Extracellular vesicles (EVs) are emerging as a potential diagnostic test for cancer. Owing to the recent advances in microfluidics, on‐chip EV isolation is showing promise with respect to improved recovery rates, smaller necessary sample volumes, and shorter processing times than ultracentrifugation. Immunoaffinity‐based microfluidic EV isolation using anti‐CD63 is widely used; however, anti‐CD63 is not specific to cancer‐EVs, and some cancers secrete EVs with low expression of CD63. Alternatively, phosphatidylserine (PS), usually expressed in the inner leaflet of the lipid bilayer of the cells, is shown to be expressed on the outer surface of cancer‐associated EVs. A new exosome isolation microfluidic device (newExoChip), conjugated with a PS‐specific protein, to isolate cancer‐associated exosomes from plasma, is presented. The device achieves 90% capture efficiency for cancer cell exosomes compared to 38% for healthy exosomes and isolates 35% more A549‐derived exosomes than an anti‐CD63‐conjugated device. Immobilized exosomes are then easily released using Ca2+chelation. The recovered exosomes from clinical samples are characterized by electron microscopy and western‐blot analysis, revealing exosomal shapes and exosomal protein expressions. ThenewExoChip facilitates the isolation of a specific subset of exosomes, allowing the exploration of the undiscovered roles of exosomes in cancer progression and metastasis.
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.
more » « less- Award ID(s):
- 2107882
- NSF-PAR ID:
- 10390921
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- ELECTROPHORESIS
- Volume:
- 44
- Issue:
- 1-2
- ISSN:
- 0173-0835
- Page Range / eLocation ID:
- p. 190-202
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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