Extracellular vesicles (EVs) derived from tumor cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. However, current methods for EV isolation have limited specificity towards tumor-derived EVs that limit their clinical use. Researchers from the Ohio State University present an approach called immunomagnetic sequential ultrafiltration (iSUF) that consists of sequential stages of purification and enrichment of EVs (nonspecifically and specifically) in 2h. In iSUF, EVs present in different volumes of biofluids (0.1 mL to 100 mL) can be significantly enriched (up to 1000 times), with 99.9 % removal of contaminating proteins (e.g., albumin). The yields of cell culture media (CCM), serum, and urine EVs corresponded to 98% ± 3.6%, 94% ± 2.0% and 95% ± 2.0%, respectively (p > 0.05). The final step of iSUF enables the separation of tumor-specific EVs by incorporating immunomagnetic beads specific to a target subpopulation of EVs. Serum from a small cohort of clinical samples from metastatic breast cancer patients and healthy donors were processed by the iSUF platform and the isolated EVs from patients showed significantly higher expression levels of breast cancer biomarkers (i.e., HER2, CD24, and miR21).
The Immunomagnetic sequential ultrafiltration (iSUF) platform
A) Schematic representation of iSUF stages 1, 2, 3, including tangential flow filtration (TFF) purification, centrifugal unit enrichment, and tEV immunoaffinity isolation, respectively. In stage 1, biofluids were processed using a 500 kDa TFF filter, EVs were retained in the retentate and enriched in 7 mL while free proteins and nucleic acids permeated through the TFF filter. Then the PBS valve was opened to start TFF diafiltration until removing 99.9 % of free proteins and nucleic acids. Finally, EVs were recovered in 2 mL of PBS after flushing the TFF system with air. In stage 2, EVs were centrifuged using a 10 kDa centrifugal unit at 3,000 x g and enriched in 100 µL. In stage 3, tEVs were captured using antibodies immobilized to streptavidin-coated magnetic beads (i.e., EGFR) and subsequently pulled out with a magnet. B) iSUF stage 1 purification performance for different biofluids. Cell supernatant (CCM), urine, and serum took 80, 100, and 120 min to obtain 99.9 % efficiency of free protein and nucleic acid removal.