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

Osteoclasts are the tissue-specific macrophage population of the bone and unique in their bone-resorbing activity. Hence, they are fundamental for bone physiology in health and disease. However, efficient protocols for the isolation and study of primary human osteoclasts are scarce. In this study, we aimed to establish a protocol, which enables the efficient differentiation of functional human osteoclasts from monocytes. To this end, human monocytes were isolated through a double-density gradient from healthy donor blood. The differentiation to macrophages was performed either in cell culture dishes or in Teflon-coated bags. The impact of these two distinct differentiation protocols on the gene expression profile of macrophages was assessed via RNA-Sequencing. The macrophages were then further differentiated to terminal osteoclasts through the addition of Receptor Activator of NF-κB Ligand (RANKL). The yield and function of multinuclear osteoclasts was significantly increased upon initial differentiation of monocytes to macrophages in Teflon-coated bags. Our study has established a novel protocol for the isolation of primary human osteoclasts that improves osteoclastogenesis in comparison to the conventionally used cultivation approach. Monocytes from 5 healthy donors were differentiated to macrophages in the presence of M-CSF. Two differentiation protocols were compared for matched samples of the same donor: 1. Macrophages derived from monocytes cultured in regular cell culture plates; 2. Macrophages derived from monocytes cultured in Teflon-coated bags