Abstract
Exosomes are important mediators of intercellular communication and modulate many physiological and pathological processes. Knowledge of secretion, content, and biological functions of fish exosomes during pathological infection is still scarce due to lack of suitable standardized isolation techniques. In this study, we aimed to isolate exosomes from the plasma of marine fish, rock bream (Oplegnathus fasciatus), by two isolation methods: differential ultracentrifugation (UC) and a commercial membrane affinity spin column technique (kit). Morphological and physicochemical characteristics of the isolated exosomes were determined by these two methods, and the efficiencies of the two methods were compared. Exosomes isolated by both methods were in the expected size range (30–200 nm) and had a characteristic cup-shape in transmission electron microscopy observation. Moreover, more intact exosomes were identified using the kit-based method than UC. Nanoparticle tracking analysis demonstrated a heterogeneous population of exosomes with a mean particle diameter of 114.6 ± 4.6 and 111.2 ± 2.2 nm by UC and a kit-based method, respectively. The particle concentration obtained by the kit method (1.05 × 1011 ± 1.23 × 1010 particles/mL) was 10-fold higher than that obtained by UC (4.90 × 1010 ± 2.91 × 109 particles/mL). The kit method had a comparatively higher total protein yield (1.86 mg) and exosome protein recovery (0.55 mg/mL plasma). Immunoblotting analysis showed the presence of exosome marker proteins (CD81, CD63, and HSP90) in the exosomes isolated by both methods and suggests the existence of exosomes. However, the absence of cytotoxicity or adverse immune responses to fish and mammalian cells by the exosomes isolated by the UC procedure indicates its suitability for functional studies in vitro. Overall, our basic characterization results indicate that the kit-based method is more suitable for isolating high-purity exosomes from fish plasma, whereas UC has higher safety in terms of yielding exosomes with low toxicity. This study provides evidence for the existence of typical exosomes in rock beam plasma and facilitates the selection of an efficient exosome isolation procedure for future applications in disease diagnosis and exosome therapy as fish medicine.
Highlights
Exosomes are nano-sized (30–200 nm), membrane-bound, extracellular vesicles (EVs) secreted by various cell types that are capable of intercellular communication [1]
Fish exosome research is at a primitive stage, with few reported studies related to aquatic animals [6,7,8,9,10,11,12]
The protein concentration of exosomes isolated by the UC method (2.65 mg/mL) was not significantly different (p > 0.05) compared to that of exosomes isolated by the kit method (2.33 mg/mL); the total yield was significantly (p < 0.05) higher for exosomes isolated by the kit method (1.86 mg; 2.33 mg/mL × 0.8 mL) compared to that of exosomes isolated by the UC method (0.40 mg; 2.65 mg/mL × 0.15 mL) (Table 1)
Summary
Exosomes are nano-sized (30–200 nm), membrane-bound, extracellular vesicles (EVs) secreted by various cell types that are capable of intercellular communication [1]. Exosomes are distributed in a variety of body fluids, and owing to their wide range of functions in normal physiological processes, as well as under pathological conditions, they are of wide scientific and clinical research interest [2,3]. Analyzing body fluids in teleosts can deepen the understanding of exosomes with regard to their secretion into the extracellular fluids, such as plasma, and their biological functions. In-depth research on fish exosomes will facilitate disease diagnosis and enhance the understanding of host–pathogen interactions and the role of EV/exosome cargos resulting from the expression of pathogenderived factors in eliciting immune defence responses and immune regulatory mechanisms during viral and bacterial infections [9,13]
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