Isolation of Human Osteal Macrophages.

Osteal macrophages ("osteomacs") are resident bone macrophages that support osteoblast differentiation and bone formation. Despite their importance in bone homeostasis, their function in human bone metabolism and osteoporosis remains poorly understood, largely due to the lack of a standardized isolation protocol. Here, we present a protocol for isolating primary human osteomacs from femoral head specimens obtained during arthroplasty. After the removal of bone marrow to minimize contamination with marrow-derived macrophages, bone fragments were enzymatically digested and osteomacs were isolated using CD14-based MACS® or CD14/CD45/ALP-based FACS. Immunofluorescence confirmed macrophage identity and revealed expression of markers associated with both M1-like and M2-like activation states. Isolated cells displayed heterogeneous morphology and could be maintained in culture. This protocol enables reproducible isolation of human osteomacs and provides a foundation for translational studies investigating osteoimmune interactions in bone disease and osteoporosis.