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However, isolation of brain\u2010derived EVs (BDEVs) from tissue remains challenging, often involving enzymatic digestion steps that may compromise the integrity of EV proteins and overall functionality. Here, we describe that collagenase digestion, commonly used for BDEV isolation, produces undesired protein cleavage of EV\u2010associated proteins in brain tissue homogenates and cell\u2010derived EVs. In order to avoid this effect, we studied the possibility of isolating BDEVs with a reduced amount of collagenase or without any protease. Characterization of the isolated BDEVs from mouse and human samples (both female and male) revealed their characteristic morphology and size distribution with both approaches. However, we show that even minor enzymatic digestion induces \u2018artificial\u2019 proteolytic processing in key BDEV markers, such as Flotillin\u20101, CD81, and the cellular prion protein (PrP\n                    <jats:sup>C<\/jats:sup>\n                    ), whereas avoiding enzymatic treatment completely preserves their integrity. We found no major differences in mRNA and protein content between non\u2010enzymatically and enzymatically isolated BDEVs, suggesting that the same BDEV populations are purified with both approaches. Intriguingly, the lack of Golgi marker GM130 signal, often referred to as contamination indicator (or negative marker) in EV preparations, seems to result from enzymatic digestion rather than from its actual absence in BDEV samples. Overall, we show that non\u2010enzymatic isolation of EVs from brain tissue is possible and avoids artificial pruning of proteins while achieving an overall high BDEV yield and purity. This protocol will help to understand the functions of BDEV and their associated proteins in a near\u2010physiological setting, thus opening new research approaches.\n                  <\/jats:p>","DOI":"10.1002\/jev2.70011","type":"journal-article","created":{"date-parts":[[2024,11,7]],"date-time":"2024-11-07T07:24:58Z","timestamp":1730964298000},"source":"Crossref","is-referenced-by-count":28,"title":["Efficient enzyme\u2010free isolation of brain\u2010derived extracellular vesicles"],"prefix":"10.1002","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4810-8091","authenticated-orcid":false,"given":"Andreu","family":"Matamoros\u2010Angles","sequence":"first","affiliation":[{"name":"Institute of Neuropathology University Medical Center Hamburg\u2010Eppendorf (UKE)  Hamburg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emina","family":"Karadjuzovic","sequence":"additional","affiliation":[{"name":"Institute of Neuropathology University Medical 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