TY - JOUR
T1 - Improved recovery of urinary small extracellular vesicles by differential ultracentrifugation
AU - Teixeira-Marques, Ana
AU - Monteiro-Reis, Sara
AU - Montezuma, Diana
AU - Lourenço, Catarina
AU - Oliveira, Miguel Carlos
AU - Constâncio, Vera
AU - Sequeira, José Pedro
AU - Carvalho-Maia, Carina
AU - Freitas, Rui
AU - Martens-Uzunova, Elena S.
AU - Vasconcelos, M. Helena
AU - Henrique, Rui
AU - Jerónimo, Carmen
N1 - Publisher Copyright: © The Author(s) 2024.
PY - 2024/5/28
Y1 - 2024/5/28
N2 - 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.
AB - 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.
UR - https://www.scopus.com/pages/publications/85194899067
U2 - 10.1038/s41598-024-62783-9
DO - 10.1038/s41598-024-62783-9
M3 - Article
C2 - 38806574
AN - SCOPUS:85194899067
SN - 2045-2322
VL - 14
JO - Scientific Reports
JF - Scientific Reports
IS - 1
M1 - 12267
ER -
