Efficient enzyme-free isolation of brain-derived extracellular vesicles

ABSTRACT: Extracellular vesicles (EVs) have gained significant attention as potential diagnostic and therapeutic tools due to their role in intercellular communication and their ability to encapsulate various biomolecules. However, the isolation of EVs from tissue remains challenging, often requiring enzymatic digestion steps that may introduce unwanted biases and affect EV integrity. In this study, we present an efficient non-enzymatic approach for the isolation of brain-derived extracellular vesicles (BDEVs). Our method employs sequential ultracentrifugation and a density-based gradient to achieve a high yield and purity of BDEVs from brain tissue without any enzymatic digestion. Characterization of the isolated EVs revealed their typical morphology and size distribution, as confirmed by transmission electron microscopy and nanoparticle tracking analysis. We show by western blot that known BDEV markers, such as Flotlin-1, CD81, Alix, and the prion protein (PrP) are present without proteolytic cuts introduced by the enzymatic digestion of the tissue. We also show that the absence ofGM130 ,widely accepted as a negative marker to assess BDEV’s purity, is due to enzymatic digestion rather than lack of this protein in the sample. Moreover, we find no differences in mRNA content between the non-enzymatically and the enzymatically isolated BDEVs, showing that the intraluminal part of the BDEVs is not affected by enzymatic isolation. Similarly, our proteomic analysis shows unaltered protein expression in BDEVs isolated with both methods. However, other proteins, potentially in the BDEVs corona and membrane, are differentially expressed, highlighting the consequences of the enzymatically-based isolation protocol.

INSTRUMENT(S): Orbitrap Fusion, Q Exactive

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

SUBMITTER: Bente Siebels  

LAB HEAD: Mohsin Shafiq

PROVIDER: PXD045737 | Pride | 2024-11-07

REPOSITORIES: Pride