Stemmacs expansion medium

The first sample contained resident SF-MSCs, and the second aspirate sample contained Sm-MSCs, both of which were retrieved and collected as previously described.^{4 (link)}
The aspirated fluid was centrifuged (500 rcf [relative centrifugal force] for 5 minutes), and cells were resuspended in 10 mL of Dulbecco’s modified Eagle medium (DMEM) containing 100 U/mL of penicillin and 100 mg/mL of streptomycin (all from Invitrogen); the SF cells were split according to the experimental design shown in Appendix Figure A1 (available in the online version of this article). For MSC expansion, cells were cultured in StemMACS expansion medium (Miltenyi Biotec), containing penicillin and streptomycin with twice-weekly media changes, and expanded for 3 or 4 passages. Moreover, donor-matched cultures were used in all experiments when they reached passage 3.

hBMSC were obtained after femoral head removal from seven patients who underwent total hip arthroplasty surgery (mean age: 64.6 ± 7.8 years, range: 50–72 years, female: 57.1%). hBMSC were harvested by density gradient centrifugation according to established protocols in this lab (Leyh et al., 2014a (link),b (link)). Subsequently, hBMSC were expanded for three passages in StemMACS Expansion Medium (Miltenyi Biotec, Germany) supplemented with 0.2% MycoZap (#923c1069, Lonza, Switzerland) before usage.
Human articular cartilage biopsies were obtained from OA patients' knee joints who underwent total knee arthroplasty surgery (mean age: 67.3 ± 7.7 years, range: 57–79 years, female: 87.5%). Cartilage was cut into small pieces after being removed from the subchondral bone and digested with 0.2% type II collagenase in Dulbecco's modified Eagle's medium (DMEM F12) (#D8437, Sigma-Aldrich, Germany). After 18 h digestion, passage 0 chondrocytes were pelleted and expanded in DMEM F12 medium, supplemented with 10% fetal calf serum (FCS) (#F7524, Sigma-Aldrich, Germany) and 1% penicillin–streptomycin (#A5955, Sigma-Aldrich, Germany).

Human bone marrow-derived mesenchymal stromal cells (hBMSC) were obtained from nine patients (mean age 62.8 ± 7.9 years, range 50–72 years, female 55.6%) who underwent total hip arthroplasty surgery due to OA. hBMSC were harvested by density gradient centrifugation according to established protocols [32 (link), 33 (link)]. Subsequently, hBMSC were expanded for three passages in StemMACS Expansion Medium (Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with 0.2% MycoZap (#923c1069, Lonza, Switzerland) before use. Human articular cartilage biopsies were obtained from fifteen OA patients (mean age 66.4 ± 7.2 years, range 56–79 years, female 73.2%) who underwent total knee arthroplasty surgery. Healthy (non-OA) articular cartilage was obtained from the knee joints of five cadavers (mean age 24 ± 6.3 years, range 17–34 years, female 20.1%). Cartilage biopsies were cut into small pieces after being dissected from subchondral bone and digested with 0.2% type II collagenase in Dulbecco’s modified Eagle’s medium (DMEM, #D8437, Sigma, UK). After 18 h digestion, chondrocytes were pelleted, seeded in T175 flasks, and expanded in DMEM F12 medium, supplemented with 10% FCS and 1% penicillin–streptomycin until 70–80% confluency.