T175 culture flask

Viable cells were plated at 500,000 cells/cm² in 40 mL of growth media ±10 ng/mL bFGF (Austral Biologicals, San Ramon, CA, cat# GF-030-5) per T-175 culture flask (Nunclon®, Naperville, IL, cat# 178883). After plating, MSCs were immediately transferred to incubators at 37 °C, 5% CO₂ and either 20% oxygen (normoxia) or 5% oxygen (hypoxia). 24 hours after initial plating, the media containing any non-adherent cells was carefully removed and replaced with 30 mL of fresh growth media ±10 ng/mL bFGF. Media was changed every 3–4 days. See details on mMSCs passaging in the Online Data Supplement. When MSCs reach p2-3, basal media for cell culture changes over from RPMI to DMEM with same supplementation.

The simulations were performed using the Microvawes & RF (Biomedical, Exposure, SAR) package of CST^® STUDIO SUITE^® 2021.05 software.
The subject of the simulation was a T175 culture flask (Nunc^®) with a layer of biological material of bone marrow cells with a volume of 10 mL and a layer thickness of 0.588 mm. In the simulation, the specific absorption rate (SAR) distribution (2): $$SAR=\frac{1}{V}{\int }_{sample}\frac{\sigma \left(r\right){\left|\mathrm{E}\left(r\right)\right|}^2}{\rho \left(r\right)}dr$$
(where σ is the sample electrical conductivity, E is the root mean square value of the electric field, $\rho$ is the sample density, and V is the volume of the sample) was calculated for a flat electromagnetic wave pulse (with an electric field magnitude of ~1 MV/m) falling perpendicularly on the culture flask (Figure 10).
The values of electric field data obtained during the experiment using the Teledyne LeCroy oscilloscope (640 Zi) (Chestnut Ridge, NY, USA) were used for the calculations made in the CST^® STUDIO SUITE^® 2021.05 software (Figure 11).

Pooled human periodontal ligament fibroblasts from several donors (HPdLF, Lonza, Basel, Switzerland) were grown in culture medium consisting of Dulbecco’s modified Eagle medium (DMEM; Thermo Fisher Scientific, Carlsbad, CA, USA) containing 4.5 g/L glucose, 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Carlsbad, CA, USA), 100 U/mL penicillin, 100 µg/mL streptomycin and 50 mg/L L-ascorbic acid at 37 °C, 5 % CO₂ and 95% humidity. Cells were regularly passaged with 0.05% Trypsin/EDTA (Thermo Fisher Scientific, Carlsbad, CA, USA) when 75% confluence was reached. HPdLF of passage four to eight were used for experimental setups. For RNA and protein expression analysis, 1 × 10⁵ cells were seeded per well of a 6-well plate and cultured to 75% confluency prior further treatment. For THP1 cell adherence assay, 50 HPdLF per mm² were seeded on coverslips in 24-well plates and cultured to 75% confluence.
THP1 monocytic cells (DMSZ, Braunschweig, Germany) were cultured in RPMI 1640 medium (Gibco) containing 10 % FBS, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 °C, 5 % CO₂ and 95 % humidity. They were passaged weekly and seeded at a density of 1 × 10⁶ cells in 20 mL medium in T175 culture flask (Thermo Fisher Scientific, Carlsbad, CA, USA).