Methods:
Synovial tissue was obtained from a total of 16 patients fulfilling the American Rheumatism Association criteria for RA [8] and 21 patients with osteoarthritis (OA) under approval of the local Ethics Committees. The tissue was placed in cell culture medium at ambient temperature and subjected to tissue digestion within 2 h. Synovectomy samples of RA and OA SM were finely minced, digested for 30 min at 37°C in phosphate-buffered saline (PBS) containing 0.1% trypsin (Sigma, Deisenhofen, Germany), and thereafter digested in 0.1% collagenase P (Boehringer Mannheim, Mannheim, Germany) in Dulbecco's modified Eagle medium (DMEM)/10% fetal calf serum (FCS) for 2 h at 37°C, 5% CO2. The cell suspension was then filtered and the cells collected by centrifugation. Cells were kept in primary culture for 7 days (DMEM/10% FCS, 25 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml amphotericin B [Gibco BRL, Eggenstein, Germany], including removal of non-adherent cells on days 1, 3, 5, and 7) and subsequently used for SFB isolation. The samples were randomly tested to exclude Mycoplasma contamination. For negative isolation of SFB from primary culture, adherent synovial cells were detached by short-term trypsinization for 2 min (0.25% trypsin/0.2% EDTA; Gibco) and 107/ml synovial cells were incubated with 4 × 107/ml Dynabeads® M-450 CD14 (clone RMO52; Dynal, Hamburg, Germany) in PBS/2% FCS for 1 h at 4°C. Nine milliliters of PBS/2% FCS were then added and the conjugated cells collected using the Dynal magnetic particle concentrator®. The compositions of magnetobead-conjugated cells and unconjugated cells were analyzed by flow cytometry. Phenotype analysis of the expression of FB markers, as well as that of SFB features previously reported at a tissue level, was conducted by flow cytometry in RA-SFB, either negatively isolated from primary culture or obtained from conventional fourth passage. The findings were compared with those of normal skin-FB (lineage control) and OA-SFB (disease control). The proliferation of RA-SFB, either negatively isolated from primary culture or obtained from conventional fourth passage, was assayed by [3H]-thymidine incorporation.