Generation of osteoclasts in vitro, and assay of osteoclast activity

Methods Mol Med. 2007;135:285-301. doi: 10.1007/978-1-59745-401-8_18.


Osteoclasts are bone-resorbing multinucleated cells derived from the monocyte-macrophage lineage. The authors have developed a mouse marrow culture system and a coculture system of mouse osteoblasts and hemopoietic cells, in which osteoclasts are formed in response to various osteotropic factors such as 1alpha,25-dihydroxyvitamin D3, parathyroid hormone, prostaglandin E2, and interleukin-11. Recent studies have revealed that osteoblasts express two cytokines essential for osteoclastogenesis: receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Using RANKL and M-CSF, we can induce osteoclasts from monocyte-macrophage lineage cells even in the absence of osteoblasts. This chapter describes the methods for osteoclast formation in vitro in the presence and absence of osteoblasts, and for pit-formation assay using dentine slices and osteoclasts formed in vitro. These culture systems have made it possible to investigate each step of osteoclast development and function separately.

MeSH terms

  • Actins / metabolism
  • Animals
  • Bone Marrow Cells / cytology
  • Bone Resorption / metabolism
  • Bone Resorption / pathology
  • Buffers
  • Cathepsin K
  • Cathepsins / metabolism
  • Cell Differentiation / drug effects
  • Cell Line
  • Cell Separation / methods
  • Coculture Techniques / methods
  • Collagen
  • Culture Media
  • Gels
  • Immunohistochemistry / methods
  • Macrophage Colony-Stimulating Factor / metabolism
  • Macrophage Colony-Stimulating Factor / pharmacology
  • Macrophages / cytology
  • Mice
  • Models, Biological
  • Osteoblasts / cytology
  • Osteoclasts / cytology*
  • Osteoclasts / drug effects
  • Osteoclasts / metabolism
  • RANK Ligand / metabolism
  • RANK Ligand / pharmacology


  • Actins
  • Buffers
  • Culture Media
  • Gels
  • RANK Ligand
  • Tnfsf11 protein, mouse
  • Macrophage Colony-Stimulating Factor
  • Collagen
  • Cathepsins
  • Cathepsin K
  • Ctsk protein, mouse