Isolation and Generation of Osteoclasts

  • Nadia Rucci
  • Alberta Zallone
  • Anna TetiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1914)


This chapter describes the isolation, culture, and staining of osteoclasts. The key advantages of this assay are that it allows direct measurement of osteoclast number, bone resorption, as well as yielding good quantities of osteoclasts at defined stages of formation for molecular analysis. An additional focus of this chapter will be the generation of osteoclasts from less conventional animal species and cell lines.

Key words

Osteoclast Bone resorption Bone remodeling Bone loss 



We are grateful to Prof Rodolfo Amprino and Prof Gastone Marotti for introducing us into the bone field, to Prof Pier Carlo Marchisio for the instrumental collaboration in the discovery of the podosomes, and to Prof Steven L. Teitelbaum and Prof Arnold J. Kahn, who believed in our chicken osteoclast isolation method and made it possible to disseminate this knowledge to the bone community.


  1. 1.
    Cappariello A, Maurizi A, Veeriah V, Teti A (2014) The great beauty of the osteoclast. Arch Biochem Biophys 558:70–78CrossRefGoogle Scholar
  2. 2.
    Walker DG (1975) Control of bone resorption by hematopoietic tissue. The induction and reversal of congenital osteopetrosis in mice through use of bone marrow and splenic transplants. J Exp Med 142:651–663CrossRefGoogle Scholar
  3. 3.
    Marks SC Jr, Walker DG (1981) The hematogenous origin of osteoclasts: experimental evidence from osteopetrotic (microphthalmic) mice treated with spleen cells from beige mouse donors. Am J Anat 161:1–10CrossRefGoogle Scholar
  4. 4.
    Pivetta E, Wassermann B, Bulian P, Steffan A, Colombatti A, Polesel J, Spessotto P (2015) Functional osteoclastogenesis: the baseline variability in blood donor precursors is not associated with age and gender. Oncotarget 6:31889–31900CrossRefGoogle Scholar
  5. 5.
    Takahashi N, Maeda K, Ishihara A, Uehara S, Kobayashi Y (2011) Regulatory mechanism of osteoclastogenesis by RANKL and Wnt signals. Front Biosci (Landmark Ed) 16:21–30CrossRefGoogle Scholar
  6. 6.
    Zhang C, Dou CE, Xu J, Dong S (2014) DC-STAMP, the key fusion-mediating molecule in osteoclastogenesis. J Cell Physiol 229:1330–1335CrossRefGoogle Scholar
  7. 7.
    Rucci N, Teti A (2016) The "love-hate" relationship between osteoclasts and bone matrix. Matrix Biol 52–54:176–190CrossRefGoogle Scholar
  8. 8.
    Holtrop ME (1975) The ultrastructure of bone. Ann Clin Lab Sci 5:264–271PubMedGoogle Scholar
  9. 9.
    Holtrop ME, King GJ (1977) The ultrastructure of the osteoclast and its functional implications. Clin Orthop Relat Res (123):177–196Google Scholar
  10. 10.
    Nelson RL, Bauer GE (1977) Isolation of osteoclasts by velocity sedimentation at unit gravity. Calcif Tissue Res 22:303–313CrossRefGoogle Scholar
  11. 11.
    de Bernard B, Stagni N, Camerotto R, Vittur F, Zanetti M, Zambonin Zallone A, Teti A (1980) Influence of calcium depletion on medullary bone of laying hens. Calcif Tissue Int 32:221–228CrossRefGoogle Scholar
  12. 12.
    Zambonin Zallone A, Teti A (1981) The osteoclasts of hen medullary bone under hypocalcaemic conditions. Anat Embryol 162:379–392CrossRefGoogle Scholar
  13. 13.
    Zambonin Zallone A, Teti A, Primavera MV (1982) Isolated osteoclasts in primary culture: first observations on structure and survival in culture media. Anat Embryol 165:405–413CrossRefGoogle Scholar
  14. 14.
    van de Wijngaert FP, Rademakers LH, Schuurman HJ, de Weger RA, Kater L (1983) Identification and in situ localization of the "thymic nurse cell" in man. J Immunol 130:2348–2351PubMedGoogle Scholar
  15. 15.
    Flynn MA, Qiao M, Garcia C, Dallas M, Bonewald LF (1999) Avian osteoclast cells are stimulated to resorb calcified matrices by and possess receptors for leukotriene B4. Calcif Tissue Int 64:154–159CrossRefGoogle Scholar
  16. 16.
    Blair HC, Kahn AJ, Crouch EC, Jeffrey JJ, Teitelbaum SL (1986) Isolated osteoclasts resorb the organic and inorganic components of bone. J Cell Biol 102:1164–1172CrossRefGoogle Scholar
  17. 17.
    Marchisio PC, Cirillo D, Naldini L, Primavera MV, Teti A, Zambonin-Zallone A (1984) Cell-substratum interaction of cultured avian osteoclasts is mediated by specific adhesion structures. J Cell Biol 99:1696–1705CrossRefGoogle Scholar
  18. 18.
    Teti A, Blair HC, Teitelbaum SL, Kahn AJ, Koziol C, Konsek J, Zambonin-Zallone A, Schlesinger PH (1989) Cytoplasmic pH regulation and chloride/bicarbonate exchange in avian osteoclasts. J Clin Invest 83:227–233CrossRefGoogle Scholar
  19. 19.
    Horne WC, Neff L, Chatterjee D, Lomri A, Levy JB, Baron R (1992) Osteoclasts express high levels of pp60c-src in association with intracellular membranes. J Cell Biol 119:1003–1013CrossRefGoogle Scholar
  20. 20.
    Chambers TJ, Magnus CJ (1982) Calcitonin alters behaviour of isolated osteoclasts. J Pathol 136:27–39CrossRefGoogle Scholar
  21. 21.
    Chambers TJ, Chambers JC, Symonds J, Darby JA (1986) The effect of human calcitonin on the cytoplasmic spreading of rat osteoclasts. J Clin Endocrinol Metab 63:1080–1085CrossRefGoogle Scholar
  22. 22.
    Malgaroli A, Meldolesi J, Zallone AZ, Teti A (1989) Control of cytosolic free calcium in rat and chicken osteoclasts. The role of extracellular calcium and calcitonin. J Biol Chem 264:14342–14347PubMedGoogle Scholar
  23. 23.
    Zaidi M, Datta HK, Patchell A, Moonga B, MacIntyre I (1989) 'Calcium-activated' intracellular calcium elevation: a novel mechanism of osteoclast regulation. Biochem Biophys Res Commun 163:1461–1465CrossRefGoogle Scholar
  24. 24.
    Chambers TJ, McSheehy PM, Thomson BM, Fuller K (1985) The effect of calcium-regulating hormones and prostaglandins on bone resorption by osteoclasts disaggregated from neonatal rabbit bones. Endocrinology 116:234–239CrossRefGoogle Scholar
  25. 25.
    Takahashi N, Akatsu T, Udagawa N, Sasaki T, Yamaguchi A, Moseley JM, Martin TJ, Suda T (1988) Osteoblastic cells are involved in osteoclast formation. Endocrinology. Ther Nova 123:2600–2602Google Scholar
  26. 26.
    Matsumoto HN, Tamura M, Denhardt DT, Obinata M, Noda M (1995) Establishment and characterization of bone marrow stromal cell lines that support osteoclastogenesis. Endocrinology 136:4084–4091CrossRefGoogle Scholar
  27. 27.
    Itzstein C, van 't Hof RJ (2012) Osteoclast formation in mouse co-cultures. Methods Mol Biol 816:177–186CrossRefGoogle Scholar
  28. 28.
    Lee SK, Lorenzo J (2006) Cytokines regulating osteoclast formation and function. Curr Opin Rheumatol 18:411–418CrossRefGoogle Scholar
  29. 29.
    Takayanagi H, Kim S, Koga T, Nishina H, Isshiki M, Yoshida H, Saiura A, Isobe M, Yokochi T, Inoue J, Wagner EF, Mak TW, Kodama T, Taniguchi T (2002) Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev Cell 3:889–901CrossRefGoogle Scholar
  30. 30.
    Rucci N, Rufo A, Alamanou M, Capulli M, Del Fattore A, Ahrman E, Capece D, Iansante V, Zazzeroni F, Alesse E, Heinegård D, Teti A (2009) The glycosaminoglycan-binding domain of PRELP acts as a cell type-specific NF-kappaB inhibitor that impairs osteoclastogenesis. J Cell Biol 187:669–683CrossRefGoogle Scholar
  31. 31.
    Capulli M, Maurizi A, Ventura L, Rucci N, Teti A (2015) Effective small interfering RNA therapy to treat CLCN7-dependent autosomal dominant osteopetrosis type 2. Mol Ther Nucleic Acids 4:e248CrossRefGoogle Scholar
  32. 32.
    Chambers TJ, Revell PA, Fuller K, Athanasou NA (1984) Resorption of bone by isolated rabbit osteoclasts. J Cell Sci 66:383–399PubMedGoogle Scholar
  33. 33.
    Teti A, Colucci S, Grano M, Argentino L, Zambonin Zallone A (1992) Protein kinase C affects microfilaments, bone resorption, and [Ca2+]o sensing in cultured osteoclasts. Am J Phys 263:C130–C139CrossRefGoogle Scholar
  34. 34.
    Collin-Osdoby P, Osdoby P (2012) RANKL-mediated osteoclast formation from murine RAW264.7 cells. Methods Mol Biol 816:187–202CrossRefGoogle Scholar
  35. 35.
    Dunford JE, Rogers MJ, Ebetino FH, Phipps RJ, Coxon FP (2006) Inhibition of protein prenylation by bisphosphonates causes sustained activation of Rac, Cdc42, and Rho GTPases. J Bone Miner Res 21:684–694CrossRefGoogle Scholar
  36. 36.
    Murillo A, Guerrero CA, Acosta O, Cardozo CA (2010) Bone resorptive activity of osteoclast-like cells generated in vitro by PEG-induced macrophage fusion. Biol Res 43:205–224CrossRefGoogle Scholar
  37. 37.
    Amoui M, Suhr SM, Baylink DJ, Lau KHW (2004) An osteoclastic protein-tyrosine phosphatase may play a role in differentiation and activity of human monocytic U-937 cell-derived, osteoclast-like cells. Am J Physiol Cell Physiol 287:C874–C884CrossRefGoogle Scholar
  38. 38.
    Caselli GF, Mantovanini M, Gandolfi CA, Allegretti M, Fiorentino S, Pellegrini L, Melillo G, Bestini R, Sabbatici W, Anacardio R, Clavenna G, Sciortino G, Teti A (1997) Tartronates: a new generation of drugs affecting bone metabolism. J Bone Miner Res 12:972–981CrossRefGoogle Scholar
  39. 39.
    Prallet R, Male P, Neff L, Baron R (1992) Identification of a functional mononuclear precursor of the osteoclast in chicken medullary bone marrow cultures. J Bone Miner Res 7:405–414CrossRefGoogle Scholar
  40. 40.
    Reddy SV (2004) Regulatory mechanisms operative in osteoclasts. Crit Rev Eukaryot Gene Expr 14:255–270CrossRefGoogle Scholar
  41. 41.
    Neri T, Muggeo S, Paulis M, Caldana ME, Crisafulli L, Strina D, Focarelli ML, Faggioli F, Recordati C, Scaramuzza S, Scanziani E, Mantero S, Buracchi C, Sobacchi C, Lombardo A, Naldini L, Vezzoni P, Villa A, Ficara F (2015) Targeted gene correction in osteopetrotic-induced pluripotent stem cells for the generation of functional osteoclasts. Stem Cell Reports 5:558–568CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biotechnological and Applied Clinical SciencesUniversity of L’AquilaL’AquilaItaly
  2. 2.Department of Basic Medical Sciences, Neuroscience and Sense OrgansUniversity of BariBariItaly

Personalised recommendations