Recombinant human ifn gamma

The co-culture of hORSCs and hHMSCs was performed as previously described [50 (link)]. hORSCs were seeded in the lower chambers of Transwell culture plates (Cell Culture Insert Companion Plate; Corning, Falcon, Franklin Lakes, NJ, USA) at a density of 1 × 10⁵ cells per well. hHMSCs were added to the upper chamber of the Transwell in separate plates at a density of 5 × 10⁴ cells per well. The upper chambers were coated with polyethylene terephthalate (pore size: 0.8 mm, six-well format, Cell Culture Inserts; Corning, Falcon). After 24 h, the media was changed to serum-free DMEM and the upper chamber containing hHMSCs was transferred to the wells where the hORSCs were cultured to produce the co-culture. The IFN-γ group was treated with recombinant human IFN-gamma at 100 ng/mL, obtained from Peprotech (315-05, Rocky Hill, NJ, USA). After different times (24 h, 48 h, and 72 h), the upper chamber was removed and washed with Dulbecco’s phosphate-buffered saline (DPBS, T&I, Bioprince, Chuncheon, Korea). Next, the hORSCs were harvested and used for analysis. The experiment was repeated three times.

We obtained PBMCs from a healthy donor with no history of systemic or chronic inflammatory diseases. Human PBMCs were plated into 24-well culture plates at a density of 5 × 10⁵ cell/mL per well and cultured in RPMI 1640 (Gibco, Grand Island, NY, USA) containing 10% FBS and IL-2 (1 μg/mL, R&D systems, Minneapolis, USA).
An AA-like microenvironment was induced by treatments with recombinant human IFN-gamma—which was obtained from Peprotech (315-05, Rocky Hill, NJ, USA)—on PBMCs at 100 ng/mL. Ruxolitinib measuring 10 nM/mL was used as a positive treatment control. For the MSC co-cultured group, the upper chamber containing hMSCs (5 × 10⁴ cell per well) was transferred to the lower chamber, where PBMCs were cultured to produce the co-culture for 3 days. After 3 days of incubation at 37 °C with 5% CO₂, the upper chamber was removed so that PBMCs could be harvested for analysis.