Experimental setup. Human articular chondrocytes were isolated from six normal donors post mortem and expanded in monolayer culture. After cryopreservation and a second monolayer expansion, the cells were encapsulated in alginate beads and cultured three-dimensionally for 14 days. Subsequently, the cartilage-like beads were stimulated for 48 hours with supernatants (sn) of SV40 T-antigen immortalized human synovial fibroblasts derived from a healthy, normal donor (NDSF) and from a patient with rheumatoid arthritis (RASF), respectively. Supernatants of RASF (RASFsn) and NDSF (NDSFsn) and medium control were analyzed for soluble mediators with the use of antibody-based protein membrane arrays. Genome-wide expression analyses of NDSFsn-stimulated and RASFsn-stimulated chondrocytes were performed with oligonucleotide microarrays. Additionally, unstimulated chondrocytes were analyzed for baseline expression. Two independent experiments (n = 2) were performed for NDSFsn-stimulated and RASFsn-stimulated and unstimulated chondrocytes; each experimental group (G1, G2) consisted of chondrocytes derived from three different donors. Expression of selected differentially expressed genes was validated by real-time RT-PCR.