TriPort tangential flow filtration: a lab-assembled system for EV isolation across diverse cell-culture sources

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.