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Extracellular vesicles (EVs) are 50–1000 nm membranous vesicles secreted from all cells that play important roles in many biological processes. Exosomes, a smaller-sized subset of EVs, have become of increasing interest in fundamental biochemistry and clinical fields due to their rich biological cargos and their roles in processes such as cell-signaling, maintaining homeostasis, and regulating cellular functions. To be implemented effectively in fundamental biochemistry and clinical diagnostics fields of study, and for their proposed use as vectors in gene therapies, there is a need for new methods for the isolation of large concentrations of high-purity exosomes from complex matrices in a timely manner. To address current limitations regarding recovery and purity, described here is a frontal throughput and recovery analysis of exosomes derived from human embryonic kidney (HEK) cell cultures and human urine specimens using capillary-channeled polymer (C-CP) fiber stationary phases via high performance liquid chromatography (HPLC). Using the C-CP fiber HPLC method for EV isolations, the challenge of recovering purified EVs from small sample volumes imparted by the traditional techniques was overcome while introducing significant benefits in processing, affordability (~5 $ per column), loading (~1012 particles), and recovery (1011–1012 particles) from whole specimens without further processing requirements. 

There is great interest in advancing methodologies for the isolation and characterization of exosomes (30–150 nm, extracellular vesicles (EVs)) for fundamental biochemical research and liquid biopsy applications. This is due to the accessibility of exosomal surface biomarkers, providing relevant biochemical information from their cells of origin. Exosome-based techniques hold potential for diagnostic applications through less invasive sampling ( versus the physical extraction methods of pathology). This study demonstrates a simple spin-down tip methodology for generic exosome capture, followed by immunoaffinity-based fluorescent labeling to classify EVs captured on a polyester capillary-channeled polymer (C-CP) fiber stationary phase. An antibody to the generic EV tetraspanin protein (CD81) is employed to confirm the presence of biologically active EVs on the fiber surface. An antibody to the CA125 protein, upregulated in the case of ovarian cell stress, is included as a cancer marker protein. Scanning electron microscopy and confocal fluorescence microscopy were performed directly on the capture fibers to visualize the morphology and assess the bioactivity/identity of captured vesicles. This report provides a proof-of-concept for an efficient means of isolating, purifying, immunolabeling, and fluorescent imaging for the biomarker assessment of extracellular vesicles on a single platform . Herein lies the novelty of the overall approach. The ability to affect the entire isolation, immunolabeling, and imaging process in <5 hours is demonstrated. The C-CP fiber spin-down tip is an efficient exosome isolation methodology for microliter samples from diverse media (human urine and cell culture media here) towards diverse means of characterization and identification. 

Exosomes, a subset of extracellular vesicles (EVs, 30–200‐nm diameter), serve as biomolecular snapshots of their cell of origin and vehicles for intercellular communication, playing roles in biological processes, including homeostasis maintenance and immune modulation. The large‐scale processing of exosomes for use as therapeutic vectors has been proposed, but these applications are limited by impure, low‐yield recoveries from cell culture milieu (CCM). Current isolation methods are also limited by tedious and laborious workflows, especially toward an isolation of EVs from CCM for therapeutic applications. Employed is a rapid (<10 min) EV isolation method on a capillary‐channeled polymer fiber spin‐down tip format. EVs are isolated from the CCM of suspension‐adapted human embryonic kidney cells (HEK293), one of the candidate cell lines for commercial EV production. This batch solid‐phase extraction technique allows 10¹²EVs to be obtained from only 100‐µl aliquots of milieu, processed using a benchtop centrifuge. The tip‐isolated EVs were characterized using transmission electron microscopy, multi‐angle light scattering, absorbance quantification, an enzyme‐linked immunosorbent assay to tetraspanin marker proteins, and a protein purity assay. It is believed that the demonstrated approach has immediate relevance in research and analytical laboratories, with opportunities for production‐level scale‐up projected.