Investigating the Effect of Small Extracellular Vesicles at Varying Concentrations on Wound Healing

Abstract

Extracellular vesicles (EVs) are nanoscale lipid bilayer particles that are secreted by cells into their surrounding environment. EVs in the extracellular space taken up by nearby cells can influence the recipient cells’ phenotype. For this reason, EVs are believed by some to influence cell‐cell communication, specifically cell motility. While previous literature has claimed EVs have a significant effect of wound healing and cell recovery, we believe these results are due to cells being dosed in super supraphysiological levels of EVs. We hypothesize that cells dosed with EVs at physiological levels will have a less significant effect on wound healing properties than what has previously been seen. This study investigates the effect of adipose derived stem cell (ADSC) derived EVs on human dermal fibroblast (hDFb) migration and wound healing speeds using time lapse imaging and cell migration analysis code.Cell number recovery (CNR) is the ratio of the number of cells which have entered the wound region to the initial number of cells in the wound region prior to scratching. Initial number of cells in the wound region was estimated from the starting density of unscratched cells outside the wound region. A CNR of 1 indicates the wound has fully recovered and reached its initial density. We compared CNR in media control (complete media) and baseline control (serum free media) to cell recovery in EV dosed media with a concentration of 5*108 EVs/mL. EV doses were calculated such that they corresponded to EVs produced by ADSCs within 24 hours at a ratio ranging from 1:1 (5*109 EVs/mL) to 1:10 (5*108 EVs/mL) ADSCs to hDFbs. These ratios were chosen as they likely mimic the upper limits of physiological ratios during wound healing. Previous studies often used very high levels of EVs that may be relevant for therapeutic approaches. A vascular endothelial growth factor (VEGF) ELISA was performed to confirm growth factors did not co‐precipitate during isolation and did not influence the scratch wound assay results. EV Depleted media, collected from the supernatant of the final ultracentrifugation spin, confirmed the presence of VEGF. Conversely, both serum free and small EVs measured below the limit of detection for VEGF. These results confirm the slight increase in CNR is not due to growth factors that co‐isolated with EVs. In conclusion, EVs may provide a therapeutic effect for wound healing when dosed at supraphysiological levels, however, at more physiological levels, only a minor difference in wound healing is seen between the EV dosed cells and baseline controls.