Name: isolation, purification, and differentiation of osteoclast precursors from rat bone marrow
Uploaded: 2019-05-19T12:30:00.000+00:00
Duration: 11 min 11 s
Description: Watch this Scientific Journal Video about Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow at JoVE.com

This work was supported by the National Natural Science Foundation of China (81473692) to Yong Ma. The authors thank all the staff of the Medical Research Center of the First College of Clinical Medicine in Nanjing University of Chinese Medicine.

All the methods involving the animals described here are approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing University of Chinese Medicine.
1. Setup
<ol>
	<li>Prepare several longitudinally cut 1 mL pipette tips (1 cm) and several 1.5 mL microcentrifuge tubes. Put the pipette tips and microcentrifuge tubes at 103 kPa and 121 &#176;C for 20 min and ensure that they are sterile.</li>
	<li>Prepare a box of ice and several sterile dishes to preserve the isolated tissues during the isolation procedure.</li>
</ol>
2. Preparation of Culture Medium
<ol>
	<li>Prepare the complete culture medium. Use minimum essential medium alpha (&#945;-MEM) that contains a final solution of 1% of penicillin/streptomycin and 10% fetal bovine serum (FBS).</li>
	<li>Filter the media from step 2.1 using a 0.22 &#956;m filter.</li>
	<li>Prepare the bone marrow induction medium. For 50 mL of the solution, add 125 &#181;L of M-CSF at a concentration of 10,000 ng/mL (Table of Materials) to 49.875 mL of complete culture medium (from step 2.2) to make a final solution of 25 ng/mL M-CSF.</li>
	<li>Prepare the osteoclast induction medium. For 50 mL of the solution, add 500 &#181;L of RANKL at a concentration of 10,000 ng/mL (Table of Materials) to 49.5 mL of bone marrow induction medium (from step 2.3) to make a final solution of 25 ng/mL M-CSF and 100 ng/mL RANKL.</li>
</ol>
3. Isolation of Bone mMarrow-derived Cells
<ol>
	<li>Euthanasia
	<ol>
		<li>Euthanize four Sprague Dawley rats by CO2 inhalation followed by cervical dislocation, and immerse them in 75% ethanol for 1 min. 
		NOTE: Ensure that animals are of the same sex and of a similar age. Animals that are 2 to 3 weeks old are recommended. Two rats are prepared for the centrifugal method, while the other two are for the traditional method.</li>
	</ol>
	</li>
	<li>Traditional method
	<ol>
		<li>Place the animals on a disinfectant board in a supine position. Make a small incision (approximately 1 cm) at the proximal femur to peel the skin, using sterile scissors.</li>
		<li>Dissect out the femurs and tibias. Cut the bilateral connection parts around the hip, knee, and ankle joints to isolate the tibias and femurs carefully and gently. Cut off part of the tissues around the bone; be thorough. Do not fracture the tibias and femurs during the whole process.</li>
		<li>Place the cleaned bones into a dish with 5 mL of the complete culture medium. Cut off the long bone with sterile scissors and use a 1 mL syringe needle to flush the marrow cavity carefully with 10 mL of complete media until the marrow cavity turns white.</li>
		<li>Transfer the cell suspension from step 3.2.3 to a 50 mL tube, and then, filter it with a 70 &#956;m strainer to remove the remaining tissue.</li>
		<li>Add 5 mL of red blood cell lysis buffer (Table of Materials) to the cell suspension. Incubate the cells for 8 min on ice. Then, centrifuge the cells at 250 x g for 5 min to yield the cell pellet.</li>
		<li>Aspirate off the medium and resuspend the cells in 10 mL of complete media. Count the cells using a hemocytometer and calculate the time spent on the steps from this section (section 3.2).</li>
	</ol>
	</li>
	<li>Improved method
	<ol>
		<li>Place the animals on a disinfectant board in a supine position. Make a small incision (approximately 1 cm) at the proximal femur with sterile scissors to peel the skin.</li>
		<li>Dissect out the femurs and tibias. Properly cut off the part of the tissues around the bone (no need to remove completely). Do not fracture the tibias and femurs during the whole process.</li>
		<li>Rinse the tibias and femurs with 12 mL of phosphate-buffered saline (PBS) and cut them in half. Place the snipped tibias and femurs into a 1 mL pipette tip (from step 1.1), which is then put into a microcentrifuge tube and is centrifuged 3x at 1,000 x g for 45 s at 4 &#176;C.</li>
		<li>After centrifugation, remove the pipette tip containing the bone, and leave the bone marrow in the tube. Add 200 &#956;L of complete media into the microcentrifuge tube and repeat pipetting to disintegrate the marrow thoroughly.</li>
		<li>Transfer the cell suspension from step 3.3.4 to a 50 mL tube, and then, filter it with a 70 &#956;m strainer to remove the remaining tissue.</li>
		<li>Count the cells using a hemocytometer, and calculate the time spent on the steps from this section (section 3.3).</li>
	</ol>
	</li>
</ol>
4. Purification by Density Gradient Centrifugation
<ol>
	<li>Adjust the cell suspension from step 3.2.6 or step 3.3.5 to 2 mL with &#945;-MEM.</li>
	<li>Prepare one sterile silicified centrifugal tube and add 8 mL of the cell separation solution (Table of Materials) to the tube.</li>
	<li>Add the cell suspension from step 4.1 to the cell separation solution. Adhere the pipette tip to the inner surface of the tube and keep a 45&#176; to the inner surface. After adding the suspension, a clear limit will appear between the layer of the cells and the separation solution. 
	NOTE: The operation requires great care and needs to be performed slowly.</li>
	<li>Centrifuge the layered solution in a horizontal centrifuge at 500 x g for 30 min. After centrifugation, aspirate off the cloudy second layer, which contains the target cells, from top to bottom. 
	NOTE: Cancel the acceleration and deceleration of the centrifuge before centrifugation. There are six layers after centrifugation; the first layer is the diluent layer, the second layer is the monocytes layer, the third layer is the layer of mononuclear cells, the fourth layer is the transparent separation liquid one layer, the fifth is the granular cell layer, and the sixth layer is the red cell layer.</li>
	<li>Transfer the target cells to a new tube. Add 5 mL of PBS to wash the cells 3x. After each wash, centrifuge the target cells at 250 x g for 5 min to yield the cell pellet.</li>
	<li>Resuspend the cell pellet with bone marrow induction medium and count the cells using a hemocytometer. Add 5&#8211;8 mL of bone marrow induction medium from step 2.3 to obtain a final cell solution of 300,000 cells/mL. Add 1 mL to each well of a 24-well plate.</li>
</ol>
5. Culture and Differentiation
<ol>
	<li>After incubating the cells at 37 &#176;C for 24 h, gently aspirate off the medium and add 1 mL of the osteoclast induction medium to each well. Agitate the plate gently and put it in an incubator at 37 &#176;C.</li>
	<li>Change the osteoclast induction medium every 48 h. While doing so, change 0.8 mL of the medium in the well and agitate the plate gently. 
	NOTE: Large, motile, and multinucleated osteoclasts should be observed in the well under an inverted microscope, typically around days 4&#8211;6.</li>
</ol>
6. Tartrate-resistant Acid Phosphatase Staining
NOTE: Multinucleate osteoclasts will be present after 4&#8211;6 days if the induction (section 5) is successful.
<ol>
	<li>Prepare the fixative solution by combining 4 mL of 37% formaldehyde, 32.5 mL of acetone, and 12.5 mL of a citrate solution. Store the fixative solution at 4 &#176;C.</li>
	<li>Prepare the tartrate-resistant acid phosphatase (TRAP) stain solution (Table of Materials) by adding 50 &#181;L of sodium nitrite, 50 &#181;L of Fast Garnet GBC base solution, 50 &#181;L of naphthol AS-BI phosphate solution, 200 &#181;L of an acetate solution, and 100 &#181;L of a tartrate solution into 4.55 mL of deionized water that is prewarmed to 37 &#176;C. Then, mix gently by inversion for 1 min, and let it stand for 2 min.</li>
	<li>After the successful induction of osteoclasts, aspirate off the medium, and gently wash the wells 3x with PBS.</li>
	<li>Bring the fixative solution to room temperature (RT). Add 2 mL of fixative solution to the wells for 30 s, and do not allow the cells to dry.</li>
	<li>Aspirate the fixative solution, gently wash it 3x with deionized water that is prewarmed to 37 &#176;C, and then, aspirate the water.</li>
	<li>Add 2 mL of TRAP stain solution for 1 h at 37 &#176;C and keep the sample in the dark.</li>
	<li>After 1 h, aspirate the stain, and gently wash the sample with deionized water that is prewarmed to 37 &#176;C, and then, aspirate the water.</li>
	<li>Counterstain the cells for 1 min in a hematoxylin solution. After staining, aspirate the hematoxylin solution and gently wash the cells 3x with deionized water.</li>
	<li>Image osteoclasts using brightfield microscopy, and TRAP+ cells with three or more nuclei will appear purple.</li>
</ol>
7. Bone Resorption Assay Using Toluidine Blue Staining
<ol>
	<li>Pretreatment of the bone slices
	<ol>
		<li>Cut the fresh bovine femoral bone cortex into 2 cm thick slices along the longitudinal axis by microelectric saw. Then, cut and grind the slices with hard tissue grinders into 80 &#181;m.</li>
		<li>Wash the slices in a beaker with deionized water and 100 Hz ultrasound for 1 h, and repeat 3x.</li>
		<li>Immerse the slices into 75% alcohol for 2 h. Then, aspirate off the alcohol and expose each side of the slices to ultraviolet light for 1 h on a clean platform.</li>
		<li>Before planting the cells, immerse the slices in the culture medium for at least 2 h.</li>
	</ol>
	</li>
	<li>Toluidine blue staining
	<ol>
		<li>Place the pretreated bone slices into the 24-well plate. Plant the cells as mentioned in step 4.6 and induce the cells as mentioned in section 5.</li>
		<li>After the appearance of osteoclasts (after 4&#8211;6 days), wash the slices with 1 mL of 0.25 M ammonium hydroxide, and sonicate them 3x for 5 min each to remove the living cells to allow the analysis of the resorption pits on the bone slices. Then, remove the ammonium hydroxide and stain the slices with 1 mL of 1% (wt/vol) toluidine blue solution per slice for 2 min.</li>
		<li>Wash the slices with PBS. Randomly select five views and perform a semiquantitative analysis of the resorption area using an image analysis software.</li>
	</ol>
	</li>
</ol>
8. Scanning Electron Microscopy
<ol>
	<li>Prefix the bone slices from step 7.2.1 with 1 mL of 2.5% glutaraldehyde per slice for 2 h at RT, and then, sonicate the slices 3x for 3 min each with 1 M ammonium hydroxide to remove the cells and remove the ammonium hydroxide.</li>
	<li>Wash the bone slices 3x for 12 min each with PBS.</li>
	<li>Fix the bone slices with 1% osmic acid for 2 h at RT.</li>
	<li>Perform ethanol gradient dehydration, with 50%, 70%, 80%, 90%, and 95% ethanol, for 15 min for every gradient, and then, replace the ethanol with isoamyl acetate for 15 min.</li>
	<li>Coat the slices with gold palladium, and then, analyze by scanning electron microscopy.</li>
</ol>
9. Immunofluorescence Staining of Calcitonin Receptor
NOTE: Multinucleate osteoclasts will be present after 4&#8211;6 days if the induction is successful (section 5).
<ol>
	<li>Aspirate off the medium and gently wash the well 3x with PBS.</li>
	<li>Fix the cells for 10 min with 4% paraformaldehyde, which is precooled to 4 &#176;C.</li>
	<li>Add 1 mL of PBS with 0.3% nonionic surfactant per well for 30 min on ice. Then, aspirate off the PBS with 0.3% nonionic surfactant and add 1 mL of PBS with 5% FBS per well for 30 min on ice.</li>
	<li>Aspirate off the PBS and incubate the membranes with anti-calcitonin receptor (anti-CTR) at a 1:100 dilution in PBS at 4 &#176;C overnight.</li>
	<li>Aspirate off the solution, and incubate the membranes with Alexa-488-conjugated anti-rabbit IgG antibodies at a 1:1,000 dilution in PBS for 1 h at RT.</li>
	<li>Aspirate off the solution and gently wash the cells 3x with PBS. Counterstain the nuclei with Hoechst 33342 stain for 3 min at RT.</li>
	<li>Aspirate off the solution, and gently wash the cells 3x with PBS. Observe the intensity of CTR by fluorescence microscopy.</li>
</ol>

<ol>
	<li>Boyle, W. J., Simonet, W. S., Lacey, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclast+differentiation+and+activation.">Osteoclast differentiation and activation.</a> Nature. 423 (6937), 337-342 (2003).</li><li>Kular, J., Tickner, J., Chim, S. M., Xu, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+the+regulation+of+bone+remodelling+at+the+cellular+level.">An overview of the regulation of bone remodelling at the cellular level.</a> Clinical Biochemistry. 45 (12), 863-873 (2012).</li><li>Park, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+of+the+reduced+enamel+epithelium+and+its+implications+for+bone+resorption+during+tooth+eruption.">Apoptosis of the reduced enamel epithelium and its implications for bone resorption during tooth eruption.</a> Journal Of Molecular Histology. 44 (1), 65-73 (2013).</li><li>Boyde, A., Ali, N. N., Jones, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resorption+of+dentine+by+isolated+osteoclasts+in+vitro.">Resorption of dentine by isolated osteoclasts in vitro.</a> British Dental Journal. 156 (6), 216-220 (1984).</li><li>Chambers, T. J., Revell, P. A., Fuller, K., Athanasou, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resorption+of+bone+by+isolated+rabbit+osteoclasts.">Resorption of bone by isolated rabbit osteoclasts.</a> Journal of Cell Science. 66, 383-399 (1984).</li><li>Takahashi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclast-like+cell+formation+and+its+regulation+by+osteotropic+hormones+in+mouse+bone+marrow+cultures.">Osteoclast-like cell formation and its regulation by osteotropic hormones in mouse bone marrow cultures.</a> Endocrinology. 122 (4), 1373-1382 (1988).</li><li>Takahashi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblastic+cells+are+involved+in+osteoclast+formation.">Osteoblastic cells are involved in osteoclast formation.</a> Endocrinology. 123 (5), 2600-2602 (1988).</li><li>Suda, T., Takahashi, N., Martin, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+osteoclast+differentiation.">Modulation of osteoclast differentiation.</a> Endocrine Reviews. 13 (1), 66-80 (1992).</li><li>Yoshida, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+murine+mutation+osteopetrosis+is+in+the+coding+region+of+the+macrophage+colony+stimulating+factor+gene.">The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene.</a> Nature. 345 (6274), 442-444 (1990).</li><li>Yasuda, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclast+differentiation+factor+is+a+ligand+for+osteoprotegerin/osteoclastogenesis-inhibitory+factor+and+is+identical+to+TRANCE/RANKL.">Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL.</a> Proceedings of The National Academy of Sciences of The United States of America. 95 (7), 3597-3602 (1998).</li><li>Lacey, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoprotegerin+ligand+is+a+cytokine+that+regulates+osteoclast+differentiation+and+activation.">Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.</a> Cell. 93 (2), 165-176 (1998).</li><li>Marino, S., Logan, J. G., Mellis, D., Capulli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+culture+of+osteoclasts.">Generation and culture of osteoclasts.</a> BoneKEy Reports. 3, 570 (2014).</li><li>Pei, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluoride+decreased+osteoclastic+bone+resorption+through+the+inhibition+of+NFATc1+gene+expression.">Fluoride decreased osteoclastic bone resorption through the inhibition of NFATc1 gene expression.</a> Environmental Toxicology. 29 (5), 588-595 (2014).</li><li>Arnett, T. R., Dempster, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pH+on+bone+resorption+by+rat+osteoclasts+in+vitro.">Effect of pH on bone resorption by rat osteoclasts in vitro.</a> Endocrinology. 119 (1), 119-124 (1986).</li><li>Arnett, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+is+a+major+stimulator+of+osteoclast+formation+and+bone+resorption.">Hypoxia is a major stimulator of osteoclast formation and bone resorption.</a> Journal of Cellular Physiology. 196 (1), 2-8 (2003).</li><li>Orriss, I. R., Arnett, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+osteoclast+cultures.">Rodent osteoclast cultures.</a> Methods in Molecular Biology. 816, 103-117 (2012).</li><li>Quinn, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcitonin+receptor+antibodies+in+the+identification+of+osteoclasts.">Calcitonin receptor antibodies in the identification of osteoclasts.</a> Bone. 25 (1), 1-8 (1999).</li></ol>

The authors have nothing to disclose.

The ability to obtain and study osteoclasts in vitro is a critical and fundamental skill for any researcher wishing to study bone metabolism, which may help understand the mechanisms of bone-absorbing diseases and develop novel therapeutic agents. The present study described a protocol with some modifications based on previous methods.
By using pipette tips and microcentrifuge tubes to obtain bone marrow, it largely reduced the operation time of obtaining bone marrow and the workload of labora...

Bone homeostasis is a complex physiological process that is regulated by bone-resorbing osteoclasts and bone-forming osteoblasts1. A balance between osteoblastic and osteoclastic activity mediated through osteoblasts and osteoclasts, respectively, is highly essential for maintaining bone health and homeostasis, because perturbations in bone homeostasis might lead to bone diseases, such as abnormal bone growth or loss of bone density. As unique bone-resorption cells, osteoclasts are important in diseases related to abnormal bone destruction, including osteoporosis, periodontitis, and periprosthetic osteolysis2,3.
The development of an osteoclast culture is mainly divided into two stages. The methods established by Boyde&#160;et al.4 and Chambers&#160;et al.5 composed the first stage in the 1980s. They obtained relatively abundant osteoclasts from the bones of newborn animals, which is the rapid remodeling period. Osteoclasts are released by fragmenting and stirring the bones in a special medium. However, the cells obtained by this method are low in quantity and purity. The second stage was the development of long-range cultures of osteoclast formation, using hematopoietic lineage cells derived from bone marrow6. Cytokines, such as 1&#945;,25-dihydroxyvitamin D3, prostaglandin E2 (PGE-2), and parathyroid hormone (PTH), which are added into the culture medium, act through the system of osteoblasts/stromal cells to stimulate osteoclast formation7,8. However, the purity and quantity of osteoclasts obtained by this method cannot meet the needs of modern molecular biology research. Then, the discovery of macrophage colony-stimulating factor (M-CSF) and receptor activator for nuclear factor-&#954;B ligand (RANKL) make osteoclastogenesis easier9,10,11, and the method of using M-CSF and RANKL to directly stimulate osteoclast formation is widely used around the world. However, there are still some details in the methodology that need to be improved.
Currently, the most commonly used osteoclast culture method, as described by Marino et al.12 and Pei et al.13, often requires the removal of the surrounding tissue around the bone and uses a sterilized needle to flush the marrow cavity with completed media. There are some drawbacks to this process, including the fact that (1) the removal of the surrounding tissue around the bone requires much time and great surgical technique, (2) the bones are fragile and can lead to bone marrow outflow, (3) the bone marrow cavity might be too tiny to flush, and (4) there is a risk of needle stick injury. To avoid these problems, we centrifuge the tubes containing the bones for bone marrow instead of needle-flushing the bone marrow. Here, we introduce a stable and safe method which isolates bone marrow in less time and with less effort compared to the traditional procedure. Together with the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts&#160;in vitro.

<table><tbody><tr><td>Acid Phosphatase, Lekocyte (TRAP) kit</td><td>Sigma-Aldrich</td><td>387A</td><td></td></tr><tr><td>Automatic Hard Tissue Slicer</td><td>Lecia</td><td>RM2265</td><td></td></tr><tr><td>Bovine femoral bone</td><td></td><td></td><td>Purchased by ourselves</td></tr><tr><td>Cell Incubator</td><td>Heraus</td><td>BB16/BB5060</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sarana</td><td>s-fbs-au-015</td><td></td></tr><tr><td>Goat Anti-Rabbit IgG H&amp;amp;L (Alexa Fluor 488)</td><td>Abcam</td><td>ab150077</td><td></td></tr><tr><td>Hard Tissue Grinders</td><td>Lecia</td><td>SP-2600</td><td></td></tr><tr><td>Histopaque Kit</td><td>TianJing Haoyang</td><td>TBD2013DR</td><td>Silicified centrifugal tube amd cell separation solution</td></tr><tr><td>Hochest33342</td><td>Sigma-Aldrich</td><td>B2261</td><td></td></tr><tr><td>Inverted Phase Contrast Microscope</td><td>Olympus</td><td>CKX31</td><td></td></tr><tr><td>Inverted Fluorescence Microscope</td><td>Lecia</td><td>DMI-3000</td><td></td></tr><tr><td>MEM, no Glutamine</td><td>Gibco</td><td>11090-081</td><td></td></tr><tr><td>Penicillin-Streptomycin, Liquid</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Rabbit Anti-Calcitonin receptor</td><td>Bioss</td><td>bs-0124R</td><td></td></tr><tr><td>Recombinant Rat M-CSF</td><td>PeproTech</td><td>400-28</td><td></td></tr><tr><td>Recombinant Rat sRANK Ligand</td><td>PeproTech</td><td>400-30</td><td></td></tr><tr><td>Red Blood Cell Lysis Buffer</td><td>Absin</td><td>abs47014932</td><td></td></tr><tr><td>Scanning Electron Microscopy</td><td>FEI</td><td>Quanta 200</td><td></td></tr><tr><td>Toluidine Blue</td><td>Sigma-Aldrich</td><td>89649</td><td></td></tr></tbody></table>

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.

The purpose of the protocol was to isolate and purify large numbers of osteoclast precursors conveniently and induce osteoclasts successfully. By supplementing with M-CSF and RANKL, giant osteoclasts were seen on days 5&#8211;6. The formation of osteoclasts was successfully identified by TRAP staining (Figure 1A). Large and purple cells were regarded as TRAP-positive cells with multiple nuclei (typically &#8805; three nuclei). Through this method, it was typi...

Watch this Scientific Journal Video about Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow at JoVE.com

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

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.

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

isolation-purification-differentiation-osteoclast-precursors-from-rat

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.0K Views.  Nanjing University of Chinese Medicine. 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.

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

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 ...

Developmental Biology

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.

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 ...

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 ...

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors.

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

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 ...

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 ...

Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous high-quality cells for experimental requirements. mBMSCs could be widely used in therapeutic applications as tissue engineering cells in case of craniofacial diseases and cranio-maxillofacial regeneration in the future due to the excellent self-renewal ability and multi-lineage differentiation potential. Therefore, it is important to obtain mBMSCs in large numbers.
In this study, bone marrow was flushed from the mandible and primary mBMSCs were isolated through whole bone marrow adherent cultivation. Furthermore, CD29+CD90+CD45&#8722; mBMSCs were purified through fluorescent cell sorting. The second generation of purified mBMSCs were used for further study and displayed potential in differentiating into osteoblasts, adipocytes, and chondrocytes. Utilizing this in vitro model, one can obtain a high number of proliferative mBMSCs, which may facilitate the study of the biological characteristics, the subsequent reaction to the microenvironment, and other applications of mBMSCs.

This article presents a method that combines whole bone marrow adherence and flow cytometry sorting for isolating, cultivating, sorting, and identifying bone marrow mesenchymal stem cells from rat mandibles.

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous ...

Medicine

Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by 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.

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 ...

Niche-Based Isolation of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a &#39;niche-based approach on stemness&#39; for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a &#39;fibroblast-like&#39; spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.

Mesenchymal stem cells from the jaw bone marrow have significant functions in diverse differentiation, self-renewal, and immune modulation. They have emerged as a crucial reservoir of precursor cells in gene therapy, tissue engineering, and regenerative medicine. Here, we present a unique method for isolating jaw bone marrow mesenchymal stem cells in rats.

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from ...