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
{"title":"新型多级富集工作流程和 HEK293F 衍生细胞外囊泡的综合表征。","authors":"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","doi":"10.1002/jev2.12454","DOIUrl":null,"url":null,"abstract":"<p>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.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":null,"pages":null},"PeriodicalIF":15.5000,"publicationDate":"2024-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11101607/pdf/","citationCount":"0","resultStr":"{\"title\":\"A novel multi-stage enrichment workflow and comprehensive characterization for HEK293F-derived extracellular vesicles\",\"authors\":\"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\",\"doi\":\"10.1002/jev2.12454\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>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.</p>\",\"PeriodicalId\":15811,\"journal\":{\"name\":\"Journal of Extracellular Vesicles\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":15.5000,\"publicationDate\":\"2024-05-17\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11101607/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Extracellular Vesicles\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/jev2.12454\",\"RegionNum\":1,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"CELL BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Extracellular Vesicles","FirstCategoryId":"3","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/jev2.12454","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CELL BIOLOGY","Score":null,"Total":0}
A novel multi-stage enrichment workflow and comprehensive characterization for HEK293F-derived extracellular vesicles
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.
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.