CNRS UMR7370, Laboratoire de PhysioMédecine Moléculaire, Faculty of Medicine, Nice, France.
University Nice Sophia Antipolis, Nice, France.
Front Immunol. 2018 Nov 2;9:2567. doi: 10.3389/fimmu.2018.02567. eCollection 2018.
Osteoclasts (OCLs) are multinucleated phagocytes of monocytic origin responsible for physiological and pathological bone resorption including aging processes, chronic inflammation and cancer. Besides bone resorption, they are also involved in the modulation of immune responses and the regulation of hematopoietic niches. Accordingly, OCLs are the subject of an increasing number of studies. Due to their rarity and the difficulty to isolate them directly , analyses on OCLs are usually performed on differentiated cells. In this state, however, OCLs represent a minority of differentiated cells. Since up to date a reliable purification procedure is still lacking for mature OCLs, all cells present in the culture are analyzed collectively to answer OCL-specific questions. With the development of in-depth transcriptomic and proteomic analyses, such global analyses on unsorted cells can induce severe bias effects in further results. In addition, for instance, analysis on OCL immune function requires working on purified OCLs to avoid contamination effects of monocytic precursors that may persist during the culture. This clearly highlights the need for a reliable OCL purification procedure. Here, we describe a novel and reliable method to sort OCLs based on cell multinucleation while preserving cell viability. Using this method, we successfully purified multinucleated murine cells. We showed that they expressed high levels of OCL markers and retained a high capacity of bone resorption, demonstrating that these are mature OCLs. The same approach was equally applied for the purification of human mature OCLs. Comparison of purified OCLs with mononucleated cells or unsorted cells revealed significant differences in the expression of OCL-specific markers at RNA and/or protein level. This exemplifies that substantially better outcomes for OCLs are achieved after the exclusion of mononucleated cells. Our results clearly demonstrate that the in here presented procedure for the analysis and sorting of pure OCLs represents a novel, robust and reliable method for the detailed examination of bona fide mature OCLs in a range that was previously impossible. Noteworthy, this procedure will open new perspectives into the biology of osteoclasts and osteoclast-related diseases.
破骨细胞（OCLs）是单核细胞来源的多核吞噬细胞，负责生理和病理骨质吸收，包括衰老过程、慢性炎症和癌症。除了骨质吸收，它们还参与免疫反应的调节和造血龛的调节。因此，OCLs 是越来越多研究的主题。由于其稀有性和直接分离的困难，对 OCLs 的分析通常在分化细胞上进行。然而，在这种状态下，OCLs 仅代表分化细胞的少数。由于迄今为止，对于成熟 OCLs 仍然缺乏可靠的纯化程序，因此所有存在于培养物中的细胞都被集体分析，以回答 OCL 特异性问题。随着深入的转录组学和蛋白质组学分析的发展，对未分选细胞的这种全面分析可能会在进一步的结果中引起严重的偏差效应。此外，例如，对 OCL 免疫功能的分析需要在纯化的 OCL 上进行，以避免在培养过程中可能持续存在的单核细胞前体的污染影响。这清楚地强调了对可靠的 OCL 纯化程序的需求。在这里，我们描述了一种基于细胞多核化同时保持细胞活力的新型可靠的 OCL 分选方法。使用该方法，我们成功地纯化了多核化的鼠细胞。我们表明，它们表达高水平的 OCL 标志物并保留高骨吸收能力，证明这些是成熟的 OCL。同样的方法也同样适用于纯化人类成熟 OCLs。与单核细胞或未分选细胞相比，对纯化的 OCLs 的比较显示在 RNA 和/或蛋白质水平上 OCL 特异性标志物的表达存在显著差异。这说明在排除单核细胞后，OCLs 的结果得到了显著改善。我们的结果清楚地表明，这里提出的用于纯 OCL 分析和分选的程序代表了一种新颖、稳健和可靠的方法，用于以前不可能的真正成熟 OCL 的详细检查。值得注意的是，该程序将为破骨细胞和破骨细胞相关疾病的生物学研究开辟新的视角。
