## 3 Results
### Transmission Electron Microscopy (TEM)  
In this study, TEM was considered as a standard imaging method for observing nanosized samples, allowed to reveal inner cell and EV structures. The TEM images of non-starved MSCs showed polymorphic and large euchromatic nuclei with prominent nucleoli, visible in Figure 4A,C,D. In cytoplasm, mainly alongside the nucleus, well-developed dilated cisternae of rough endoplasmic reticulum (RER), formed by broad vesicles, were observed (Figure 4A,B). Multivesicular bodies (MVBs) loaded with intraluminal vesicles were in appreciable number inside the cell cytoplasm, as shown in Figure 4A, and closely packed-vesicles were detectable in Figure 4B. Protrusions of the plasma membranes, filopodia, were detectable in Figure 4B,D. The presence of wide vacuoles and vesicles gave the periphery of the cytoplasm a multivocal appearance (Figure 4D). Figure 4: Representative TEM images of non-starved MSCs. (**A–C**) the cisternae of rough endoplasmic reticulum (RER) are clearly detectable (black arrows). In (**A**,**B**), different MVBs with a characteristic cup shape in the cytoplasm are contained within a membrane boundary (white arrows). (**B**,**D**) the protrusion of the plasmatic membrane (filopodia) is evidenced by arrowhead. In (**C**,**D**), a convoluted nucleus (black arrows) and some MVBs in (**D**) (white arrows) are also detectable. Bar = 2 \(\upmu\)m.  
Figure 3: MSC functionality. (**a**) PHA-activated PBMC modulation by MSCs and S-MSCs at different ratios. Black bar= PHA-activated PBMC alone; white bars = PHA-activated PBMC in presence of MSCs at different ratios; gray bars= PHA-activated PBMC in presence of S-MSCs at different ratios. Results are expressed as SI = cpm stimulated/cpm unstimulated. (**b**) Mitochondrial activity evaluated by MTT assay in MSC and S-MSC. Results are expressed as OD. No statistical significance was observed. In order to evaluate the cellular structure and EV release, we performed TEM analysis after 12, 24, and 48 h of starvation (Figure 5). It was possible to highlight the budding process of cell membranes that released EVs with the typical morphology characterized by a lipid bilayer covering a specific payload of biomolecules, and a dilated RE was also observable. Ultrastructural differences due to an increase in the starvation time were detectable, such as EV heterogeneous populations in terms of size and density (Figure 5). After 12 h starvation (Figure 5A), the amount of EVs was lower, and EVs seem to be less preserved compared to those observed after 24 h (Figure 5B). The same results were observed in cells starved for 48 h (Figure 5C). Moreover, after 48 h starvation, EVs seemed \"wilted\", and in the area observed, a significant cell membrane blebbing was evident. In non-starved condition, EV release was not evaluable in MSCs, as already shown in Figure 4.  
In order to evaluate if it is possible to restore S-MSCs after starvation, we added complete medium for further 24 h, and the restoration of culture conditions allowed us to find restored cells with detectable RER, mitochondria, and nucleus (Figure 6A). MVBs are smaller and less numerous with respect to those observed in MSCs, as described in Figure 4. Multiple projection extending from plasma membrane and some EVs in the extracellular space were also detectable (Figure 6B,C). However, the 48 h starvation exhibited detrimental results, demonstrated by \"ghosts\" outside the plasma membrane (Figure 7A) and by mitochondria with enlarged cristae (Figure 7B). No EVs were detectable inside cell cytoplasm or in the extracellular space. Figure 5: Representative TEM of S-MSCs releasing EVs after different times of starvation.