Joris van Dongen

109 tSVF acts anti-inflammatory on chondrocytes in vitro Fisher Scientific). Real-time polymerase chain reactions were performed using iScript universal SYBR Green (Biorad) in a LightCycler 96 (Roche). Primers used are shown in Table 1. Sulphated glycosaminoglycans analysis After 28 days of culture, cocultures of tSVF-derived cells and chondrocytes (n = 3 in triplicates) were digested at 60° C for 18h in a papain enzyme solution consisting of 5 mML-cysteine, 50 mMNa2 EDTA, 0.1MNaAc, pH 5.53 with 2% (v/v) papain (Sigma). To measure the concentration of sulphated glycosaminoglycans (GAGs), a dimethylene blue (DMMB) spectrophotometric analysis was performed. The papain digests were 1 : 10 diluted and mixed with the DMMB solution and absorbance was read at 540 nm and 595 nm. Known concentrations of chondroitin sulphate C (sigma) were used as a reference. To correct for the number of cells, DNA amount in the papain digests was measured. Papain digests were diluted 1:20 and Quant-iT Picogreen (Invitrogen) reagent was added. This was incubated for 5 min. at ambient temperature in the dark whereafter the fluorescence was measured at 480 nm excitation and 520 nm emission. Known concentrations of lambda DNAwere used as a standard. In vitro inflammation assay Chondrocytes (n = 3 in triplicates) were cultured in monolayer at 37°C and 5% CO2 at a seeding density of 25,000 cells/cm 2 in expansionmedium.After pre-incubation for 24h, cells were treated with 10 ng/mL tumor necrosis factor (TNF)-alpha (Immunotools). After 24h of treatment, the cells were washed and medium was refreshed with either control medium, conditioned medium (CM) from cultured chondrocytes or cultured tSVF-derived cells or chondrocytes or tSVF-derived cells were added. After 24h, RNA was isolated from the monolayers and gene expression of interleukin (IL)-1 β and cyclooxygenase-2 (COX2) were measured as described in 2.7.1. 18S was again used as housekeeping gene. Primers used are shown in table 1.