Efficient isolation and characterization of Serum-Derived Exosomes: evaluating ultracentrifugation and Total Exosome Isolation Reagent based precipitation

Exosomes are extracellular vesicles that carry biomolecular cargos such as proteins, lipids, RNA, and DNA. These molecules play crucial roles in cell-to-cell communication and are involved in various physiological and pathological processes. Due to their potential as biomarkers for disease diagnosis and prognosis, research has increasingly focused on developing more efficient methods for their isolation and characterization. In this study, blood samples were collected from 30 participants, and serum was subsequently isolated. Serum-derived exosomes (SDEs) were extracted using ultracentrifugation (UC) as well as the Total Exosome Isolation Reagent. Modifications to the UC method were implemented to improve yield and purity, and a detailed description of the method is also provided. The exosomes were characterized by Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Dynamic Light Scattering (DLS), and Western Blotting (WB) to evaluate their size, morphology, and protein content. The exosome yields from both isolation methods were evaluated using the BCA assay. Protein estimation suggested that the Total Exosome Isolation Reagent produced exosome concentrations that were 10-fold higher compared to those obtained through ultracentrifugation. Morphological analysis showed that exosomes exhibited circular, spherical, and irregular shapes, with diameters ranging from 30 to 200 nm. Western Blotting confirmed the presence of exosomal markers (TSG101, ALIX, LAMP2, and CD63) in the SDEs. In conclusion, both ultracentrifugation and the Total Exosome Isolation Reagent effectively isolate SDEs. Thus, although both methods are viable, modified ultracentrifugation is the preferred choice for applications due to its cost-effectiveness and suitability for achieving pure protein yields.