Streptomycin

Isolation of human PBMCs from buffy coats from healthy donors (Dutch Blood Bank, Netherlands) was performed by collecting the enriched fraction of cells after density gradient centrifugation (Greiner Bio-One, The Netherlands) as previously described (18 (link)). Monocytes were isolated from PBMCs through magnetic separation via negative selection according to the manufacturer’s instructions (Miltenyi Biotec, Germany). Subsequently, monocytes were cultured for 7 days in RPMI 1640 (Lonza, Switzerland) with 10% FCS, penicillin (100 U/mL), and streptomycin (100 μg/mL) at 2×10^6 cells/mL. Human recombinant IL4 (100 ng/mL) and GM-CSF (60 ng/mL) (Prospec, Israel) were added to differentiate monocytes into monocyte-derived dendritic cells (moDCs). Every other day half of the medium was refreshed and new cytokines were added until the moDCs were collected for coculture. Naïve CD4+ T cells were isolated from PBMCs using a negative selection MACS kit according to the manufacturer’s instructions (Miltenyi Biotec). The collected T cells were frozen in 90% FCS and 10% DMSO at −80°C until use during coculture. Upon gentle thawing, the cells were resuspended in T cell medium (IMDM with 10% FCS, penicillin (100 U/mL), streptomycin (100 μg/mL), 20 μg/mL apotransferrin (Sigma-Aldrich), and 50 μM β-mercaptoethanol) and directly used.

Monocytes were cultured at a concentration of 0.75 × 10⁶ cells/mL in a 6-well plate in RPMI 1640 supplemented with 10% heat-inactivated FCS, penicillin (100 U/mL)/streptomycin (100 μg/mL), IL4 (10 ng/mL; ProSpec-Tany TechnoGene Ltd., Ness Ziona, Israel) and GM-CSF (5 ng/mL; ProSpec-Tany TechnoGene Ltd., Ness Ziona, Israel). The cells were kept for 7 days in an incubator at 37°C and 5% CO₂. At days 2, 3, and 6, 1 mL medium was refreshed. At day 7, the imDC were suitable to use in the transwell IEC-DC coculture assay.

After isolated chondrocytes were plated for 72 h for cell recovery, chondrocytes were washed with sterile PBS (Ca²⁺/Mg²⁺ free) twice. Then, chondrocytes were trypsinized for 5 min at 37 °C and centrifuged at 433 × g for 5 min to collect chondrocytes for making pellets following the procedure below^{54 (link)}. After a cell wash and cell counting with trypan blue, 5 × 10⁵ chondrocytes were resuspended in 500 µL defined chondrogenic SFM [high glucose DMEM, HEPES (10 mM), human serum albumin (125 mg/mL), ascorbic acid 2-phosphate (365 lg/mL), dexamethasone (100 nM), and L-proline (40 lg/mL) (Sigma-Aldrich), ITS + 1 premix (5 µL, 100x) (Corning, Discovery Labware, Inc.), 100 units/mL penicillin, 100 µg/mL streptomycin, TGF-b3 10 ng/mL; ProSpec, NJ, USA] in a 1.5 mL sterile conical microtube (Bio Basic Inc, Ontario, Canada). Then, chondrocytes were centrifuged at 433 × g and 22 °C for 5 min to form a pellet at the bottom of the microtube using an Allegra X-22R centrifuge (Beckman Coulter, US). The pellets were cultured in the SFM under 3% O₂ and 5% CO₂ at 37 °C in a humidified incubator for 21 days, with SFM changes twice a week.