Background

Osteoclast lineage cells (OLCs) derived from bone marrow monocytes/macrophages (BMMs) govern bone resorption to regulate bone remodeling (Quinn et al., 1998). BMMs first differentiate into the committed osteoclast precursors (OCPs), which further fuse together to form mature osteoclasts (MOCs) (Asagiri and Takayanagi, 2007). Mounting evidence demonstrates that the dysregulation of osteoclasts could cause skeletal disorders, such as osteoporosis and Paget’s disease (Feng and McDonald, 2011; Novack and Mbalaviele, 2016). The excessive osteoclast activity and aberrant osteoclastic resorption in subchondral bone could also contribute to the progression of osteoarthritis (Zhen et al., 2013; Zhu et al., 2019; Liu et al., 2021). Thus, it is essential to directly isolate live OLCs in vivo, for studying their biological features and pathologic changes.

The laboratory mouse is a commonly used animal model for investigating osteoclast function (Elefteriou and Yang, 2011). The conventional protocol for obtaining osteoclasts relies on stimulating the primary mouse BMMs from bone marrow cells (BMCs) with macrophage colony stimulating factor (M-CSF) and receptor activator of NFκB-ligand (RANKL) in vitro (Glantschnig et al., 2003; Marino et al., 2014). This protocol requires more than one week to form the MOCs in vitro, which could not capture their real-time status in vivo. Alternatively, fluorescence-activated cell sorting (FACS) has been previously employed to directly isolate the live OLCs from BMCs in vivo (Atkins et al., 2006; Zhang et al., 2012; Madel et al., 2018). Nevertheless, the disadvantages of FACS, including time-consuming procedures, low yield, and high cost, significantly limit its widespread use (Lee and Lufkin, 2012; Almeida et al., 2014). Therefore, it is necessary to introduce more efficient isolation techniques for directly obtaining live OLCs in vivo.

Magnetic-activated cell sorting (MACS) is another frequently used technique for sorting live cells in vivo, which enables rapid cell isolation with high yield and low cost (Lee and Lufkin, 2012; Almeida et al., 2014). This technique has been previously applied to recognize single surface markers of osteoclasts, for isolating OLCs from chicken or mouse BMCs in vivo (Collin-Osdoby and Osdoby, 2012; Liu et al., 2015). Since no specific surface marker for defining OCPs or MOCs has been found until now, it is not feasible to directly distinguish OCPs and MOCs from OLCs through one single surface marker. Calcitonin receptor (CTR), which is highly expressed on osteoclasts, is commonly used for characterizing MOCs, while osteoclast-associated receptor (OSCAR) was considered a typical cell surface marker for OLCs. We have previously employed MACS to harvest the OSCAR⁺ OLCs from mouse bone marrow in vivo for downstream gene and miRNA expression assay (Liu et al., 2015; Li et al., 2016). Considering the distinctive expression of OSCAR and CTR throughout the differentiation stages of OLCs, OSCAR may serve as an ideal surface marker for identifying OLCs among BMCs, while CTR could be used to distinguish the MOCs (OSCAR⁺CTR⁺) from the OCPs (OSCAR⁺CTR^–) within the OSCAR⁺ OLCs. Therefore, we further developed a two-step MACS method for distinguishing OCPs and MOCs from OLCs in BMCs, which demonstrated high efficiency in the direct capture of OCPs and MOCs from mice that had undergone surgery-induced osteoarthritis in our most recent study (Liu et al., 2021).

Here, we described this novel two-step MACS protocol for rapidly isolating OCPs and MOCs directly from mouse bone marrow with high yield and purity. This protocol consists of three main steps: BMC preparation, antibody-microbead labelling and magnetic sorting, and osteoclast characterization (Figure 1). In brief, the CTR⁺ and CTR^– cell fractions were first separated from mouse BMCs by magnetic positive and negative selection. Thereafter, the OSCAR⁺CTR⁺ MOCs, among the CTR⁺ cell fractions, and the OSCAR⁺CTR^– OCPs, among the CTR^– cell fractions, were enriched by another magnetic positive selection. We employed this protocol to rapidly isolate the MOCs and OCPs from both ovariectomized (OVX) and sham-operated (Sham) mice. The sorting procedures can be carried out in four mice concurrently within ~5 h.

Figure 1. Schematic diagram of isolation for osteoclast lineage cells (OLCs) from mouse bone marrow