Chromatographic purification of small extracellular vesicles using an affinity column for phospholipid membranes.

IF 2 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Biotechnology Letters Pub Date : 2023-12-01 Epub Date: 2023-10-03 DOI:10.1007/s10529-023-03430-7

Kanako Masaki, Abo Bakr F Ahmed, Takenori Ishida, Yuuki Mikami, Hisakage Funabashi, Ryuichi Hirota, Takeshi Ikeda, Akio Kuroda

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Abstract

Objectives: This study aimed to investigate whether chromatography using an ExoPUA column, an affinity column for phospholipid membranes, could potentially serve as an efficient, rapid, scalable, and reproducible method for purifying small extracellular vesicles (sEVs).

Results: We used the ExoPUA column connected to a fast-performance liquid chromatography system. One-step chromatographic purification of sEVs from culture supernatant using the ExoPUA protocol resulted in an 82 ± 16-fold increase in purity with a yield of 38 ± 5% of sEVs. The purified sEVs contained CD9, CD63, TSG101, and miRNA (miR-21), but not the endoplasmic reticulum protein Calnexin. Transmission electron microscopy indicated that the purified sEVs were intact. The purification performance of the ExoPUA protocol showed superior results in terms of yield compared to that of the differential ultracentrifugation method, the most commonly used method for purifying sEVs in laboratories, and purity compared to that of the DEAE chromatography protocol.

Conclusion: The sEVs were effectively purified in the bind-elute mode and the ExoPUA column can be refreshed and sterilized with sodium hydroxide (NaOH), having high potential for multiple sEV purification in a scalable and industrial manner.

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使用磷脂膜亲和柱对细胞外小泡进行色谱纯化。

目的：本研究旨在研究使用ExoPUA柱（一种磷脂膜亲和柱）的色谱法是否有可能成为一种高效、快速、可扩展和可重复的纯化细胞外小泡（sEV）的方法。使用ExoPUA方案从培养上清液中一步色谱纯化sEV得到82 ± 纯度提高16倍，产量达到38 ± sEV的5%。纯化的sEV包含CD9、CD63、TSG101和miRNA（miR-21），但不包含内质网蛋白Calnexin。透射电子显微镜显示纯化的sEV是完整的。与实验室中最常用的纯化sEV的方法差速超速离心法相比，ExoPUA方案的纯化性能在产率和纯度方面均优于DEAE色谱方案。结论：sEV在结合-洗脱模式下得到了有效的纯化，ExoPUA柱可以用氢氧化钠（NaOH）进行刷新和灭菌，具有可扩展和工业化的多重sEV纯化潜力。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Biotechnology Letters 工程技术-生物工程与应用微生物

CiteScore

5.90

自引率

3.70%

发文量

108

审稿时长

1.2 months

期刊介绍： Biotechnology Letters is the world’s leading rapid-publication primary journal dedicated to biotechnology as a whole – that is to topics relating to actual or potential applications of biological reactions affected by microbial, plant or animal cells and biocatalysts derived from them. All relevant aspects of molecular biology, genetics and cell biochemistry, of process and reactor design, of pre- and post-treatment steps, and of manufacturing or service operations are therefore included. Contributions from industrial and academic laboratories are equally welcome. We also welcome contributions covering biotechnological aspects of regenerative medicine and biomaterials and also cancer biotechnology. Criteria for the acceptance of papers relate to our aim of publishing useful and informative results that will be of value to other workers in related fields. The emphasis is very much on novelty and immediacy in order to justify rapid publication of authors’ results. It should be noted, however, that we do not normally publish papers (but this is not absolute) that deal with unidentified consortia of microorganisms (e.g. as in activated sludge) as these results may not be easily reproducible in other laboratories. Papers describing the isolation and identification of microorganisms are not regarded as appropriate but such information can be appended as supporting information to a paper. Papers dealing with simple process development are usually considered to lack sufficient novelty or interest to warrant publication.