细胞外囊泡 (EV) 是脂膜封闭结构，与多种疾病相关，包括泌尿生殖道疾病。尿液中含有源自尿路细胞的 EV。由于其非侵入性收集，尿液是泌尿生殖系统疾病（包括癌症）生物标志物的一个有前途的来源。尿液 EV 分离最常用的方法是差速超速离心 (UC)，但目前的方案会导致 EV 大量损失，从而影响其效率。此外，UC方案是劳动密集型的，进一步限制了临床应用。在此，我们寻求优化 UC 协议，减少花费的时间并提高小型电动汽车 (SEV) 的产量。通过测试不同的 200,000g 超速离心时间来沉淀 SEV，我们发现与 25 分钟相比，48 分钟和 60 分钟可以提高 SEV 的回收率。还评估了沉淀大型 EV（LEV）的步骤，并与过滤尿液上清液进行了比较。我们发现，尿液上清液过滤导致 SEV 回收率增加 1.7 倍，而洗涤步骤导致 SEV 产量减少 0.5 倍。在全球范围内，优化的 UC 协议被证明比 Exoquick-TC (EXO) 更省时，回收的 SEV 数量更多。此外，优化的 UC 方案保留了 RNA 的质量和数量，同时减少了 SEV 分离时间。© 2024。作者。

Extracellular vesicles (EVs) are lipid-membrane enclosed structures that are associated with several diseases, including those of genitourinary tract. Urine contains EVs derived from urinary tract cells. Owing to its non-invasive collection, urine represents a promising source of biomarkers for genitourinary disorders, including cancer. The most used method for urinary EVs separation is differential ultracentrifugation (UC), but current protocols lead to a significant loss of EVs hampering its efficiency. Moreover, UC protocols are labor-intensive, further limiting clinical application. Herein, we sought to optimize an UC protocol, reducing the time spent and improving small EVs (SEVs) yield. By testing different ultracentrifugation times at 200,000g to pellet SEVs, we found that 48 min and 60 min enabled increased SEVs recovery compared to 25 min. A step for pelleting large EVs (LEVs) was also evaluated and compared with filtering of the urine supernatant. We found that urine supernatant filtering resulted in a 1.7-fold increase on SEVs recovery, whereas washing steps resulted in a 0.5 fold-decrease on SEVs yield. Globally, the optimized UC protocol was shown to be more time efficient, recovering higher numbers of SEVs than Exoquick-TC (EXO). Furthermore, the optimized UC protocol preserved RNA quality and quantity, while reducing SEVs separation time.© 2024. The Author(s).