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A novel asymmetric depth filtration (DF) approach for isolation of extracellular vesicles (EVs) from biological fluids is presented, and its performance is compared with established methods. The developed workflow is simple, inexpensive, and relatively fast. Compared with ultracentrifugation and size-exclusion chromatography, the developed method isolates EVs with higher purity and yield. Only standard laboratory equipment is needed for its implementation, which makes it suitable for low-resource locations. The described implementation of the method is suitable for EV isolation from small biological samples in diagnostic and treatment guidance applications. Following the scale-up routes adopted in the biomanufacturing of therapeutics, which routinely rely on DF as one of the product purification steps, the developed method may be scaled up to harvesting therapeutic EVs from large volumes of growth medium.

The aim of the study was to explore the polyethylene glycol–dextran two-phase polymer system formed in human plasma to isolate the exosome-enriched fraction of plasma extracellular nanovesicles (ENVs). Systematic analysis was performed to determine the optimal combination of the polymer mixture parameters (molecular mass and concentration) that resulted in phase separation. The separated phases were analyzed by nanoparticle tracking analysis and Raman spectroscopy. The isolated vesicles were characterized by atomic force microscopy and dot blotting. In conclusion, the protein and microRNA contents of the isolated ENVs were assayed by flow cytometry and by reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR), respectively. The presented results revealed the applicability of a new method for plasma ENV isolation and further analysis with a diagnostic purpose.

A novel asymmetric depth filtration (DF) approach for isolation of extracellular vesicles (EVs) from biological fluids is presented, and its performance is compared with established methods. The developed workflow is simple, inexpensive, and relatively fast. Compared with ultracentrifugation and size-exclusion chromatography, the developed method isolates EVs with higher purity and yield. Only standard laboratory equipment is needed for its implementation, which makes it suitable for low-resource locations. The described implementation of the method is suitable for EV isolation from small biological samples in diagnostic and treatment guidance applications. Following the scale-up routes adopted in the biomanufacturing of therapeutics, which routinely rely on DF as one of the product purification steps, the developed method may be scaled up to harvesting therapeutic EVs from large volumes of growth medium.

The concept of liquid biopsy has emerged as a novel approach for cancer screening, which is based on the analysis of circulating cancer biomarkers in body fluids. Among the various circulating cancer biomarkers, including Food and Drug Administration (FDA)-approved circulating tumor cells (CTC) and circulating tumor DNA (ctDNA), exosomes have attracted tremendous attention due to their ability to diagnose cancer in its early stages with high efficiency. Recently, surface-enhanced Raman spectroscopy (SERS) has been applied for the detection of cancer exosomes due to its high sensitivity, specificity, and multiplexing capability. In this article, we review recent progress in the development of SERS-based technologies for in vitro identification of circulating cancer exosomes. The accent is made on the detection strategies and interpretation of the SERS data. The problems of detecting cancer-derived exosomes from patient samples and future perspectives of SERS-based diagnostics are also discussed.