Biophysical Characterization and Proteomic Analysis of Small Extracellular Vesicles Derived From Different Neural Cell Lines

Small extracellular vesicles (sEVs) are nanosized membrane‐bound particles facilitating intercellular communication in the nervous system. Although previous studies investigated sEVs released from various neural cell lines, a comprehensive comparison among various immortalized neural lineages has not yet been performed. Herein, we isolated sEVs from four representative immortalized neural cell lines, including SH‐SY5Y (neuroblastoma), differentiated SH‐SY5Y neurons, 1321N1 (astrocytes), and BV2 (microglia), in order to elucidate cell‐type‐specific characteristics under controlled in vitro conditions. The results reveal that all isolated sEVs exhibited characteristic morphology under transmission electron microscopy, while the particle diameters were also within the expected range of 30–200 nm as determined by nano‐flow cytometry. A significant variation in size was observed between the different cell‐line‐derived sEVs where neuron‐derived sEVs exhibited the smallest size and the surface charge was less negative than other sEVs. Microglia‐derived sEVs, on the other hand, were largest in size and presented the most negative surface charge and highest RNA content. Such variations in the different types of sEV suggest the variability in the composition of the different types of neural cell types. The presence of sEV markers like CD9, CD63, CD81, and Alix was confirmed by Western blot analysis. Additional proteomic analysis helped in identifying the composition of the sEVs, revealing the characteristic protein fingerprinting of the neural origin of the sEVs. Neuron‐derived sEVs were enriched in proteins linked to synaptic activity and neurogenesis, whereas astrocyte‐derived sEVs expressed proteins involved in metabolic and antioxidant pathways. Microglial sEVs expressed proteins linked with immune regulatory mechanisms. Interestingly, sEVs originating from differentiated neurons expressed a distinctive proteomic profile that was different from their undifferentiated SH‐SY5Y counterparts. Collectively, this study provides for the first time an in‐depth characterization profile for sEVs derived from neurons, which were differentiated from SH‐SY5Y cells, and, furthermore, provides a comparative framework of immortalized neural cell‐derived sEVs, allowing for the provision of a reproducible and physiologically relevant reference model to further neurodegenerative disease research.