## 4 Discussion  
It is known that MSCs act by paracrine components represented by bioactive molecules that are secreted in response to the surrounding microenvironment. Over the last decade, the role of MSC secretoma, with particular attention provided to extracellular vesicles (EVs), has emerged as a convenient alternative to cell therapy. Several functions of EVs have been demonstrated, such as the capability to regulate the balance between pro-inflammatory and anti-inflammatory cytokines [16] and to modulate fibrosis and tissue regeneration after damage [17]. Different preconditioning strategies, such as oxygen percentage and nutrient composition of medium, can affect the metabolic activities of MSCs and the characteristics of the produced and released biomolecules [5; 6; 7; 8]. For example, EVs derived from MSCs cultured under hypoxia showed an increased tube formation capacity, becoming more potent in angiogenesis compared to EVs obtained under normoxic conditions [18]. In addition, EVs from hypoxic MSCs regulated miRNA and proteins involved in extracellular matrix-receptor interaction, focal adhesion, leukocyte transendothelial migration, protein digestion, absorption, and metabolic pathways [19]. Moreover, EVs secreted by MSCs after  
Figure 6: Representative TEM images showing 24 h S-MSCs after restoration with complete medium. (**A–C**) show a well-conserved cellular morphology with a prominent nucleus (arrow); multiple projection (arrowhead) extending to the nearby cells, and some EVs in the extracellular space are detectable (white arrow); some mitochondria in B and MVBs (white arrow). Bar = 2 \(\upmu\)m.  
Figure 7: Representative TEM images showing 48 h S-MSCs after restoration. (**A**) A restored cell with “ghosts” detectable outside the plasmatic membrane (arrow) and bleb (arrowhead), and many vacuoles are also detectable inside cytoplasm. (**B**) Multiloculated vacuoles (arrowhead) and mitochondria with enlarged cristae (arrow), bar = 1 \(\upmu\)m.  
serum starvation showed immunomodulatory capacity, eliciting the switch from M1 to M2 phenotype of bone marrow-derived macrophages [16]. Similar results were reported for EVs released by MSCs cultured in hypoxia and in presence of TNF\(\alpha\) and IL1\(\alpha\), to mimic the inflammatory state of the injured environment. The EVs obtained under these culture conditions modulated the macrophages, exerting an anti-inflammatory activity [20]. Moreover, it has been reported that EVs from umbilical cord-derived MSCs (UC-MSCs), cultured in serum-free conditions, were loaded with increased levels of regeneration-related cytokines, compared to EVs derived from UC-MSCs cultured in normal conditions [21]. Even if recent studies related to enhanced EV production by the application of mechanical stimuli, including flow and stretching, in bioreactors to engineered tissues seeded with stem cells [22], or commercially accessible track-etched membranes to generate EVs containing therapeutic mRNAs by cellular nanoelectroporation [23], we underline the safety and accessibility of starvation as a more suitable method for EV bioproduction. On the other hand, all the preconditioning culture conditions described for EV release have been demonstrated to promote and increase proliferation, viability, and potency of MSCs, favoring the expression of cytoprotective genes, as well as improving the secretion of reparative factors [24; 25]. In particular, it has been demonstrated that MSCs in the absence of oxygen can survive using anaerobic ATP production. Further observations, such as the rapid death of MSCs following glucose deprivation but the slower depletion of cellular ATP under hypoxic conditions, suggested that glycolysis has specific pro-survival functions. Moreover, exposed MSCs maintained the differentiation capacity, highlighting their metabolic flexibility, which makes them able to survive in an ischemic environment [26].