Fig. 2

EV and LPS-EV particle size, yield, protein content and surface marker profile from flasks versus bioreactor. A Mean and mode (± SEM) of EV particle diameters from multiple flask production runs of EVs from conditioned media collected after 24-h (24H Flask) or after LPS stimulation (24H + LPS Flask) (N = 10 biological replicates) compared to EVs produced in multiple a hollow–fiber bioreactor collected after four 24-h cycles (24H-96H Bioreactor (N = 4 biological replicates) and after 24-h of LPS stimulation (24H + LPS Bioreactor). Overall, the mean and mode particle diameters of EVs or LPS-EVs between production methods were reproducible and not significantly different from each other. B Comparison of mean particle yields per 10 ⁵ cells (± SEM) from conditioned media of multiple flask runs (24H Flask and 24H + LPS Flask), (N = 10 biological replicates) bioreactor runs (24H-96H Bioreactor) (N = 4 biological replicates) and (24H + LPS Bioreactor) or with LPS stimulation and 24H + LPS Bioreactor). There was a significant (t-test) increase (p ≤ 0.05) in yield produced in the bioreactor runs for EVs (24H-96H Bioreactor) compared to the respective flask runs. C Mean protein content (± SEM) of flasks (N = 10 biological replicates) and bioreactor EVs (N = 4 biological replicates) or LPS-EVs based on mg protein / 10¹¹ EV particles. The EVs of the 24H-Flask production runs had significantly more protein/ 10¹¹ particles compared to the 24H-96H-Bioreactor (t-test ** p < 0.005). D Characterization of surface markers (mean (± SEM)) present on EVs (24H Flask) or LPS-EVs produced in flasks from multiple runs from MSC F1 and F2 MSC isolates (N = 2 biological replicates preformed in duplicate) and bioreactor MSC isolate (96H Bioreactor and 24H + LPS Bioreactor) from MSC isolate B as determined by MACSPlex flow cytometry. The EVs were stained with 37 different bead surface marker populations and compared by mean fluorescence intensity. The same set of surface markers were expressed in both EVs and LPS-EVs produced at both scales. However, when the expression levels in flask EVs and flask LPS-EVs were compared by Kruskal–Wallis with a Dunn post-test and several surface markers (CD146, CD29, CD44, MCSP, CD9 and CD49e) were found to be higher in the flask, *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005