Isolation of Extracellular Vesicles From Mouse Brown Adipose Tissue Secretome Using Size-Exclusion Chromatography

Brown adipose tissue (BAT) is a thermogenic tissue that functions in an endocrine, paracrine, and autocrine manner. BAT secretes extracellular vesicles (EVs) that carry miRNA and proteins involved in mitochondrial function, lipid metabolism, and the β-oxidation pathway. EVs are small lipid membrane-bound structures that are secreted by all eukaryotic cells for various purposes like cell signaling, organ crosstalk, and discarding of waste materials. Because EV populations reflect the unique molecular composition, physiological state, and functional roles of the tissues from which they originate, and therefore exhibit substantial heterogeneity across organs, it is essential for mechanistic and therapeutic studies to use an organ-specific EV isolation method to ensure that the isolated vesicles accurately represent the biology of the source tissue. However, the literature surrounding BAT-EVs lacks a uniform method for their isolation, with major drawbacks being inconsistent incubation time with ECM-degrading enzymes and varied media additives. To address this, we systematically subjected mouse BAT to temporal explant culture with dose-dependent digestion by collagenase type 1 and optimized a protocol to consistently and reproducibly isolate EVs from the BAT-secretome using size-exclusion chromatography. This study presents a standardized, efficient, and accessible method with optimized digestion duration and collagenase type 1 concentration to achieve an appropriate yield of BAT-derived extracellular vesicles (BAT-EVs).