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Optimizing in vitro osteoclastogenesis: bone marrow-derived macrophages differentiation and cell density as critical determinants

biotechnology
cell-biology
molecular-biology
orthopedics
metabolic-sciences

Abstract

Background. Osteoclasts are multinucleated cells essential for bone resorption and remodeling, and their activity is closely linked with bone formation. In vitro models of osteoclastogenesis are crucial for studying the mechanisms of osteoclast differentiation and related bone diseases. Optimizing these models is vital for advancing research in bone metabolism and therapeutic strategies.

Methods. In this study, we compared three methods for inducing osteoclast differentiation from mouse bone marrow-derived monocyte/macrophage (BMMs). Method 1 involved direct isolation of BMMs, Method 2 differentiated BMMs into bone marrow-derived macrophages (BMDM), and Method 3 incorporated Ficoll-Paque density gradient centrifugation prior to M-CSF-induced differentiation. Osteoclastogenesis efficiency was assessed using RT-qPCR, Western blot, TRAP staining, F-actin ring staining, and bone resorption assays. Flow cytometry analysis was performed to evaluate cell purity and osteoclast precursor enrichment.

Results. We found that Method 2, which involved differentiating BMMs into BMDM, yielded the highest proportion of live cells and osteoclast precursors, and exhibited the most efficient osteoclast differentiation. The optimal cell density for osteoclastogenesis was 1–2 × 10 5 cells/mL for Methods 2 and 3. In contrast, Method 3, despite the additional purification step, did not significantly improve precursor purity compared to Method 2, indicating that the extra purification did not enhance differentiation efficiency.

Conclusions. This study highlights the importance of precursor cell purity and seeding density in osteoclast differentiation. Method 2 (BMMs to BMDM) provides a simplified and effective approach for in vitro osteoclastogenesis, optimizing conditions for studying bone resorption and related diseases.

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