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. In this study, 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 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 toward osteoclasts. Osteoclastogenesis was monitored longitudinally using nondestructive 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. Impact statement Research related to bone biology requires a standardized and reliable method for in vitro generation of human osteoclasts. We here describe such a procedure which avoids shortcomings of previously published protocols. Further, we report on nondestructive methods to qualitatively and quantitatively monitor osteoclastogenesis longitudinally, and on analysis of osteoclast generation and functionality after 14 days. Specifically, we recommend assessment of morphological human osteoclast characteristics using combinatory staining of three markers to confirm successful osteoclast generation.