Cell culture insert companion plate

We obtained human bone marrow-derived MSCs (Catholic MASTER Cells from the Catholic Institute of Cell Therapy (CIC, Seoul, Korea). Allogenic MSCs that had been donated from a healthy, young donor in the GMP facility were produced to prevent possible immune reactions and transmissible infectious diseases. The hMSCs in passages 3 to 5 were used for the experiments. hMSCs were seeded at a density of 5 × 10⁴ cells per well for 24 h in the polyethylene terephthalate-coated upper chamber (pore size: 0.8 mm, 24-well format, Cell Culture Inserts; Corning, Falcon, Franklin Lakes, NJ, USA) of the Transwell culture plates (Cell Culture Insert Companion Plate; Corning, Falcon, Franklin Lakes, NJ, USA).
The culture medium was Dulbecco’s Modified Eagle Medium (DMEM high glucose, Gibco BRL, Life Technology, Karlsruhe, Germany) containing 10% fetal bovine serum (FBS; Gibco BRL, Life Technology, Karlsruhe, Germany) and 1% penicillin/streptomycin (Gibco, BRL, Life Technology, Karlsruhe, Germany). After 24 h, the upper chamber containing hMSCs was transferred to the wells for co-culture with PBMCs.

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.