Pt 3273

In selected experiments, ADSC were grown to 70% confluence, washed three times with serum-free medium and cultured in ADSC basal medium (catalog no. PT-3273, Lonza) supplemented with 2% fetal bovine serum for 24 h. Medium was removed and cells were then incubated in ADSC basal medium supplemented with 2% fetal bovine serum and recombinant human TGFβ1 (10 ng/mL; PeproTech) or 10% serum from patients with early dcSSc (n = 6) and healthy individuals (n = 6) for 72 and 96 h for subsequent analyses of mRNA and protein expression levels of relevant markers, respectively. Each serum sample was tested individually. The medium was changed, and additives replenished, every day.

ADSCs (PT-5006, Lonza) (<6th passage) were cultured in 175 cm² flasks in ADSC Basal Medium (ADSC-BM) (PT-3273, Lonza) supplemented with ADSC Growth Medium (ADSC-GM) SingleQuots (PT-4503, Lonza). Cultures were maintained in a humidified incubator at 37°C and 5% CO₂.
AC cells (CC-2550, Lonza) were cultured at the recommended seeding density (10,000 cells/cm²) from passage 2 to passage 6 in 25, 75 and 175 cm² culture flasks. The expansion medium was composed of Chondrocyte Basal Medium (CBM) (CC-3217, Lonza) plus SingleQuots of Growth Supplements (CC-4409, Lonza) containing R3-IGF-1, bFGF, transferrin, insulin, FBS and gentamicin/amphotericin-B. Cultures were maintained in a humidified incubator at 37°C and 5% CO₂.