Abstract
Introduction: Scalable approaches to prepare off-the-shelf extracellular vesicles (EVs) products are mandatory for clinical uses. Here, the possible effects of storage temperature were studied in human EVs.
Methods: Human serum Exos were isolated and characterized using western blotting, SEM, and DLS methods. The isolated Exos were pooled, randomly classified into two main groups [normal EVs (4°C) and frozen EVs (-20°C)], and stored for 10 days. After that, the cytoprotective effects of stored Exos were studied on HUVECs using an MTT assay. The uptake of Dil-labeled Exos was monitored by HUVECs using flow cytometry. Reciprocal interaction of EVs with HUVECs and EV fatty acid profile were assessed using surface plasmon resonance (SPR) and gas chromatography (GC).
Results: Western blotting confirmed the existence of specific tetraspanins (CD8, 63, and 81) on the samples. Based on the data, isolated EVs exhibited an average size of 40.1±25.6 nm with a zeta potential value of -11 mV. Compared to freshly isolated EVs and EVs stored at 4°C, frozen EVs increased the viability of HUVECs (P<0.05) with the lack of significant difference in the internalization rate of normal and frozen EVs (P>0.05). Of note, SPR analysis revealed the increase of EV-HUVEC interaction in EV stored at -20°C indicated higher relative response levels (RU) values (~1.245-fold). GC showed an increase in unsaturated fatty acid content in frozen EVs compared to EVs maintained at 4°C.
Conclusion: These data indicate that the storage temperature can influence the cytoprotective potential, cell interaction capacity, and fatty acid profile of human EVs.