Tangential Flow Filtration (TFF) Provides an Easy and Robust Method for the Isolation of Urine EVs
TFF cartridges can be used in combination to isolate EVs from urine with high yield and purity, while minimizing abundant proteins like THP.
Introduction
The study of Urine EVs (uEVs) and their associated biomarkers requires an efficient separation of vesicles from common contaminants. In particular, the attention is paid to the presence of the Tamm-Horsfall protein (THP), a human uromodulin that often co-isolates with the vesicles. Numerous attempts have been made to remove the THP contamination, some using chemical reagents, such as DTT or CHAPS [1,2], or trying to raise the solubility of the uromodulin [3] so as to avoid co-precipitation with vesicles. This application note shows the effectiveness and versatility of Tangential Flow Filtration (TFF) in obtaining high yield of uEVs, free from THP contamination, in a short time and with an extremely simple, scalable and reproducible procedure.
Workflow
Urine was subjected to two sequential TFF steps, using two sterile cartridges with pore size of different cut-off: a) TFF-MV (Product code: HBM-TFF-MV), with pore size of 200 nm, and b) TFF-EVs (Product code: HBM-TFF-EVs-S), with pore size of 50 nm. After filtration with TFF-MV (1), the THP-containing TFF-MV retentate (TFF-MV-RE) was recovered and stored for further analyses. The TFF-MV-derived filtrate (TFF-MV-FT) was then processed by TFF-EVs (2), followed by uEV-containing retentate (TFF-EVs-RE) washing (3) before collection and storage for downstream characterization (4).
Results
Isolation and purification of uEVs with 2-step TFF can be performed with:
- - High particle yield
- - High purity from soluble protein contaminants
Table 1: Analysis of retentates and filtrates from TFF cartridges
Conclusion
Compared with SEC, the 2-step TFF method is a simpler, faster and more scalable protocol, which can be carried out manually on the lab bench or under the safety cabinet. It provides high uEV yield (i.e., 59%) with typical EV morphology and EV-associated tetraspanin expression, enabling great purification from soluble proteins (i.e., 99.9% removal), including THP. The total turnaround time to process 40 mL of urine is 15 min, and neither chemical treatment nor dilution of the sample is required.
References
[1] Fernández-Llama, Patricia, et al. "Tamm-Horsfall protein and urinary exosome isolation." Kidney International 77.8 (2010): 736-742.
[2] Musante, Luca, et al. "A simplified method to recover urinary vesicles for clinical applications and sample banking." Scientific Reports 4.1 (2014): 1-11.
[3] Puhka, M., et al. "KeepEX, a simple dilution protocol for improving extracellular vesicle yields from urine." European Journal of Pharmaceutical Sciences 98 (2017): 30-39.