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.

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人类破骨细胞体外生成及其表征的实用程序

破骨细胞是源自造血单核细胞/巨噬细胞谱系的多核细胞，具有独特的骨吸收能力。由于破骨细胞在维持骨稳态和病理方面的关键作用，这种细胞类型在致力于健康和疾病骨骼生理学的多个研究领域中至关重要。尽管已经有许多产生人类破骨细胞的方法，但这些方法要么依赖于基于细胞标记的纯化，要么依赖于中间粘附步骤，之后细胞直接向破骨细胞分化。虽然前者需要额外的试剂和设备，但后者由于可用于不同供体的破骨细胞前体数量不同而存在形成可变破骨细胞的风险。在这项研究中，我们报告了一种从血液源性前体细胞生成人类破骨细胞的简便可靠的三步法。将外周血来源的单核细胞粘附到塑料基质上，然后进行巨噬细胞诱导和增殖，从而产生同质的破骨细胞前体群，从而获得单核细胞。最后，将巨噬细胞接种到合适的培养容器中并分化为破骨细胞。使用非破坏性技术纵向监测破骨细胞生成，而成熟破骨细胞的功能在培养 14 天后通过功能分析（例如，升高的抗酒石酸酸性磷酸酶 [TRAP] 活性、吸收）和形态学（例如，存在 TRAP 、肌动蛋白环和整合素 β 将外周血来源的单核细胞粘附到塑料基质上，然后进行巨噬细胞诱导和增殖，从而产生同质的破骨细胞前体群，从而获得单核细胞。最后，将巨噬细胞接种到合适的培养容器中并分化为破骨细胞。使用非破坏性技术纵向监测破骨细胞生成，而成熟破骨细胞的功能在培养 14 天后通过功能分析（例如，抗酒石酸酸性磷酸酶 [TRAP] 活性升高、吸收）和形态学（例如，存在 TRAP 、肌动蛋白环和整合素 β 将外周血来源的单核细胞粘附到塑料基质上，然后进行巨噬细胞诱导和增殖，从而产生均质的破骨细胞前体群，从而获得单核细胞。最后，将巨噬细胞接种到合适的培养容器中并分化为破骨细胞。使用无损技术纵向监测破骨细胞生成，而成熟破骨细胞的功能在培养 14 天后通过功能分析（例如，抗酒石酸酸性磷酸酶 [TRAP] 活性升高、吸收）和形态学（例如，存在 TRAP 、肌动蛋白环和整合素 β₃）特点。此外，我们建议使用三种形态破骨细胞标记的组合染色，而不是先前报道的单个或最多两个标记的染色，以清楚地区分破骨细胞与未分化的单核细胞。