Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary culture - primary culture cells markedly differ from fourth-passage cells

To reduce culture artifacts by conventional repeated passaging and long-ter m culture in vitro, the isolation of synovial fibroblasts (SFB) was attempt ed from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation usi ng magnetobead-coupled anti-CD14 monoclonal antibodies. This method yielded highly enriched SFB (85% prolyl-4-hydroxylase(+)/74% Thy-1/CD90(+) cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in c omparison with conventional fourth-passage RA-SFB, showed a markedly differ ent phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1 beta. This isolation method is simple and reliable, and may yield cells with features closer to the in vi vo configuration of RA-SFB by avoiding extended in vitro culture.