A novel multi-stage enrichment workflow and comprehensive characterization for HEK293F-derived extracellular vesicles

IF 15.5 1区 医学 Q1 CELL BIOLOGY
Nhan Vo, Chau Tran, Nam H. B. Tran, Nhat T. Nguyen, Thieu Nguyen, Duyen T. K. Ho, Diem D. N. Nguyen, Tran Pham, Tien Anh Nguyen, Hoa T. N. Phan, Hoai-Nghia Nguyen, Lan N. Tu
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Abstract

Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompatible and capable of targeted delivery. However, clinical translation of EVs remains challenging due to the lack of standardized and scalable manufacturing protocols to consistently isolate small EVs (sEVs) with both high yield and high purity. The heterogenous nature of sEVs leading to unknown composition of biocargos causes further pushback due to safety concerns. In order to address these issues, we developed a robust quality-controlled multi-stage process to produce and isolate sEVs from human embryonic kidney HEK293F cells. We then compared different 2-step and 3-step workflows for eliminating protein impurities and cell-free nucleic acids to meet acceptable limits of regulatory authorities. Our results showed that sEV production was maximized when HEK293F cells were grown at high-density stationary phase in semi-continuous culture. The novel 3-step workflow combining tangential flow filtration, sucrose-cushion ultracentrifugation and bind-elute size-exclusion chromatography outperformed other methods in sEV purity while still preserved high yield and particle integrity. The purified HEK293F-derived sEVs were thoroughly characterized for identity including sub-population analysis, content profiling including proteomics and miRNA sequencing, and demonstrated excellent preclinical safety profile in both in-vitro and in-vivo testing. Our rigorous enrichment workflow and comprehensive characterization will help advance the development of EVs, particularly HEK293F-derived sEVs, to be safe and reliable drug carriers for therapeutic applications.

Abstract Image

新型多级富集工作流程和 HEK293F 衍生细胞外囊泡的综合表征。
细胞外囊泡(EVs)具有良好的生物相容性和靶向给药能力,正在成为一种前景广阔的给药载体。然而,由于缺乏标准化和可扩展的生产规程来持续分离出高产率和高纯度的小EVs(sEVs),EVs的临床转化仍面临挑战。sEVs 的异质性导致生物卡戈的成分不明,从而引发了更多的安全问题。为了解决这些问题,我们开发了一种稳健的多阶段质量控制流程,用于从人类胚胎肾脏 HEK293F 细胞中生产和分离 sEV。然后,我们比较了消除蛋白质杂质和无细胞核酸的不同 2 步和 3 步工作流程,以满足监管机构的可接受限值。我们的结果表明,当 HEK293F 细胞在半连续培养的高密度静止期生长时,sEV 的产量最大。新颖的三步工作流程结合了切向流过滤、蔗糖垫超速离心法和碱性排阻色谱法,在保持高产率和颗粒完整性的同时,在 sEV 纯度方面优于其他方法。我们对纯化的 HEK293F 衍生 sEV 进行了全面的特性鉴定,包括亚群分析、蛋白质组学和 miRNA 测序等内容分析,并在体外和体内测试中证明了其出色的临床前安全性。我们严格的富集工作流程和全面的特征描述将有助于推动 EVs(尤其是 HEK293F 衍生的 sEVs)的发展,使其成为安全可靠的药物载体,用于治疗应用。
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来源期刊
Journal of Extracellular Vesicles
Journal of Extracellular Vesicles Biochemistry, Genetics and Molecular Biology-Cell Biology
CiteScore
27.30
自引率
4.40%
发文量
115
审稿时长
12 weeks
期刊介绍: The Journal of Extracellular Vesicles is an open access research publication that focuses on extracellular vesicles, including microvesicles, exosomes, ectosomes, and apoptotic bodies. It serves as the official journal of the International Society for Extracellular Vesicles and aims to facilitate the exchange of data, ideas, and information pertaining to the chemistry, biology, and applications of extracellular vesicles. The journal covers various aspects such as the cellular and molecular mechanisms of extracellular vesicles biogenesis, technological advancements in their isolation, quantification, and characterization, the role and function of extracellular vesicles in biology, stem cell-derived extracellular vesicles and their biology, as well as the application of extracellular vesicles for pharmacological, immunological, or genetic therapies. The Journal of Extracellular Vesicles is widely recognized and indexed by numerous services, including Biological Abstracts, BIOSIS Previews, Chemical Abstracts Service (CAS), Current Contents/Life Sciences, Directory of Open Access Journals (DOAJ), Journal Citation Reports/Science Edition, Google Scholar, ProQuest Natural Science Collection, ProQuest SciTech Collection, SciTech Premium Collection, PubMed Central/PubMed, Science Citation Index Expanded, ScienceOpen, and Scopus.
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