## 2 Materials and Methods
### Human BM-MSC Culture  
The release obtained from PEF exposure was tested for bioactivity using hMSCs isolated from bone marrow (BM-MSC, donor 00055; RoosterBio, Frederick, MD, USA). For routine cell culture, BM-MSCs were thawed and propagated in MesenchymalTM human MSC media according to the manufacturer's instructions. For bioactivity experiments, MSCs were grown in (i) standard culture medium composed of DMEM, low glucose, pyruvate, no glutamine, no phenol red (GibcoTM, ThermoFisher, Waltham, MA, USA) supplemented with 10% (\(\
u\)/\(\
u\)) FBS, 1\(\times\) GlutaMAXTM supplement (GibcoTM, ThermoFisher), 2 IU/mL heparin (Stem cell technologies, Vancouver, BC, Canada), 100 U/mL penicillin, and 100 \(\upmu\)g/mL streptomycin (GibcoTM, ThermoFisher); (ii) medium supplemented with PR PEF composed of DMEM supplemented with 6% (\(\
u\)/\(\
u\)) PR, 1\(\times\) GlutaMAXTM supplement (GibcoTM, ThermoFisher), 2 IU/mL heparin (Stem cell technologies), 100 U/mL penicillin, and 100 \(\upmu\)g/mL streptomycin (GibcoTM, ThermoFisher), supplemented with either 6% PR PEF or PL F&T. For all conditions, MSCs were inoculated at a density of 4500 cell/cm\({}^{2}\) in adherent static culture systems (12- and 48-well plates) and cultured for up to 7 days at 37 \({}^{\circ}\)C in a humidified atmosphere of 5% CO\({}_{2}\) and 95% air. MSCs were cultured inside of an incubator at 37 \({}^{\circ}\)C, 5% CO\({}_{2}\). The medium was exchanged every 3 days and cultures were monitored as follows:Cell adhesion: Cell adhesion was evaluated by determining the number of cells 24 h after inoculation via the CellToxTM green cytotoxicity assay (Promega, Madison, WI, USA) using the manufacturer's instructions. Cell proliferation: Cell number was determined using the CellToxTM green cytotoxicity assay (Promega) according to the manufacturer's protocol for live cells. Briefly, CellToxTM green was added to cells and incubated for 15 min before fluorescence measurement, using an Inucucyte(r) Zoom, Essen BioScience, Ann Arbor, MI, USA, 10\(\times\) objective. At each time point, the initial number of positive CellToxTM green cells was acquired, followed by the fluorescence measurement of the total CellToxTM green positive cells (total cell number) determined 10 min after 0.5% (_v/v_) Triton X-100 in PBS treatment. The number of live cells over time was obtained by subtracting (Initial CellToxTM green positive cells) from (total CellToxTM green positive cells). Cell viability: MSC viability was determined by double-staining the MSCs with 20 \(\upmu\)g/mL fluorescein diacetate (FDA; Sigma, St. Louis, MO, USA) and 10 \(\upmu\)g/mL propidium iodide (PI; Sigma), which stains viable and non-viable cells, respectively. Briefly, the MSCs were incubated for 5 min with FDA/PI in PBS, and cell imaging was acquired using an Inucyte(r) Zoom, Essen BioScience. MSC Immunophenotype: MSC phenotype was evaluated by measuring the expression of cell surface antigens through flow cytometry. After 7 days of culture in different medium formulations, cells were detached using TrypLETM Select Enzyme (GibcoTM, ThermoFisher), and 2 \(\times\) 10\({}^{5}\) were resuspended in PBS with 5% (_v/v_) FBS. Cells were incubated with the following antibodies: CD73 (Biological, San Diego, CA, USA), CD90 (Biological), CD105 (Miltenyi Biotec), CD29 (Biological), CD34 (Miltenyi Biotec, Cologne, Germany), CD45 (BD PharmingenTM, Franklin Lakes, NJ, USA), and HLA-DR (Biological) for 30 min. Data were acquired using a BD FACSCelestaTM Cell Analyzer--BD Biosciences. A total of 5000 events were acquired per sample. Multilineage differentiation potential of BM-MSC: BM-MSC cells were seeded in 48-well plates at 4500 cells/cm\({}^{2}\) and expanded for 6 days in DMEM supplemented with 6% PR or 10% FBS, as mentioned above. After this period, cells were analyzed for their potential to differentiate into adipocytes, osteocytes, and chondrocytes.