Synovial tissues were obtained from synovial biopsies of six patients with RA undergoing joint surgery (synovectomy or joint replacement by prosthesis implantation), who all met the criteria of the American College of Rheumatology [27 (link)]. The tissue samples were obtained during routine surgery at the Department of Orthopedics of the University of Regensburg, where approved by the local ethics committee and patients involved gave informed consent. Culture of SF was performed as described recently [5 (link)]. Following enzymatic digestion, fibroblasts were grown in DMEM (Biochrom, Berlin, Germany) containing 10% heat inactivated FCS (Gibco Life Technologies, Grand Island, NY, USA), 100 U/ml penicillin and streptomycin (PAA Laboratories GmbH, Linz, Austria) and cultured for four passages at 37°C in 10% carbon dioxide. The SF were stained for a fibroblast marker by immunohistochemistry. More than 95% could be stained positively for the fibroblast enzyme prolyl 4-hydroxylase and none were positive for the macrophage marker CD68 or the neutrophil marker cathepsin G after the second passage of cultivation with enzymatic digestion equalling passage 0 (data not shown). Routine tests for mycoplasms were negative. At 85 to 95% confluency, cells were passaged 1:2 and a part of the cells was harvested. Total RNA was extracted and stored at -70°C. Culture conditions were (and have to be) kept constant during the experiments. Passaging of the cells was performed at 85 to 95% confluency as fibroblasts exhibit contact inhibition (unpublished observations) and were passaged 1:2 to provide cell-cell contacts between the cells.
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