## 4 Discussion  
In this work, we evaluated the use of several PEF protocols applied to platelets with no therapeutic value to produce a platelet derivative component that could be used as an alternative to FBS and PL as a media supplement for MSC cultures. Taken together, our data show that the application of PEF to expired PCs leads to a controlled released of GFs from platelets (Figure 2), and this PR PEF supports BM-MSC renewal and differentiation, while cell characteristics are maintained, as seen in Figures 3 and 4. It was observed that PR features are dependent on the PEF parameters applied, as different levels of GFs are released upon PEF application, as well as the percentage of platelet activation (Figure 2). These data are in accordance with previous studies demonstrating that PEF parameters influence the platelet rich plasma characteristics [19; 31; 32; 33; 34]. The application of PEF to cells induces the opening of pores on cell membranes leading to a release of proteins into and out of the cells [17; 18]. Depending on PEF conditions, it can permeabilize internal membranes, such as mitochondria or endoplasmic reticulum membrane [35; 36; 37]. Consequently, the PEF parameters applied can regulate the bioactive components that are released from the cells, due to their protein size and location within the cell compartments. The performed analysis of PDGF, VEGF, TGF-\(\beta\)1, and IGF in the PR obtained after PEF stimulation demonstrates that TGF-\(\beta\)1 was released in a higher amount than VEGF, PDGF, or IGF, when compared with the basal levels characterized by the untreated PR (Figure 2c-e). Interestingly, only TGF-\(\beta\)1 achieved similar levels to PL F&T, which causes the breakage of all platelets and the release of all proteins stored in the cell (Figure 2d). Previously, [PERSON] et al. characterized the GF release from platelet rich plasma upon PEF application and correlated it with the expression of several platelet granule markers, observing that GFs were differently localized in the platelets [31]. PDGF and VEGF distribution correlates with \(\alpha\)-granules and T granules, indicating a similar distribution in the cell [31]. In this study, an analogous release of PDGF and VEGF was observed, while TGF-\(\beta\)1 was highly released, which might indicate that these GFs were stored differently in the platelets, and that different PEF parameters affect platelets differently. Besides this, TGF-\(\beta\)1 is the GF with the lowest protein size analyzed in the present study,  
Figure 4: Differentiation potential of BM-MSCs cultured in media supplemented with PR obtained after PEF application and FBS. **(a)** Flow cytometry analysis of MSC markers, CD29, CD73, CD90, CD105, CD34, CD45, and HLA-DR. **(b)** Differentiation potential to adipogenic, osteogenic, and chondrogenic lineages by oil red O, alizarin red S, and Alcian blue staining, respectively. thus it requires small pores in the cell membrane to be able to cross the cell membrane [38]. Previously, studies have shown that PDGF-BB and TGF-beta are stored in the \(\alpha\)-granules of platelets; however, it might be differently distributed within the granules, as TGF-beta is highly released upon PEF application in comparison to PDGF [31; 39]. Moreover, the TGF-beta protein is smaller than PDGF which also could lead to these differences in protein release from platelets. In addition, our data show that the IGF levels obtained in the PR PEF I and C, and PL F&T were significantly lower than in the untreated condition, which might indicate IGF degradation caused by the freezing cycles or by the application of PEF [14; 40]. Currently, there are multiple ongoing clinical trials using MSC which have raised concerns about the use of animal-derived components as a medium supplement for cell expansion in vitro [4]. One alternative proposed is the use of chemical-defined media; however, studies have demonstrated that it fails to sustain the MSC features [41].