Extracellular vesicles including exosomes from animal mesenchymal stem/stromal cells

Mammalian mesenchymal stem/stromal cells (MSCs) produce extracellular vesicles (EVs) associated with the plasma cell membrane, which may contain growth factors, chemokines, cytokines, and microRNAs. Currently, EVs are widely used to develop new regenerative strategies in the treatment of numerous diseases, since they convey most of the therapeutic properties of MSCs. This work shows for the first time that EVs enriched with exosomes can be isolated from conditioned media (CM) of MSCs from five different animal species using the method of differential centrifugation (DC) followed by ultracentrifugation (UC). The purpose of the work is to obtain EVs from conditioned media (CM) of MSCs of bone marrow (BM), umbilical cord blood (UCB) and adipose tissue (AT) of agricultural (cattle, sheep, horses) and small domestic animals (dogs, cats). We used MSCs that were previously obtained from the bovine and ovine BM, equine UCB, ovine, bovine, equine, canine and feline AT. MSCs were thawed and seeded into 25 cm2 growth area flasks, after 48 hours they were reseeded into 175 cm2 growth area flasks in a ratio 1:7 and incubated for 10 days until the cells reached a complete monolayer. Then the CM from all MSC samples was poured into 50 ml sterile centrifuge tubes and EVs were isolated. For this purpose, we used the method of DC followed by UC. In all samples, electron microscopy revealed round or irregularly shaped microparticles of different sizes. The diameters of individual EVs did not differ statistically between different animal species (p = 0.1). When comparing the number of particles isolated from 50 ml of CM from MSCs of different animal species, no statistically significant differences were detected (p = 0.1). Thus, on one mesh of bovine MSC(BM) and MSC(AT), 3±0.1 and 6±0.07 particles with a size of 50-100 nm, 7±0.02 and 4±0.03 particles of 100- 150 nm, as well as 3±0.4 and 2±0.06 particles larger than 150 nm. EVs from CM of canine MSC(AT) were the most homogeneous in both shape (round) and size, and the main part was found in the range of 50-100 nm (12±0.02 particles). In samples isolated from the CM of ovine, bovine, equine MSC(AT) and equine MSC(UCB), the number of particles with a diameter of 50-100 nm was 7±0.2; 7±0.01; 5±0.7 and 8±0.02. Analysis of the obtained electron diffraction patterns showed that more than 70 % of EVs had a diameter from 50 to 100 nm, that is, they were classified as exosomes. EVs isolated from CM canine MSC were positively stained with antibodies against the TSG101 antigen (cytoplasmic protein, exosome marker). The results obtained demonstrate that the CM of animals MSCs, isolated from BM, AT and UCB contains EVs, including exosomes. The DC method does not exclude the possibility that other particles are present in the preparation, so we propose to designate the result obtained as micro explosives enriched with exosomes. Obtaining EVs of a certain composition from the CM of agricultural and domestic animal MSCs opens up broad prospects for the use of exosomes in the diagnosis of diseases and treatment of agricultural and domestic animals.