3.1. Characterization of stem cell-derived EVs
EVs were isolated from both BMSCs and PSCs cultured in growth media (D0)
as well as osteogenic media at 7 days (D7) and 21 days (D21) of culture.
The yield of the BMSC-derived EVs was 71.0±38.3, 149.3±22.9, and
123.6±16.9 μg/mL for D0, D7, and D21, respectively. BMSCs exhibited
similar yields to the values reported in the literature [44], but
PSCs consistently yielded higher EV protein quantities in comparison to
BMSCs. The PSC-derived EVs had yields of 394.6±38.2, 959.3±59.7, and
695.7±90.8 μg/mL at D0, D7, and D21, respectively (Figure 2A ).
The lipid concentration of the EVs was also quantified so that a
comparison could be made between the protein to lipid ratios of each EV
group as is standard according to ISEV to ensure EVs are pure and the
isolation method does not co-isolate unbound proteins. The BMSC-derived
EVs had lipid concentrations of 185.2±17.2, 495.2±32.9, and 1339.37±74.2
μg/mL at D0, D7, and D21 respectively. The PSC-derived EVs had lipid
concentrations of 3486.5±227.1, 818.1±85.8, and 247.7±8.2 μg/mL at D0,
D7, and D21 respectively (Figure 2B ). This means that for the
protein to lipid ratios, BMSC-derived EVs had a ratio of 0.383, 0.302,
and 0.092 at D0, D7, and D21 respectively, while the PSC-derived EVs
were 0.113, 1.172, and 2.808 at D0, D7, and D21, respectively.
EVs were characterized by TEM and DLS to confirm that the size and
morphology were consistent with the values and shape reported by other
groups isolating EVs. The TEM images showed that all six groups of
examined EVs had a cup-shaped morphology and an average hydrodynamic
diameter between 120-150 nm (Figure 2C) . The TEM images
revealed that the different groups of isolated EVs featured similar
shape and size ranges, confirming that the techniques for isolation
yielded consistent batches of EVs.
DLS was also performed to quantitatively confirm the EV size(Figure 2D) . DLS is able to calculate the hydrodynamic diameter
of EVs from the Stokes-Einstein Equation, which is used to characterize
how large particles move slower due to Brownian motion. From this
experiment, we found that the BMSC-derived EVs had diameters of
137.4±11.97, 158.7±29.41, and 218.3±20.3 nm on D0, D7, and D21,
respectively. The PSC-derived EVs had diameters of 135.5±1.17,
129.3±25.22, and 197.5±34.85 nm on D0, D7, and D21, respectively.