Conditioned media (CM) from MSC in 3D and 2D culture platforms

Conditioned media (CM) was generated from 3D culture pellets as previously described [34]. Briefly, MSCs were expanded in expansion media until 70–80% confluency before being subjected to pellet formation (2.5 × 10⁵ cells per pellet) and left in expansion media overnight. The following day the expansion media was replaced with 0.5 ml of low-glucose DMEM (Life Technologies) media without FBS. PEMF treatment was applied for a duration of 10 min at 1, 2, 3, and 4 mT, or 2 mT for 30 min. CMs from pellet culture were collected 24 h post-PEMF exposure and pooled for later use.

To generate CM from 2D culture platforms, MSCs were cultured in T75 flasks at a seeding density of 1.5–2 × 10⁵ in expansion media. At 50–60% confluency, the expansion media was replaced with 10 ml of low-glucose DMEM media without FBS. PEMF exposure was applied at 1, 2, 3, and 4 mT for 10 min, or 2 mT for 30 min, and the CM generated (PCM) was collected at 24 h post-PEMF exposure, pooled, and used for further application and analysis. Control CM (CCM) from 2D and 3D culture platform was generated from similarly cultured MSC without PEMF treatment (0 mT).

In either culturing platform, CM was collected and subsequently concentrated 10× by high centrifugation force using a protein concentrator with a molecular weight cut-off of 3 kDa (Thermo Fisher Scientific, USA). In subsequent experiments, the concentrated CM was diluted 1:10 with low-glucose DMEM media without FBS prior to application to the experimental culture to achieve a final working strength of 1× CM.