FEATURES OF EXTRACELLULAR VESICLE PRODUCTION BY THP-1 CELLS DURING IN VITRO STIMULATION

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Highlights Extracellular vesicles (EVs) are membrane objects with inner and surface molecular cargo performing various biological functions associated with intercellular connection. Therefore, they are a promising object for diagnostic and therapeutic purposes. THP-1 is a human leukemia monocytic cell line and a classic model for monocyte/macrophage function and differentiation. Spectrum of THP-1-derived extracellular vesicles are heterogenic and understudied. The review presents the comparison of EVs produced by stimulated THP-1 cells. Aim. To quantitatively and qualitatively characterize THP-1-derived extracellular vesicles in depending on type and dose of used stimulators. Methods. THP-1 cells were stimulated by PMA (4-phorbol 12-myristate 13-acetate), LPS (lipopolysaccharide) and TNF (tumor necrosis factor). Cell activation and viability were assessed by the expression of CD54 and DAPI (4′,6-diamidino-2-phenylindole) staining respectively. Size and quantitative characteristics were assessed using nanoparticle tracking analysis (NTA). Qualitative assessment of surface molecular markers (CD9, CD63, CD54) was performed by high-sensitivity multicolor flow cytometry (hs-FCM). Results. TNF and PMA stimulation in medium and high doses led to an increase in quantity of small EVs (30–150 nm) that express tetraspanins CD9 and CD63. Low doses of TNF and PMA did not lead to a significant increase of EVs compared to control. There was a decrease in amount of small EVs (30–150 nm) in LPS-stimulated probes. However, medium and high doses of LPS stimulated selective CD63-positive EVs secretion with absence of CD9-positive events. Cell activation assessed by CD54 expression have been dose-dependent and was most prominent under PMA stimulation. Conclusion. Every type of THP-1 cell activators promotes changes in the functional activity of cells towards production of extracellular vesicles. Moreover, we have demonstrated quantitative and qualitative difference in THP-1-derived vesicles under different stimulation conditions. Medium and high doses of PMA and TNF were the most relevant in stimulation of vesiculation. LPS-stimulation could be used to selectively obtain CD63-positive fraction of EV.

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