Abstract 82P
Background
Microvesicles (MVs) are a subset of extracellular vesicles secreted by most body cells. They are being actively studied as cell-free biosimilar drugs. Many ways of inducing MVs are being investigated - natural (passive secretion, cell activation) and stressful (hypoxia, chemical impact, etc.).
Methods
The culture of T-lymphocytes was activated by CD3/CD28 antibodies with subsequent expansion. Some of the cells were reactivated. Cytochalasin B (CHB) was used to chemically induce MVs formation. Ultrasound (US) was used as a physical inductor. The resulting MVs were purified by differential centrifugation. The samples were studied by immunoblotting and nanoparticle tracking analysis (NTA).
Results
The NTA method was used to determine the concentration of MVs (for all samples, about 108 particles/mL) and the dispersity of the particle size. The induction of MVs by activation with antibodies, treatment with CHB or US increased the monodispersity of the particles without affecting their average size. The application of US to activated cells led to a decrease in monodispersity and the appearance of a fraction of larger MVs (up to 700nm). As a result of Western blot analysis, an increase in the level of CD3 in samples of natural MVs, as well as induced by activation and US, was revealed compared with the cell control. In the samples obtained by US, an increase in the content of granzyme B was detected. In MVs induced by CHB, these proteins were not detected. With an equal amount of total protein, a decrease in the content of beta-actin was noted in the series cells>natural MVs>USinduction>CHB induction. This observation can be explained by a change in the cytoplasm/membrane ratio due to a difference in the size of cells and MVs (10μm and 0.1μm, respectively), as well as the mechanism of action of CHV (inhibition of actin filament synthesis).
Conclusions
All studied methods of induction, except for CHB, make it possible to obtain MVs carrying functionally significant molecules of T-lymphocytes. Further study the structure and functions of the MVs obtained by these methods is needed. The work was supported by the Russian Science Foundation grant 19-74-20026, with the support of the Strategic Academic Leadership Program of the Kazan (Volga Region) Federal University (PRIORITET- 2030).
Legal entity responsible for the study
Kazan Federal University.
Funding
The work was supported by the Russian Science Foundation grant 19-74-20026, with the support of the Strategic Academic Leadership Program of the Kazan (Volga Region) Federal University (PRIORITET- 2030).
Disclosure
All authors have declared no conflicts of interest.