Precise isolation of tumor-relevant EV sub-populations using the Kairos cLDEP particle sorter

Extracellular vesicles (EVs) are cell-derived fragments released by all cell types and are promising platforms for cancer detection and nanomedicine. Isolating and enriching ultra-pure EV sub-populations from patient biofluids based on cell- or cancer-specific biomarkers can reveal tumor-derived molecular signatures with diagnostic and therapeutic implications. However, most existing EV isolation methods rely on size and density, lacking specificity for defined EV subtypes. Existing nano flow cytometry (nFC) platforms (e.g., Astrios EQ) for EV sub-population sorting are also unsatisfactory due to lengthy preparation, limited specificity, and unknown recovery of target sub-populations. To overcome these limitations, we utilized the Apogee Kairos particle sorter, which applies centripetal liquid dielectrophoretic (cLDEP) force to redirect fluids around optically identified EVs, enabling high-speed, high-precision EV sub-population sorting. Operational protocols were optimized using polystyrene beads of defined sizes, achieving 97% purity and 40% recovery. EVs were isolated from cell culture conditioned media (CM) and prostate cancer patient plasma based on size (200-300 nm, 300-400 nm, 600-900 nm, >900 nm), and further sorted post nucleic acid staining, immunolabeling for EV membrane biomarkers (CD9, CD63, etc.) and cancer-associated biomarkers (PSMA, EpCAM, etc.), achieving 83.5-89.4% purity of target EV sub-populations. Transmission electron microscopy confirmed preserved EV vesicular integrity post-sorting. Protein and RNA analyses revealed the presence of molecular cargo in sorted sub-populations consistent with EV identity and disease relevance. We established a robust standard operating protocol (SOP) for the Kairos cLDEP sorter, providing a powerful approach for isolating cancer-derived EV sub-populations with high precision. This platform advances the utility of EV-based liquid biopsies by enabling tumor-selective EV capture for downstream molecular profiling.