Osteoclast Formation in the Mouse Coculture Assay

The murine coculture assay originally described by Takahashi et al. (1 ), was the first culture system developed that generated genuine, bone-resorbing osteoclasts. In this assay, osteoblasts are stimulated with 1,25-dihydroxyvitamin D₃ (D3) to stimulate RANKL and macrophage colony-stimulating factor (M-CSF) expression. These factors then stimulate early osteoclast precursors present in the spleen or bone marrow cell populations to differentiate into mature osteoclasts. At the end of the culture, osteoclasts can be identified by tartrate-resistant acid phosphatase (TRAP) staining, and, when the cultures are performed on dentine slices, resorption activity can be measured as well. Even though today it is possible to generate osteoclasts from bone marrow cells alone by treating the cultures with RANKL and M-CSF, the coculture system is still a useful model for studying osteoblast-osteoclast interactions. It has been widely used to study the origin of the osteoclast (2 ) and the effects of growth factors and drugs on osteoclast formation (3 ,4 ). In studies with osteopetrotic mice, the coculture assay has been used to determine whether the underlying mechanism was due to a defect in the osteoblasts or in the osteoclast precursors (5 ).