Exploration of serum- and cell culture-derived exosomes from dogs

Abstract

BackgroundExosomes are defined as extracellular membrane vesicles, 30–150 nm in diameter, derived from all types of cells. They originate via endocytosis and then they are released through exocytosis to the extracellular space, being found in various biological fluids as well as in cell culture medium. In the last few years, exosomes have gained considerable scientific interest due to their potential use as biomarkers, especially in the field of cancer research. This report describes a method to isolate, quantify and identify serum- and cell culture-derived exosomes from dog samples, using small volumes (100 μL and 1 mL, respectively).ResultsQuantification and sizing of exosomes contained in serum and cell culture samples were assessed by utilizing nanoparticle tracking analysis, transmission electron microscopy and immunoelectron microscopy. Detected particles showed the normal size (30–150 nm) and morphology described for exosomes, as well as presence of the transmembrane protein CD63 known as exosomal marker.ConclusionsBased on a validated rapid isolation procedure of nanoparticles from small volumes of different types of dog samples, a characterization and exploration of intact exosomes, as well as facilitation for their analysis in downstream applications was introduced.

Canine colostrum exosomes: characterization, influence on canine mesenchymal stem cell secretory profile and fibroblast anti-oxidative capacity

“…Ultracentrifugation techniques allowed isolating abundant CCM exosomes, in a similar manner to that already described in the isolation of other origins [27,29,33]. The presence of CCM exosomes were confirmed by TEM, size determination and western blot analysis, expressing ALIX, Hsp70 and TSG101 exosomal markers, according to the recommendation of the International Society for Extracellular Vesicles [45,46].…”

“…It has been described in human milk and some domestic species such as pig, cow, horse, buffalo, yak or camel [19][20][21][22][23][24][25][26]. Although exosomes have been isolated from different cell types in canine species [27,28,29], they have not been described in canine colostrum milk.…”

Deregulation of miR‐27a may contribute to canine fibroblast activation after coculture with a mast cell tumour cell line

“…Primary fibroblasts (PFs) were isolated and cultured as previously described [22], and passage number 5 was used for all experiments. C2 cells, a canine mast cell tumour cell line, were kindly provided by P. Dubreuil (Centre de Recherche en Cancérologie de Marseille, Inserm U1068, Marseille, France), after previous consent of the cell line originator, W. Gold (University of California, San Francisco, School of Medicine, CA, USA), and were cultured as described earlier [22].…”

“…PFs were first seeded in the lower compartment, and then, 2.2 × 10 5 C2 cells were seeded into the insert (upper compartment). For the Exo‐PF group, exosomes were isolated from C2 cell culture media as described earlier [22], using the commercial kit Total Exosome Isolation Reagent – from cell culture media (Invitrogen, Vilnius, Lithuania), after a previous 48‐h incubation period in exosome‐depleted medium. Exosomes from 2.5 mL medium were pelleted (10 000 g , 60 min, 4 °C), resuspended in PBS and added to the culture medium of all wells of the Exo‐PF group.…”

“…Some of this intercellular crosstalk occurring within the tumour microenvironment is also attributed to exosomes [21]. Exosomes are small membrane vesicles secreted by all types of cells capable of mediating cell‐to‐cell communication by means of exchanging DNA, proteins, mRNAs and miRNAs [22,23]. For instance, in vitro and in vivo models for the human disease have shown that exosome‐derived miR‐27a may regulate the transformation of normal fibroblasts into CAFs and also modulate the growth and metastasis of tumour cells [21].…”