Monoject 29g insulin syringes

Treatments were 15 μl concentrated CM-MSC or 5.0 × 10⁵ MSC (P4-P5) resuspended in 15 μl of SF-IMDM. 15 μl of concentrated CM-MSC corresponded to cell secretions from 5.08 × 10⁵ cells on average. Treatments or SFM control were injected intra-articularly 1 day post arthritis induction with 0.5 ml monoject (29 G) insulin syringes (BD Micro-Fine, Franklyn Lakes, USA) through the patellar ligament into the right knee joint. Joint diameters were measured at 2, 3, 7 and 14 days post injection. Blood, joints, spleen, inguinal and popliteal lymph nodes were collected immediately post-mortem. Three independent experiments were performed. All measures were taken to reduce animal numbers (n = 5–11 per time point).

Treatments comprising 15 μL of EVs suspension in PBS corresponding to EV secretions from ~5.0 × 10⁵ cells or PBS alone controls were injected intra-articularly 1 day post arthritis induction with 0.5 mL monoject (29G) insulin syringes (BD Micro-Fine, Franklyn Lakes, NJ, USA) through the patellar ligament into the right knee joint. Joint diameters were measured at 1-, 2- and 3-days post-injection. Blood, joints, spleen, inguinal and popliteal lymph nodes were collected immediately post-mortem. Four independent experiments were performed to assess primed EVs impact on joint swelling and histopathology (Control n = 21, EV-NormO₂n = 10, EV-2%O₂ and EV-Pro-Inflam n = 6). A further independent experiment was conducted to address the potential for EV immunomodulation of T cells in the in vivo environment in AIA (EV-NormO₂ versus PBS controls, n = 4 per condition). All measures were taken to reduce animal numbers wherever possible.