Time-dependent release of extracellular vesicle subpopulations in tumor CABA I cells.
Investigations into extracellular vesicles (EVs) have significantly increased since their role in physiological and pathological processes has become more clearly understood. Furthermore, it has become increasingly clear that several subpopulations of EVs exist, such as exosomes (EXOs) and microvesicles (MVs). Various methods and techniques used to identify and isolate the specific EVs subpopulations exist. However, these methods should be further elucidated. A deep understanding of the different factors that affect the EVs release may therefore be useful for the standardization of protocols and to establish guidelines for a more adequate analysis and correct inter‑laboratory comparison. In the present study, we investigated whether composition and molecular features of EVs altered over time following a trigger stimulus. Starved CABA I cells were stimulated with FBS and conditioned medium was collected after different time intervals (30 min and 4, 8 and 18 h). The dynamic of EVs release was time-dependent, as shown by the results of scanning electron microscopy. Additionally, the time elapsed from the stimulus affected the size distribution (as highlighted by transmission electron microscopy and NanoSight assay), amount (in terms of the number of particles and protein amount) and molecular composition (CD63, HLA, Ago-2, gelatinases, and plasminogen activators) suggesting that, different EVs subpopulations were released at different time intervals following cell stimulation. Collectively, the results suggested that, parameters useful to standardize procedures for EVs isolation, including stimulation time should be considered.
Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.
Ilaria
I
Giusti
Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.
Marianna
M
Di Francesco
Department of Clinical Sciences and Community Health, Molecular Epidemiology and Environmental Epigenetics Laboratory, Università degli Studi di Milano, Milan, Italy.
Laura
L
Cantone
Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.
Sandra
S
D'Ascenzo
Department of Clinical Sciences and Community Health, Molecular Epidemiology and Environmental Epigenetics Laboratory, Università degli Studi di Milano, Milan, Italy.
Valentina
V
Bollati
Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.
Gaspare
G
Carta
Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.
Vincenza
V
Dolo
D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D017077:Culture Media, Conditioned / Q000494:pharmacology; D000067128:Extracellular Vesicles / Q000378:metabolism*; D006801:Humans; D009369:Neoplasms / Q000378:metabolism*; D013997:Time Factors
D017077:Culture Media, Conditioned