Enrichment of Extracellular Vesicle Subpopulations Via Affinity Chromatography

Abstract

Extracellular vesicles (EVs) are secreted nanoscale particles that transfer biomolecular cargo between cells in multicellular organisms. EVs play a variety of roles in intercellular communication and are being explored as potential vehicles for delivery of therapeutic biomolecules. However, EVs are highly heterogeneous in composition and biogenesis route, and this poses substantial challenges for understanding the role of EVs in biology and for harnessing these mechanisms for therapeutic applications, for which purifying therapeutic EVs from mixed EV populations may be necessary. Currently, technologies for isolating EV subsets are limited by overlapping physical properties among EV subsets. To meet this need, here we report an affinity chromatography-based method for enriching a specific EV subset from a heterogeneous EV starting population. By displaying an affinity tagged protein (tag-protein) on the EV surface, tagged EVs may be specifically isolated using simple affinity chromatography. Moreover, recovered EVs are enriched in the tag-protein relative to the starting population of EVs and relative to EVs purified from cell culture supernatant by standard differential centrifugation. Furthermore, chromatographically enriched EVs confer enhanced delivery of a cargo protein to recipient cells (via enhancing the amount of cargo protein per EV) relative to EVs isolated by centrifugation. Altogether, affinity chromatographic enrichment of EV subsets is a viable and facile strategy for investigating EV biology and for harnessing EVs for therapeutic applications.

An Emerging Frontier in Intercellular Communication: Extracellular Vesicles in Regeneration

“…Combining methods like UC or precipitation with size exclusion chromatography also yields higher purity, despite sometimes lower yields of EVs within narrower size ranges ( Sidhom et al, 2020 ). Immunoprecipitation-based approaches that target EV surface molecules like CD63 or phosphatidylserine are more selective ( Nakai et al, 2016 ; Liangsupree et al, 2021 ), and transgenic affinity tagging enables purification of EV subtypes expressing specific proteins ( Hung et al, 2018 ). Newer methods attempt to analyze EV preparations at the single-particle level, such as digital PCR, flow cytometry, and multiplexed immunolabeling, but their use remains limited due to expense and complexity ( Hilton and White, 2021 ).…”

Bioengineering multifunctional extracellular vesicles for targeted delivery of biologics to T cells

“…Affinity chromatography isolation was performed as previously reported. 46 Briefly, an anti-FLAG affinity column was prepared by loading anti-FLAG M2 affinity gel (Sigma A2220-1ML) in a 4 mL 1 x 5 cm glass column (Bio-Rad) and drained via gravity flow.…”

Exploiting Microfluidics for Extracellular Vesicle Isolation and Characterization: Potential Use for Standardized Embryo Quality Assessment

“…AC is based on interactions with immobilized ligands on the stationary phase, while AIEX uses a positively charged matrix to attract negatively charged EVs (54). The AC strategy involves tagging EVs for capture in the affinity columns, which can then be eluted to recover enriched tag-specific EVs (67). The AIEX protocol has been recently improved to enable the isolation from up to 1 L of cell media within 3 h with fewer protein contaminants than UC and TFF (68) and sucrose-DGUC (69).…”