2.2. Preparation of Human Synovial Cells

RA synoviocytes were isolated from fresh synovial biopsies obtained from four RA patients undergoing finger arthroplasty. All patients fulfilled the 1987 American Rheumatism Association criteria for RA [29]. The mean age of the patients was 67.4 ± 3.2 years (range 53–81 years). The mean disease duration was 8.7 ± 2.3 years. At the time of surgery, the disease activity score (DAS 28) was greater than 3.2. These activities were approved by local institutional review boards, and all subjects gave written informed consent. Synovia were minced and digested with 1.5 mg/mL collagenase-dispase for 3–4 h at 37 °C as previously described [30]. After centrifugation, cells were resuspended in DMEM supplemented with 10% FCS, 4.5 g/L D-glucose, 25 mM Hepes, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco BRL) in a humidified atmosphere containing 5% (v/v) CO₂ at 37 °C. After 48 h, nonadherent cells were removed. Adherent cells (macrophage-like and FLS) were cultured in complete medium, and, at confluence, cells were trypsinized and only the FLS were passed. These cells were used between passages 4 and 8, when they morphologically resembled FLS after an indirect immunofluorescence study (see Culture of human RA FLS). RA FLS were cultured 45–60 days before experimentation. This delay allowed for the elimination of all possible interactions resulting from any preoperative treatment (with nonsteroidal anti-inflammatory drugs, analgesics, disease-modifying antirheumatic drugs, or steroids).