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The separation of different subpopulations of EVs from mixtures is critical but challenging. Epididymosomes are secreted by the epididymal epithelium and play a key role in sperm maturation and function. However, limited access to human epididymal tissue and epididymal fluid has hampered further study of epididymosomes and their potential clinical applications. Here, we established a novel strategy based on flow cytometry sorting to isolate human CD63\u2010positive epididymosomes from ejaculate. We identified CD52, a membrane\u2010located protein expressed exclusively in the epididymis, as the sorting marker for human epididymosomes. Then, CD63\u2010positive epididymosomes were isolated from human semen using a flow cytometry sorting instrument and concentrated. Additionally, we observed that isolated CD63\u2010positive epididymosomes improved sperm function more than other CD63\u2010positive seminal EV subpopulations did, demonstrating the successful isolation of a subpopulation of epididymosomes from human semen and their potential application in the clinic.<\/jats:p>","DOI":"10.1002\/jev2.70006","type":"journal-article","created":{"date-parts":[[2024,10,17]],"date-time":"2024-10-17T08:39:53Z","timestamp":1729154393000},"source":"Crossref","is-referenced-by-count":13,"title":["Isolation of CD63\u2010positive epididymosomes from human semen and its application in improving sperm function"],"prefix":"10.1002","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7994-7154","authenticated-orcid":false,"given":"Jingwen","family":"Luo","sequence":"first","affiliation":[{"name":"Center of Reproductive Medicine, Union Hospital, Tongji Medical College Huazhong University of Science and Technology  Wuhan 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