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Extracellular vesicles (EVs) are membrane-bound nanoparticles (50–1000 nm) secreted by all cell types and play critical roles in various biological processes. Among these, exosomes, a smaller subset of EVs, have attracted considerable interest due to their potential applications in diagnostics and therapeutics. However, conventional EV isolation methods are often limited by inefficiencies in processing time, recovery, and scalability. Hydrophobic interaction chromatography utilizing capillary-channeled polymer (C–CP) fiber stationary phases offers a promising alternative, enabling rapid (<15 min), cost-effective (~$5 per column) EV isolation with high loading capacities (~1010–10¹² particles) and minimal sample pre-processing. Despite these advantages, achieving high-throughput EV isolation for larger-scale applications using the C–CP fiber platform is the present challenge. To this end, further optimization of stationary phase packing and adsorption conditions is necessary to maximize the available binding surface area in the current microbore column format. This study systematically investigates the influence of interstitial fraction (i.e. packing density) in polyester (PET) C–CP fiber columns on the dynamic binding capacity (DBC) of EVs isolated from human urine using a high-performance liquid chromatography platform. Microbore columns (0.76 mm i.d. × 300 mm) packed with PET C–CP fibers in both an eight-channel (PET-8) and a novel trilobal (PET-Y) configuration were evaluated using breakthrough curves and frontal analysis. The results reveal that lower packing densities correlate with higher mass- and surface areabased EV binding capacities, with a maximum DBCs of 2.86 × 10¹³ EVs g-1 fiber and 1.22 × 10¹⁴ EVs m⁻² fiber achieved in <2 min of sample loading. Under optimum conditions, surface utilization of >50 % is realized. These results establish a framework for optimizing C–CP fiber-based platforms to enhance EV capture efficiency, facilitating the development of scalable EV isolation techniques for biomedical research and therapeutic applications.
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Rapid isolation and quantification of extracellular vesicles from suspension‐adapted human embryonic kidney cells using capillary‐channeled polymer fiber spin‐down tips
Abstract 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.
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- Award ID(s):
- 2107882
- PAR ID:
- 10390920
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- ELECTROPHORESIS
- Volume:
- 44
- Issue:
- 1-2
- ISSN:
- 0173-0835
- Format(s):
- Medium: X Size: p. 190-202
- Size(s):
- p. 190-202
- Sponsoring Org:
- National Science Foundation
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