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,, 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 10 12 EVs 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.

Quantitative Recoveries of Exosomes and Monoclonal Antibodies from Chinese Hamster Ovary Cell Cultures by Use of a Single, Integrated Two-Dimensional Liquid Chromatography Method

“…Due to the variety of surface chemistries offered, C–CP fibers have been implemented across a variety of separation modalities including reversed phase (RP), , ion exchange (IEX), − affinity, − and hydrophobic interaction chromatography (HIC). , A key physical attribute resulting from their cross-sectional shapes, C–CP fibers interdigitate and self-align in a column format to yield a plurality (1000s) of 1–4 μm diameter open capillaries when packed into column format, resulting in very low mass transfer resistance and reduced backpressure at high linear flow velocities. , More recently, columns and spin-down tips consisting of PET C–CP fibers have been demonstrated toward the rapid and effective isolation of exosomes from matrices ranging from human urine, plasma, and breastmilk, and CHO- and HEK-cell culture supernatants as well as 20 different plant materials, via an HIC capture/elution method. − The HIC separation utilizes high-to-low salt concentration gradients along with the introduction of an organic modifier to assist in eluting the species within 10 min separation times and yielding high purity EV eluates. The capture efficiency, product purity, processing times, and operational costs have been determined, and compare extremely well to the previously cited EV isolation methodologies …”

“…The refractive index of the eluate was experimentally determined to be 1.34649 by a Reichert AR7 Series Automatic Refractometer at 22 °C in the 40% ACN eluate in 1× PBS. Transmission electron microscopy (TEM) was performed using a Hitachi HR7830, using the same sample preparation method as previously reported …”

“…The capture efficiency, product purity, processing times, and operational costs have been determined, and compare extremely well to the previously cited EV isolation methodologies. 64 While efficient isolations of IgG and exosomes have been demonstrated independently from CHO cell supernatants on PP C−CP fibers modified with ProA 52−54 and via HIC on native PET fibers, 65 respectively, the simultaneous production of both entities in the same bioreactor begs the question as to how both species might be efficiently produced and harvested, providing greater overall pharmaceutical yields from the same bioprocess. As EV production is known to reflect the stress/ productivity of cell lines, 66,67 it may be that the dual-analysis capability could contribute as a means of providing fundamental insights or as a process monitoring tool.…”

“…Transmission electron microscopy (TEM) was performed using a Hitachi HR7830, using the same sample preparation method as previously reported. 64 Methods. Individual separations were performed before coupling the two modalities for comprehensive 2DLC separations.…”

Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications—Updated and completely revised edition

“…Although not specifically applied to CE, Ken Marcus' group has recently presented several examples illustrating the advantages of capillary-channeled fibers for the preconcentration of extracellular vesicles from various sources [462,463].…”