TriPort tangential flow filtration: a lab-assembled system for EV isolation across diverse cell-culture sources
- 1School of Medical Sciences, University of Sydney, Sydney, NSW, Australia
Abstract
Tangential flow filtration (TFF) is an effective method for scalable EV isolation. Here, we describe and validate a reference protocol for TriPort-TFF, a laboratory-assembled TFF system featuring a three- port reservoir design enabling sample circulation, retentate return, and diafiltration. Constructed from individually sourced, commercially available components with detailed assembly and operating parameters, TriPort-TFF can be built at a lower cost than commercially integrated TFF platforms. We evaluated the system using conditioned media (CM) from human MDM cultured with EV-depleted human serum, human cerebral organoids in serum-free medium, and mouse BMDM cultured with EV-depleted foetal bovine serum (FBS) unstimulated or LPS-stimulated. The system reproducibly isolated EVs from all three sources, and biological replicates of BMDM showed distinct proteomic clusters between LPS-stimulated and unstimulated conditions. The requirement for subsequent size-exclusion chromatography (SEC) after TFF depended on the sample serum-presence. In serum-containing CM, TFF alone co-concentrated serum proteins alongside EVs, subsequent SEC improved the particle-to- protein ratio, reduced inter-replicate variability and improved proteomic depth. TFF alone in serum- free organoid CM already yielded higher particle-to-protein ratios than TFF-only preparations from serum-containing CM, with SEC further improving this ratio. TriPort-TFF offers an accessible system for EV-isolation that can be combined with SEC for diverse sample types.
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BibTeX
@misc{mainali2026triport,
title = {{TriPort tangential flow filtration: a lab-assembled system for EV isolation across diverse cell-culture sources}},
author = {D. Mainali and M. D. Hade and A. Ge and H. Ezzarouli and S. Maksour and L. Thompson and C. L. Moreno and G. G. Neely and S. Hallal and C. Goldsbury and S. Magana and L. Piccio},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.03.756465},
url = {https://www.biorxiv.org/content/10.64898/2026.10.03.756465v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - TriPort tangential flow filtration: a lab-assembled system for EV isolation across diverse cell-culture sources
AU - D. Mainali
AU - M. D. Hade
AU - A. Ge
AU - H. Ezzarouli
AU - S. Maksour
AU - L. Thompson
AU - C. L. Moreno
AU - G. G. Neely
AU - S. Hallal
AU - C. Goldsbury
AU - S. Magana
AU - L. Piccio
PY - 2026
DA - 2026/10/03
PB - bioRxiv
DO - 10.64898/2026.10.03.756465
UR - https://www.biorxiv.org/content/10.64898/2026.10.03.756465v1
AB - Tangential flow filtration (TFF) is an effective method for scalable EV isolation. Here, we describe and validate a reference protocol for TriPort-TFF, a laboratory-assembled TFF system featuring a three- port reservoir design enabling sample circulation, retentate return, and diafiltration. Constructed from individually sourced, commercially available components with detailed assembly and operating parameters, TriPort-TFF can be built at a lower cost than commercially integrated TFF platforms. We evaluated the system using conditioned media (CM) from human MDM cultured with EV-depleted human serum, human cerebral organoids in serum-free medium, and mouse BMDM cultured with EV-depleted foetal bovine serum (FBS) unstimulated or LPS-stimulated. The system reproducibly isolated EVs from all three sources, and biological replicates of BMDM showed distinct proteomic clusters between LPS-stimulated and unstimulated conditions. The requirement for subsequent size-exclusion chromatography (SEC) after TFF depended on the sample serum-presence. In serum-containing CM, TFF alone co-concentrated serum proteins alongside EVs, subsequent SEC improved the particle-to- protein ratio, reduced inter-replicate variability and improved proteomic depth. TFF alone in serum- free organoid CM already yielded higher particle-to-protein ratios than TFF-only preparations from serum-containing CM, with SEC further improving this ratio. TriPort-TFF offers an accessible system for EV-isolation that can be combined with SEC for diverse sample types.
ER -