Isolation and Characterization of Plasma-Derived Exosomes from the Marine Fish Rock Bream (Oplegnathus fasciatus) by Two Isolation Techniques

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.