We acknowledge the support of NIH grants R01 DE021683, R01 DE019434, U01 HL099776, The Oak Foundation and The Hagey Laboratory for Pediatric Regenerative Medicine.

NOTE: Ethical statement: All research involving vertebrate animals was performed in accordance protocols approved by the Stanford Administrative Panel on Laboratory Animal Care (APLAC).
1. Preparation
<ol>
	<li>Allow 10 ml of commercially available density gradient cell separation media (which contains polysucrose and sodium diatrizoate, adjusted to a density of 1.077 g/ml) to come to RT in a 50 ml conical tube.</li>
	<li>Prepare flow cytometry (FACS) buffer with 1x phosphate buffered saline (PBS) and 2% fetal c...

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+resorption+restored+in+osteopetrotic+mice+by+transplants+of+normal+bone+marrow+and+spleen+cells.">Bone resorption restored in osteopetrotic mice by transplants of normal bone marrow and spleen cells.</a> Science. 190, 784-785 (1975).</li><li>Burger, E. 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=In+vitro+formation+of+osteoclasts+from+long-term+cultures+of+bone+marrow+mononuclear+phagocytes.">In vitro formation of osteoclasts from long-term cultures of bone marrow mononuclear phagocytes.</a> The Journal of experimental medicine. 156, 1604-1614 (1982).</li><li>Underwood, J. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Denosumab+in+patients+with+cancer-a+surgical+strike+against+the+osteoclast.">Denosumab in patients with cancer-a surgical strike against the osteoclast.</a> Nature reviews. Clinical oncology. 9, 110-118 (2012).</li><li>Khosla, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+the+OPG/RANKL/RANK+system.">Minireview: the OPG/RANKL/RANK system.</a> Endocrinology. 142, 5050-5055 (2001).</li><li>Boyle, 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=Differential+roles+of+MAPK+kinases+MKK3+and+MKK6+in+osteoclastogenesis+and+bone+loss.">Differential roles of MAPK kinases MKK3 and MKK6 in osteoclastogenesis and bone loss.</a> PloS one. 9, (2014).</li><li>Hofbauer, L. C., Heufelder, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+receptor+activator+of+nuclear+factor-kappaB+ligand+and+osteoprotegerin+in+bone+cell+biology.">Role of receptor activator of nuclear factor-kappaB ligand and osteoprotegerin in bone cell biology.</a> Journal of molecular medicine (Berlin, Germany). 79, 243-253 (2001).</li><li>Teramachi, 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=Increased+IL-6+Expression+in+Osteoclasts+is+Necessary+but+not+Sufficient+for+the+Development+of+Paget's+Disease+of+Bone.">Increased IL-6 Expression in Osteoclasts is Necessary but not Sufficient for the Development of Paget's Disease of Bone.</a> Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. , (2013).</li><li>Catalfamo, 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=Hyperglycemia+induced+and+intrinsic+alterations+in+type+2+diabetes-derived+osteoclast+function.">Hyperglycemia induced and intrinsic alterations in type 2 diabetes-derived osteoclast function.</a> Oral diseases. 19, 303-312 (2013).</li><li>Schueler, 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=Intratibial+injection+of+human+multiple+myeloma+cells+in+NOD/SCID+IL-2Rgamma(null)+mice+mimics+human+myeloma+and+serves+as+a+valuable+tool+for+the+development+of+anticancer+strategies.">Intratibial injection of human multiple myeloma cells in NOD/SCID IL-2Rgamma(null) mice mimics human myeloma and serves as a valuable tool for the development of anticancer strategies.</a> PloS one. 8, (2013).</li><li>Xing, L., Boyce, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RANKL-Based+Osteoclastogenic+Assays+from+Murine+Bone+Marrow+Cells.">RANKL-Based Osteoclastogenic Assays from Murine Bone Marrow Cells.</a> Methods in molecular biology (Clifton, N.J). 1130, 307-313 (2014).</li><li>Weischenfeldt, J., Porse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+Marrow-Derived+Macrophages+(BMM):+Isolation+and+Applications.">Bone Marrow-Derived Macrophages (BMM): Isolation and Applications.</a> CSH protocols. , (2008).</li><li>Yamamoto, Y., 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=Osteoblasts+provide+a+suitable+microenvironment+for+the+action+of+receptor+activator+of+nuclear+factor-kappaB+ligand.">Osteoblasts provide a suitable microenvironment for the action of receptor activator of nuclear factor-kappaB ligand.</a> Endocrinology. 147, 3366-3374 (2006).</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, 3597-3602 (1998).</li><li>Lacey, D. 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J., Udagawa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal+regulation+of+osteoclast+function.">Hormonal regulation of osteoclast function.</a> Trends in endocrinology and metabolism. 9, 6-12 (1998).</li><li>Nakamura, T., 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=Estrogen+prevents+bone+loss+via+estrogen+receptor+alpha+and+induction+of+Fas+ligand+in+osteoclasts.">Estrogen prevents bone loss via estrogen receptor alpha and induction of Fas ligand in osteoclasts.</a> Cell. 130, 811-823 (2007).</li><li>Bellido, T., 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=Regulation+of+interleukin-6,+osteoclastogenesis,+and+bone+mass+by+androgens.+The+role+of+the+androgen+receptor.">Regulation of interleukin-6, osteoclastogenesis, and bone mass by androgens. 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None of the authors has disclosure or conflict of interest to declare.

An ability to easily isolate and cultivate large numbers of osteoclasts in vitro has been responsible for helping to advance understanding of bone biology and osteoclast-mediated diseases. It was the identification of RANKL that lead to this, when it was recently identified as the major regulator of osteoclast formation, differentiation and survival16-18.
It has been our experience that the in vitro cultivation of osteoclasts from bone marrow is largely dependent o...

Bone remodeling is dynamic and involves the coupling of bone formation with bone resorption1. This tightly regulated process is responsible for maintaining the skeleton during normal homeostasis, and in response to injury and disease.
Osteoclasts are unique, multinucleated cells that are capable of resorbing both the organic and inorganic matrices of bone. Osteoclasts are derived from the monocyte/macrophage lineage of the bone marrow2-5. Abnormalities in the function or formation of osteoclasts can result in a variety of clinical pathologies, including common conditions like osteoporosis.
The ability to generate osteoclasts in vitro has allowed for significant advances in our understanding of bone biology6. As a result, new therapeutic agents are emerging to treat osteoclast-related diseases which are responsible for significant morbidities and mortalities7. Homeostatic maintenance of bone mass and strength requires the concerted action of bone-forming osteoblasts and bone-resorbing osteoclasts8,9. Bone homeostasis is altered in a number of diseases, including post-menopausal osteoporosis, in which increased osteoclast activity leads to pathogenic loss of bone mass and density10. With increasing availability of transgenic murine models of human disease, there is more opportunity to decipher the role of the osteoclasts in human bone disease11-13.
Numerous protocols for osteoclast culturing techniques appear in the literature, with many variations described9,12,14. Xing and colleagues describe similar methodology to the protocol described below, in their description of osteoclastogenic assays from murine bone marrow cells. However to release the bone marrow cells following long bone harvest, Xing et al. flush the marrow cavity with &#945;-MEM complete media14. Catalfamo examines the effect of hyperglycemia on osteoclast function and describes a method in which all cells mobilized by bone marrow flushing are cultured for 24 hr, at which point the non-adherent cells are discarded12, a technique also used by Boyle et al.9 These previously published protocols necessitate the practice of flushing the bone marrow, a tedious practice, which also introduces the risk of a needle stick injury and loss of valuable bone marrow, as one must cut both ends of the bone. The protocol, which we describe, implements the use of a mortar and pestle to isolate osteoclasts, which is similar to the method of macrophage isolation described by Weischenfeldt et al.15 
Our experience, however, is that osteoclast isolation and in vitro culture using previously published techniques results in variable outcomes in terms of osteoclast production, often resulting in an inability to cultivate osteoclasts. Therefore, we have devised a protocol that allows for the consistent isolation of mouse bone marrow to produce large numbers of multinucleated osteoclasts in vitro, with an approximate yield of 70-80% of cells initially plated forming macrophages and subsequently osteoclasts, in the presence of osteoclast induction media.

<table border="0" cellpadding="0" cellspacing="0" width="1017">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:503px;">Name of the Material/Equipment</td>
			<td style="width:165px;">Company</td>
			<td style="width:175px;">Catalog Number</td>
			<td style="width:175px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM, no glutamine, no phenol red</td>
			<td>Gibco</td>
			<td>51200-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">M-CSF, recombinant mouse</td>
			<td>Gibco</td>
			<td>PMC2044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Mouse TRANCE/RANK L/TNFSF11 (E. coli expressed)</td>
			<td>R&#38;D Systems</td>
			<td>462-TEC-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prostaglandin E2</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Histopaque-1077</td>
			<td>Sigma-Aldrich</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acid Phosphatase, Lekocyte (TRAP) kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Osteoassay bone resorption plates, 24 well plates</td>
			<td>Corning Life Sciences</td>
			<td>3987</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The aim of this method was to easily isolate large numbers of osteoclasts in vitro, typically in one week. Successful isolation of large numbers of osteoclasts was confirmed using tartrate-resistant acid phosphatase staining (Figure 1A). Large osteoclasts are visualized as large purple cells with multiple nuclei (typically &#8805; 3 nuclei). Using this protocol, it is common to isolate osteoclasts with as many as 30 nuclei per osteoclast (Figure 1B).
...

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Osteoclast Derivation from Mouse Bone Marrow

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 ...

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23.9K visualizações.  Stanford University School of Medicine.  Os osteoclastos são as células de reabsorção óssea principais no corpo. Uma capacidade de isolar os osteoclastos em grandes números resultou em avanços significativos na compreensão da biologia dos osteoclastos. Neste protocolo, nós descrevemos um método para o isolamento, cultivo e quantificar a actividade dos osteoclastos in vitro. 