A Novel Reliable and Efficient Procedure for Purification of Mature Osteoclasts Allowing Functional Assays in Mouse...

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 ex vivo, analyses on OCLs are usually performed on in vitro 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.