AbstractOsteoarthritis (OA) is characterized by an abundance of inflammatory M1‐like macrophages damaging local tissues. The search for new potential drugs for OA suffers from the lack of appropriate methods of long‐lasting inflammation. Here we developed and characterized an in vitro protocol of long‐lasting culture of primary human monocyte‐derived macrophages differentiated with a combination of M‐CSF+GM‐CSF that optimally supported long‐cultured macrophages (LC‐Mϕs) for up to 15 days, unlike their single use. Macrophages repeatedly stimulated for 15 days with the TLR2 ligand Pam3CSK4 (LCS‐Mϕs), showed sustained levels over time of IL‐6, CCL2, and CXCL8, inflammatory mediators that were also detected in the synovial fluids of OA patients. Furthermore, macrophages isolated from the synovia of two OA patients showed an expression profile of inflammation‐related genes similar to that of LCS‐Mϕs, validating our protocol as a model of chronically activated inflammatory macrophages. Next, to confirm that these LCS‐Mϕs could be modulated by anti‐inflammatory compounds, we employed dexamethasone and/or celecoxib, two drugs widely used in OA treatment, that significantly inhibited the production of inflammatory mediators. This easy‐to‐use in vitro protocol of long‐lasting inflammation with primary human macrophages could be useful for the screening of new compounds to improve the therapy of inflammatory disorders.