Comparison of Methods for Isolating Exosomes from Plasma in Subjects with Normal and High-Fat Percentage

The adipose tissue is responsible for fat storage and is an important producer of extracellular vesicles (EVs). The biological content of exosomes, one kind of EVs, provides information such as immunometabolic alterations. This study aimed to compare three plasma exosome isolation methods and select the best. Plasma from 118 individuals, categorized by normal and high body fat percentage, and developing a 3T3-L1 cell line as an obesogenic model through continuous glucose exposure and a prolonged hypoxic microenvironment, were used. We perform three exo-some isolation methods: commercial kit (CK), size exclusion chromatography (SEC), and differ-ential centrifugation (DC), and characterized by DLS, cryo-TEM, TEM, and western blot CD9 and CD81 markers. In the DLS, cryo-TEM, and TEM analysis showed similar quality and mor-phology of exosomes. The CK and DC proved to comply with most of the advantages of an exo-some isolation method. Still, we emphasize the importance of selecting the appropriate method-ology depending on the specific research objectives. At the same time, no differences in exosome morphology, total protein, nor microRNAs concentration were observed between individuals categorized by body fat percentage, so we suggest that exosome cargo is the one that varies in in-dividuals with normal and high-fat percentages.