Evaluation of culture conditions for osteoclastogenesis in RAW264.7 cells

Abstract

Osteoclasts are the only multinucleated cells in vivo responsible for bone resorption and are vital for regulating bone remodeling and maintaining bone mass. The RAW264.7 cell line is widely used to study osteoclastic differentiation and biological molecular mechanism. However, protocols for inducing osteoclast formation in RAW264.7 cells vary considerably between laboratories, hindering the replication of results. Therefore, we tested the influence of culture conditions on osteoclast differentiation, including cell density and receptor activator of nuclear factor kappa-B ligand (RANKL) concentrations with or without macrophage colony-stimulating factors (M-CSF). Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the morphology of osteoclasts. qPCR was used to detect gene expression of osteoclast-specific gene marker cathepsin K (CTSK), osteoclast transcription factors c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1). The bone resorption function was evaluated by a scanning electron microscope (SEM). RANKL treatment increased multinucleated osteoclasts formation and increased CTSK, c-Fos and NFATc1 gene expression. Compared with RANKL treatment, M-CSF significantly decreased multinucleated osteoclasts formation, reduced CTSK gene expression and had little effect on c-Fos and NFATc1 gene expression. Concerning bone resorption activity, RANKL treatment increased bone resorption pits on bovine bone slices. Significantly higher levels of osteoclastogenesis were observed with RAW264.7-cell density of 2×104 cells/well in 24-well plates. Our results suggest that the addition of 50 ng/ml M-CSF has no positive effect on osteoclastogenesis. RANKL treatment and cell density contribute to osteoclast formation, and the optimal conditions are beneficial when exploring osteoclast function and mechanism.

In Vitro Effects of Weissella cibaria CMU and CMS1 on Receptor Activator of NF-κB Ligand (RANKL)-Induced Osteoclast Differentiation

“…Treating RAW 264.7 cells with RANKL initiates osteoclast differentiation signaling through RANK, thereby inducing the expression of key differentiation-related genes, including c-Fos, NFATc1, OSCAR, DC-STAMP, cathepsin K, and TRAP [ 7 , 8 , 9 , 10 , 11 , 12 , 13 ]. Furthermore, c-Fos and NFATc1 work synergistically to induce the expression of several key osteoclastogenesis-related genes [ 7 , 8 , 9 , 10 ].…”

“…DC-STAMP is known to play a role in cell–cell fusion and is one of the fusion mediator molecules directly regulated by NFATc1 [ 13 ]. Meanwhile, cathepsin K and TRAP are cysteine proteases secreted by osteoclasts that are responsible for decomposing matrix proteins during bone resorption [ 12 ].…”

“…RANKL binds to RANK present in osteoclast precursors, activating the mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways and sequentially activating nuclear factor of activated T cells c1 (NFATc1), which is essential for osteoclast differentiation [ 8 , 9 ]. Furthermore, it is known to promote osteoclast differentiation and bone resorption by inducing the expression of several genes [ 9 , 10 , 11 , 12 , 13 ]. Therefore, inflammatory periodontal disease associated with osteoclasts can be alleviated by suppressing osteoclast differentiation [ 14 ].…”

A novel method to efficiently differentiate human osteoclasts from blood-derived monocytes

“…[ 13 ], we observed that the cell density has a major effect on the efficiency of osteoclastogenesis and measured the most efficient differentiation at an intermediate cell number. The fact that seeding density significantly impacts osteoclast differentiation has not only been observed for osteoclasts of human origin, but also for differentiation protocols based on murine/rat origin [ 26 , 27 ], thus indicating that cell density is a critical parameter in determining efficient osteoclastogenesis across various culture models. Another critical factor is the concentration of the two cytokines that reprogram monocytes towards osteoclasts, M-CSF and RANKL.…”

Extracellular Proteins Isolated from L. acidophilus as an Osteomicrobiological Therapeutic Agent to Reduce Pathogenic Biofilm Formation, Regulate Chronic Inflammation, and Augment Bone Formation In Vitro

“…[ 113 ] During osteoclastic activation, both RANKL and CTSK are typically upregulated. [ 114 ] To distinguish between the M1, and M2/osteoclastic phenotype, macrophage morphology, the expression of RANKL and CTSK , and the presence of TRAP + staining were assessed. Our results show the cells remained mononucleated, with reduced levels of RANKL and CTSK on day 1, and became elongated and spindle‐shaped in a dose‐ and time‐dependent manner.…”