for 5 min at room temperature. Drain the excess substrate and expose the blots to X-ray film.</li>
</ol>

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All authors state that they have no conflicts of interest.

The osteoclastogenic assay is the most widely used method to isolate and culture osteoclasts in vitro12,13. While several RANKL-based osteoclast inductions have been described13,14,15, the present study described a protocol with some modifications based on the previous methods.
In the previous study, BMMs were plating immediately after isolation14,15. We recommended that BMMs were cultured with basic medium overnight before plating to isolate BMMs from mesenchymal stem cells and fibroblasts which immediately adhere to the dish. M-CSF can promote the proliferation of monocytes to osteoclast precursors and stimulate osteoclast precursors to express RANK that binds RANKL and then induces the formation of mature osteoclasts6,16,17. Therefore, in our study, the BMMs were induced with 10 ng/mL M-CSF for 3 days before osteoclast induction, instead for osteoclast induction on the second day after plating15. The cell density is critical for osteoclast formation in vitro. It is recommended that 25,000 BMMs were seeded in each well of a 96-well plate following by 3 days incubation with 10 ng/mL M-CSF. When the cell confluence reached 80&#8211;90%, it is a suitable time for osteoclast induction of RANKL. Usually, mature osteoclasts form on day 6. The most common reason for few osteoclasts is suboptimal cell density, which can be the result of a low seeding density or low cell viability. BMMs are delicate primary cells and need to be treated gently. Keeping the bone on ice for a long time or resuspending vigorously decreases the viability of BMMs and subsequently impairs the formation of osteoclasts. Thus, the optimal seeding density of viable BMMs is critical for obtaining a large number of osteoclasts.
The activity of secretory TRAP is critical for osteoclastic bone resorption. In this study, we describe a method to quantitatively analyze secretory TRAP activity in the culture medium. The change of TRAP activity in the culture medium can result from changes of osteoclast number or secretory TRAP of osteoclast or a combination of both. Thus, this may be a useful method for analyzing osteoclast function in vitro together with the osteoclast resorption assay described previously18.
mTOR is an evolutionarily-conserved protein kinase that can regulate cell metabolism and differentiation9. In this study, we found that mTORC1 played an important role in bone resorption by regulating osteoclast formation. mTORC1 may be a potential pharmacological target for the treatment of bone metabolic disorders such as osteoporosis in the future.
In summary, our protocol for osteoclast induction successfully resulted in a large number of giant osteoclasts in vitro and our data suggest that mTORC1 is critical for osteoclast formation.

Bone is an ever-changing organ and is remodeled by osteoblasts and osteoclasts throughout life. Osteoclasts are responsible for mineralized matrix resorption and osteoblasts synthesize and secrete new bone matrices1. The balance between bone resorption and bone formation is crucial for bone health including maintenance of bone mass and response to stimulation and injury. If this balance is disrupted, a series of bone metabolic diseases may occur, including osteoporosis and periodontal diseases. In these diseases, bone mass loss resulting from osteoclastic bone resorption exceeds the bone forming capacity of osteoblasts2,3. Thus, in order to develop pharmaceutical targets to treat skeletal disorders such as osteoporosis, it is critical to understand the generation and biology of osteoclasts4.
Osteoclasts are unique giant multinucleated cells located at or near the bone surface, and belong to the monocyte/macrophage family1. Ibbotson K. J. et al. reported a method to generate osteoclast-like cells in vitro with medium containing 1,25-dihydroxy-vitamin D35. The identification of macrophage-colony stimulating factor (M-CSF) and receptor activator for nuclear factor-&#954; B ligand (RANKL) as essential factors of osteoclast formation has dramatically increased the efficiency of osteoclastogenesis in vitro1,6,7. The ability to culture osteoclasts in vitro has improved our understanding of the generation and regulation of osteoclasts.
The mammalian/mechanistic target of rapamycin (mTOR) functions in two structurally and functionally distinct complexes, namely mTORC1 and mTORC28,9. The two multi-protein complexes are distinct from each other due to their different components and downstream substrates. mTORC1 contains the unique regulatory-associated protein of mTOR (Raptor), while mTORC2 contains the rapamycin-insensitive companion of mTOR (Rictor)9. mTORC1 can integrate and transmit important signals to regulate cell growth, proliferation and differentiation. Recently, we demonstrated that mTORC1 plays a key role in the network of catabolic bone resorption by deletion of Raptor to inactivate mTORC1 in osteoclasts10. However, the underlying mechanisms require further study. In the present study, a RANKL-based osteoclastogenic method was used to generate osteoclasts from bone marrow-derived macrophages (BMMs) of wild-type (WT) and RapCtsk mice, and to study the influence of mTORC1 inactivation on osteoclast formation.

<table><tbody><tr><td>&lt;em&gt;Raptor&lt;sup&gt;fl/fl&lt;/sup&gt;&lt;/em&gt; mice</td><td>The Jackson Laboratory</td><td>013188</td><td></td></tr><tr><td>&lt;em&gt;Ctsk-cre&lt;/em&gt; mice</td><td></td><td></td><td>a gift from S. Kato, University of Tokyo, Tokyo, Japan</td></tr><tr><td>&amp;alpha;-MEM</td><td>Corning</td><td>10-022-CVR</td><td></td></tr><tr><td>Glutamine</td><td>Gibico</td><td>25030081</td><td></td></tr><tr><td>Penicillin streptomycin</td><td>Gibico</td><td>15140122</td><td></td></tr><tr><td>Fetal calf serum</td><td>BioInd</td><td>04-001-1A</td><td></td></tr><tr><td>Recombinant mouse M-CSF protein</td><td>R&amp;amp;D</td><td>Q3U4F9</td><td></td></tr><tr><td>Recombinant mouse RANKL protein</td><td>R&amp;amp;D</td><td>Q3TWY5</td><td></td></tr><tr><td>RBC lysis buffer</td><td>Beyotime</td><td>C3702</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma-Aldrich</td><td>302643</td><td></td></tr><tr><td>Acetone</td><td>Shanghai Chemical Co. Ltd.</td><td></td><td></td></tr><tr><td>Citrate solution</td><td>Sigma-Aldrich</td><td>915</td><td></td></tr><tr><td>Formaldehyde solution</td><td>Shanghai Chemical Co. Ltd.</td><td></td><td></td></tr><tr><td>Acid Phosphatase, Leukocyte (TRAP) Kit</td><td>Sigma-Aldrich</td><td>387A-1KT</td><td></td></tr><tr><td>Fast Garnet GBC Base solution</td><td>Sigma-Aldrich</td><td>3872</td><td></td></tr><tr><td>Sodium Nitrite Solution</td><td>Sigma-Aldrich</td><td>914</td><td></td></tr><tr><td>Naphthol AS-BI Phosphate Solution</td><td>Sigma-Aldrich</td><td>3871</td><td></td></tr><tr><td>Acetate solution</td><td>Sigma-Aldrich</td><td>3863</td><td></td></tr><tr><td>Tartrate solution</td><td>Sigma-Aldrich</td><td>3873</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline</td><td>Corning</td><td>21-031-CVR</td><td></td></tr><tr><td>L-tartaric acid</td><td>Sigma-Aldrich</td><td>251380</td><td></td></tr><tr><td>Sodium tartrate dibasic dehydrate</td><td>Sigma-Aldrich</td><td>s4797</td><td></td></tr><tr><td>Glycine</td><td>Shanghai Chemical Co. Ltd.</td><td></td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Shanghai Chemical Co. Ltd.</td><td></td><td></td></tr><tr><td>ZnCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Shanghai Chemical Co. Ltd.</td><td></td><td></td></tr><tr><td>NaOH</td><td>Shanghai Chemical Co. Ltd.</td><td></td><td></td></tr><tr><td>Phosphatase substrate</td><td>Sigma-Aldrich</td><td>P4744</td><td></td></tr><tr><td>anti-Raptor</td><td>Cell Signaling Technology</td><td>2280</td><td></td></tr><tr><td>anti-P-ribosomal protein S6 (S235/236)</td><td>Cell Signaling Technology</td><td>2317</td><td></td></tr><tr><td>anti-ribosomal protein S6</td><td>Cell Signaling Technology</td><td>2211</td><td></td></tr><tr><td>anti-&amp;beta;-actin</td><td>Santa Cruz Biotechnology</td><td>sc-130300</td><td></td></tr><tr><td>37% formaldehyde</td><td>Xilong scientific</td><td></td><td></td></tr><tr><td>polyvinylidene fluoride (PVDF) membrane</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Western Chemiluminescent HRP Substrate (ECL)</td><td>Millipore</td><td>00000367MSDS</td><td></td></tr><tr><td>IX71</td><td>Olympus</td><td></td><td></td></tr><tr><td>Envision</td><td>Perkin Elmer</td><td></td><td></td></tr><tr><td>0.45-mm Syringe</td><td></td><td></td><td></td></tr><tr><td>Scissor</td><td></td><td></td><td></td></tr><tr><td>Mosquito forcep</td><td></td><td></td><td></td></tr></tbody></table>

a rankl-based osteoclast culture assay of mouse bone marrow to investigate the role of mtorc1 in osteoclast formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

Using the present protocol, a large number of giant osteoclasts were seen on day 6; if giant osteoclasts are not seen, one more day of osteoclast differentiation may be needed (Figure 1). Successful osteoclast formation was confirmed by TRAP staining (Figure 2A). Osteoclasts were giant wine red/purple cells with more than 3 nuclei. More than 250 osteoclasts were obtained in each well of the 96-well plate in WT BMMs (Figure 4A).
Deletion of Raptor was confirmed by western blotting analysis and the inactivation of mTORC1 was determined by a decrease in ribosomal protein S6 phosphorylation (P-S6), which is widely used as the readout for mTORC1 (Figure 3).
The number of TRAP-positive osteoclasts decreased in RapCtsk BMMs compared with the WT group (Figure 4A), which indicated that Raptor deficiency impaired osteoclast formation.
The activity of secretory TRAP is important for bone resorption, and TRAP activity in the culture medium was analyzed in the present study. As shown in Figure 4B, TRAP activity increased due to stimulation of RANKL in both WT and RapCtsk BMMs. In addition, TRAP activity decreased in RapCtsk BMMs compared with the corresponding WT group (P &#60;0.05).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56468/56468fig1.jpg" /> 
Figure 1: Schematic diagram of the osteoclast induction protocol. BMMs were isolated on day 1 and incubated with basic &#945;-MEM medium overnight. On day 0, BMMs were collected and plated onto a 96-well plate and 24-well plate followed by incubation in &#945;-MEM medium with 10 ng/mL M-CSF. On day 3, the medium was changed to &#945;-MEM medium with 20 ng/mL M-CSF and 20 ng/mL RANKL for induction of osteoclast differentiation. On day 5, the osteoclast differentiation medium was changed. On day 6, a large number of osteoclasts were visible and osteoclast assays were performed. If osteoblasts were not visible, the osteoclast induction medium was changed and incubation continued for one more day. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56468/56468fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56468/56468fig2.jpg" /> 
Figure 2: Representative view of osteoclasts. (A) Giant osteoclasts observed in bright field on day 6. The red line outlined the border of osteoclasts. (B) A typically giant, multinucleated, TRAP positive (wine red) osteoclast. The black arrow indicated two nuclei of the multinucleated osteoclast. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56468/56468fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56468/56468fig3.jpg" /> 
Figure 3: mTORC1 inactivation in RapCtsk BMMs. Western blotting analysis of Raptor, P-S6 and S6 expression in WT and RapCtsk BMMs on day 6. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56468/56468fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56468/56468fig4.jpg" /> 
Figure 4: Deletion of Raptor impaired osteoclast formation. (A) TRAP staining of BMMs on day 6. High magnification images of the squares in WT and RapCtsk groups are shown, respectively. There were less giant, TRAP positive osteoclasts formed in the RapCtsk BMMs in comparison with WT group. (B) TRAP activity of cultured WT and RapCtsk BMMs. Data represent means &#177; S.D.* P &#60;0.05, n = 3. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56468/56468fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

a-rankl-based-osteoclast-culture-assay-mouse-bone-marrow-to

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

12.8K Views.  National Clinical Research Center of Stomatology. Dit manuscript wordt beschreven van een protocol bij isolaat en cultuur osteoclasten in vitro uit beenmerg van de muis, en te bestuderen van de rol van het zoogdier/mechanistische doelwit van rapamycin complexe 1 in osteoclast vorming.

Video: A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Cancer Research

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Medicine

Effect of Anti-c-fms Antibody on Osteoclast Formation and Proliferation of Osteoclast Precursor In Vitro

Bone remodeling is a complex process and it involves periods of deposition and resorption. Bone resorption is a process by which bone is broken down by osteoclasts in response to different stimuli. Osteoclast precursors differentiate into multinuclear osteoclasts in response to macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor Kappa-B ligand (RANKL). Under pathologic conditions, the cytokine profile is different and involves a mixture of inflammatory cytokines. Tumor necrosis factor alpha (TNF-&#945;) is one of the most important cytokines as it is found in large amounts in areas involved with inflammatory osteolysis. The purpose of this protocol is to provide a method by which murine bone marrow is isolated to generate osteoclasts through induction with M-CSF and either RANKL or TNF-&#945; which will be subsequently inhibited by increasing doses of anti-c-fms antibody, the receptor for M-CSF. This experiment highlights the therapeutic value of anti-c-fms antibody in diseases of inflammatory bone resorption.

This protocol includes dissection of murine long bones and bone marrow isolation, to generate bone marrow macrophages and produce osteoclasts using macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor Kappa-B ligand (RANKL) or tumor necrosis factor alpha (TNF-&#945;) and their arrest by anti-c-fms antibody, M-CSF receptor.

Bone remodeling is a complex process and it involves periods of deposition and resorption. Bone resorption is a process by which bone is broken down by ...

Immunology and Infection

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused by increased differentiation and activation of OCs, so the inhibition of this pathobiology is a key therapeutic principle.Two key factors drive the differentiation of OCs from myeloid precursors: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Human circulating CD14+ monocytes have long been known to differentiate into OCs in vitro. However, the exposure time and the concentration of RANKL influence the differentiation efficiency. Indeed, protocols for the generation of human OCs in vitro have been described, but they often result in a poor and lengthy differentiation process. Herein, a robust and standardized protocol for generating functionally active mature human OCs in a timely manner is provided. CD14+ monocytes are enriched from human peripheral blood mononuclear cells (PBMCs) and primed with M-CSF to upregulate RANK. Subsequent exposure to RANKL generates OCs in a dose- and time-dependent manner. OCs are identified and quantified by staining with tartrate acid-resistant phosphatase (TRAP) and light microscopy analysis. Immunofluorescence staining of nuclei and F-actin is used to identify functionally active OCs. In addition, OSCAR+CD14&#8722; mature OCs are further enriched via flow cytometry cell sorting, and OC functionality quantified by mineral (or dentine/bone) resorption assays and actin ring formation. Finally, a known OC inhibitor, rotenone, is used on mature OCs, demonstrating that adenosine triphosphate (ATP) production is essential for actin ring integrity and OC function. In conclusion, a robust assay for differentiating high numbers of OCs is established in this work, which in combination with actin ring staining and an ATP assay&#160;provides a useful in vitro model to evaluate OC function and to screen for novel therapeutic compounds that can modulate the differentiation process.

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts are key bone-resorbing cells in the body. This protocol describes a reliable method for the in vitro differentiation of osteoclasts from human peripheral blood monocytes. This method can be used as an important tool to further understand osteoclast biology in homeostasis and in diseases.

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused ...

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

Biology

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases (e.g., osteoporosis and bone cancer) and are therefore important objects of research. In vitro, their activity can be analyzed by the formation of resorption pits. In this protocol, we describe a simple pit assay method using calcium phosphate (CaP) coated cell culture plates, which can be easily visualized and quantified. Osteoclast precursors derived from human peripheral blood mononuclear cells (PBMCs) were cultured on the coated plates in the presence of osteoclastogenic stimuli. After 9 days of incubation, osteoclasts were fixed and stained for fluorescence imaging while the CaP coating was counterstained by calcein. To quantify the resorbed area, the CaP coating on plates was stained with 5% AgNO3 and visualized by brightfield imaging. The resorption pit area was quantified using ImageJ.

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Here, we present a simple and effective assay procedure for resorption pit assays using calcium phosphate coated cell culture plates.

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

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