Osteoclasts and their activity are key regulators of bone formation. However, studying osteoclasts is difficult. Primary osteoclast cultures are difficult to maintain and isolate. Also, the amount of cells that are isolated and their properties depend on the origin and differentiation protocols. These protocols are usually developed in a distinct lab and multiple protocols exist. A cell line to study osteoclasts and a thorough study of osteoclast differentiation and culturing is currently lacking. The RAW264.7 cell line is most commonly used to study osteoclast differentiation and its signaling pathways. RAW264.7 cells are not a homogenous cell line. They don't often exclusively differentiate into osteoclast but also into other multinucleated cells as well including macrophage polykaryons. A challenge of culturing RAW264.7 cells are culture conditions. Different conditions can affect survival, proliferation, and differentiation of RAW264.7 cells. Currently published protocols of culturing RAW264.7 cells often assume multinucleated cells that have three or more nuclei with distinguished osteoclast characteristics (such as TRAP+) as osteoclasts. However, osteoclasts and macrophage polykaryons are almost indistinguishable under a light microscope (TRAP+ with three or more nuclei). The goal of this paper is to examine the effect of culture conditions on the osteoclastogenesis ability of RAW264.7 cells. The focus will be on establishing the crucial parameters for culture density, time of stimulation, RANKL, and L-Gln concentrations. Although we are unable to establish the condition that offers a homogenous population of osteoclasts; nevertheless, we are able to identify the optimal conditions at which osteoclasts are found to be more than macrophage polykaryons. Finally, this article also demonstrates that osteoclasts and macrophage polykaryons can be distinguished by immunofluorescence staining for cathepsin K.
Keywords: Macrophage Polykaryons; Osteoclast Culture Protocol; Osteoclastogenesis; RAW264.7.