A practical procedure for the in vitro generation of human osteoclasts and their characterization.

Osteoclasts are multinucleated cells derived from the hematopoietic monocyte/macrophage lineage that possess the unique capacity to resorb bone. Due to the crucial role of osteoclasts in maintaining bone homeostasis and pathologies, this cell type is pivotal in multiple research areas dedicated to bone physiology in health and disease. Although numerous methods for generation of human osteoclasts are already available, those rely either on cell labeling-based purification, or an intermediate adhesion step after which cells are directly differentiated toward osteoclasts. While the former requires additional reagents and equipment, the latter harbors the risk of variable osteoclast formation due to varying numbers of osteoclast precursors available for different donors. Herein we report a facile and reliable three-step method for the generation of human osteoclasts from blood-derived precursor cells. Monocytes were obtained after adhering peripheral blood-derived mononuclear cells (PBMCs) to plastic substrates followed by macrophage induction and proliferation resulting in a homogeneous population of osteoclast precursors. Finally, macrophages were seeded into suitable culture vessels and differentiated towards osteoclasts. Osteoclastogenesis was monitored longitudinally using non-destructive techniques, while the functionality of mature osteoclasts was confirmed after 14 days of culture by analysis of functional (e.g. elevated tartrate-resistant acid phosphatase (TRAP)-activity, resorption) and morphological (e.g. presence of TRAP, actin ring and integrin β3) characteristics. Furthermore, we propose to use combinatory staining of three morphological osteoclast markers, rather than previously reported staining of a single or maximal two markers, to clearly distinguish osteoclasts from undifferentiated mononuclear cells.