75 cm2 culture flask

HEK‐293T cells were purchased from the American Type Culture Collection (ATCC). MSMSCs were isolated from normal human MSM according to our previously published methods.6 Briefly, tissues were minced and digested with 3 mg/mL collagenase type I (Sigma, St. Louis, Missouri) and 4 mg/mL dispase (Roche, Mannheim, Germany) for 1 h at 37°C. Then the samples were passed through a 70 μm strainer (Falcon, BD Labware, Franklin Lakes, New Jersey). Cells were supplemented into 75‐cm² culture flasks (Costar, Cambridge, Massachusetts) with alpha modification of Eagle's medium (GIBCO BRL, Grand Island, New York), and then incubated in 5% CO₂ and 37°C.

The study protocol was approved by the ethics committee of The First Affiliated Hospital of Soochow University. Human cartilage samples were carefully cut into fragments of 1 mm³ from tibial plateaus of six patients (four females and two males) undergoing knee replacement surgery. Cartilage fragments were washed in phosphate-buffered saline (PBS) for 5 min and digested with 0.4% collagenase II (Gibco, Invitrogen) for 12 h at 37°C. The released cells were filtered through a 100-μm nylon mesh (BD, Biosciences, San Jose, CA, USA). Cells were seeded into 75-cm² culture flasks (CoStar, Tewksbury, MA, USA) and cultured in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 with 10% fetal bovine serum (Gibco, heat-inactivated), penicillin (100 U/mL), and streptomycin (100 U/mL) at 37°C in a 5% carbon dioxide atmosphere. Primary cells were trypsinized with 0.25% trypsin-ethylenediamine tetraacetic acid (EDTA, Invitrogen) followed by centrifugation. For the following experiments, chondrocytes at passage one were used.

Mouse microglia cells, BV2, were grown in suspension in 75 cm² culture flasks (Corning, Corning, NY, USA) in DMEM, supplemented with 10% FBS, 2 mM L-glutamine and 1% PSN antibiotics (Biological Industries, Beit Haemek, Israel). Cells were grown to 70–80% confluence before experiments were conducted, and maintained at 37 °C in a humidified atmosphere containing 5% CO₂.
For lipopolysaccharide (LPS)-induced microglia activation, 300,000 BV2 cells were seeded in 6-well plate. Microglia activation was induced by exposing cells to 1 µg/mL LPS (Sigma-Aldrich, Rehovot, Israel), a non-infectious component of Gram-negative bacterial cells wall, for 20 h. In parallel to LPS stimulation, 10¹⁰ MSC-EVs were added to the culture media. Anti-inflammatory activity was then evaluated using RT-PCR for pro-inflammatory cytokines.

Hip synovial membrane was collected from three male adult patients with clinical osteoarthritis (OA) at the time of hip replacement surgery. This protocol was conducted under the approval of the local Ethics Committee (CCER, Geneva, Switzerland) (Authorization #2017-02234) and with informed and consenting patients. Once collected, tissue samples were processed, according to previously described protocols [26 (link)]. Briefly, samples were finely minced and digested for 3 h (37 °C, 5% CO₂ incubation) in a 3 mg/mL collagenase IX-RPMI 1640 solution. After centrifugation (200× g) and supernatant removal, the resuspended pellet was cultured (37 °C, 5% CO₂) in medium containing RPMI 1640, M199 (1:1), 1% penicillin/streptomycin (100 IU/mL:100 g/mL), 2 mM l-glutamine and 20% fetal bovine serum. After overnight culture, non-adherent cells were removed. At confluence, cells were trypsinized and passaged to 75 cm² culture flasks (Corning, NY, USA) in complete medium containing 10% fetal bovine serum. A double control was performed to confirm presence of HFLS after isolation: visual morphology evaluation (Figure S3) and flow cytometry with synoviocyte specific surface markers (CD14⁺ and VCAM-1). A representative sample of all cell populations was found to have above 90% HFLS markers. All cellular assays were performed from passage 3 to 9. Experiments were conducted twice per donor (n = 6).

The method for BMSC preparation has been reported previously [9 (link), 11 (link)]. Briefly, bone marrow cells were obtained by flushing out the femur shafts of 7-week-old male Fisher 344 rats with 10 mL culture medium. The cells were collected in two 75-cm² culture flasks (Corning, NY, USA) containing 15 mL minimal essential medium (Nacalai Tesque, Kyoto, Japan) with 15% fetal bovine serum (Gibco, Life Technologies, Carlsbad, CA, USA) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin; Nacalai Tesque). The cells were cultured in an incubator at 37°C with 5% CO₂. At confluency, the primary cultured cells were trypsinized from the flasks using trypsin/EDTA (Nacalai Tesque).

AW excretory-secretory products (AW-ESP) were obtained as described in Nuñez et al. [20 (link)]. At necropsy, the upper half of the small intestine of rats infected at 3 dpi was slit open lengthwise and incubated over a double layer of cheesecloth in saline at 37 °C for Baermann separation. After recovery for 2 h, the worms were washed several times by settling through RPMI supplemented with antibiotics (ATB, 500 IU/ml penicillin and 500 μg/ml streptomycin; Gibco) and counted. Worms were placed into 75 cm² culture flasks (Corning) at a concentration of 6000 AW/ml in RPMI supplemented with ATB (100 IU/ml penicillin and 100 μg/ml streptomycin; Gibco) and kept for 24 h at 37 °C in 10% CO₂ in air. The procedure then continues as for the obtention of ML-ESP.