Development of an optimized and scalable method for isolation of umbilical cord blood-derived small extracellular vesicles for future clinical use
Autor(a) principal: | |
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Data de Publicação: | 2021 |
Outros Autores: | , , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
DOI: | 10.1002/sctm.20-0376 |
Texto Completo: | http://hdl.handle.net/10316/105481 https://doi.org/10.1002/sctm.20-0376 |
Resumo: | Extracellular vesicles (EV) are a promising therapeutic tool in regenerative medicine. These particles were shown to accelerate wound healing, through delivery of regenerative mediators, such as microRNAs. Herein we describe an optimized and upscalable process for the isolation of EV smaller than 200 nm (sEV), secreted by umbilical cord blood mononuclear cells (UCB-MNC) under ischemic conditions and propose quality control thresholds for the isolated vesicles, based on the thorough characterization of their protein, lipid and RNA content. Ultrafiltration and size exclusion chromatography (UF/SEC) optimized methodology proved superior to traditional ultracentrifugation (UC), regarding production time, standardization, scalability, and vesicle yield. Using UF/SEC, we were able to recover approximately 400 times more sEV per mL of media than with UC, and upscaling this process further increases EV yield by about 3-fold. UF/SEC-isolated sEV display many of the sEV/exosomes classical markers and are enriched in molecules with anti-inflammatory and regenerative capacity, such as hemopexin and miR-150. Accordingly, treatment with sEV promotes angiogenesis and extracellular matrix remodeling, in vitro. In vivo, UCB-MNC-sEV significantly accelerate skin regeneration in a mouse model of delayed wound healing. The proposed isolation protocol constitutes a significant improvement compared to UC, the gold-standard in the field. Isolated sEV maintain their regenerative properties, whereas downstream contaminants are minimized. The use of UF/SEC allows for the standardization and upscalability required for mass production of sEV to be used in a clinical setting. |
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Development of an optimized and scalable method for isolation of umbilical cord blood-derived small extracellular vesicles for future clinical useangiogenesiscell signalingcellular therapyclinical translationmicroRNAstem cellstissue regenerationumbilical cord bloodAnimalsBiomarkersMiceMicroRNAsExosomesExtracellular VesiclesFetal BloodExtracellular vesicles (EV) are a promising therapeutic tool in regenerative medicine. These particles were shown to accelerate wound healing, through delivery of regenerative mediators, such as microRNAs. Herein we describe an optimized and upscalable process for the isolation of EV smaller than 200 nm (sEV), secreted by umbilical cord blood mononuclear cells (UCB-MNC) under ischemic conditions and propose quality control thresholds for the isolated vesicles, based on the thorough characterization of their protein, lipid and RNA content. Ultrafiltration and size exclusion chromatography (UF/SEC) optimized methodology proved superior to traditional ultracentrifugation (UC), regarding production time, standardization, scalability, and vesicle yield. Using UF/SEC, we were able to recover approximately 400 times more sEV per mL of media than with UC, and upscaling this process further increases EV yield by about 3-fold. UF/SEC-isolated sEV display many of the sEV/exosomes classical markers and are enriched in molecules with anti-inflammatory and regenerative capacity, such as hemopexin and miR-150. Accordingly, treatment with sEV promotes angiogenesis and extracellular matrix remodeling, in vitro. In vivo, UCB-MNC-sEV significantly accelerate skin regeneration in a mouse model of delayed wound healing. The proposed isolation protocol constitutes a significant improvement compared to UC, the gold-standard in the field. Isolated sEV maintain their regenerative properties, whereas downstream contaminants are minimized. The use of UF/SEC allows for the standardization and upscalability required for mass production of sEV to be used in a clinical setting.Oxford University Press2021-06info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articlehttp://hdl.handle.net/10316/105481http://hdl.handle.net/10316/105481https://doi.org/10.1002/sctm.20-0376eng2157-65642157-6580Cardoso, Renato M. S.Rodrigues, Sílvia C.Gomes, Claudia F.Duarte, Filipe V.Romao, MaryseLeal, Ermelindo C.Freire, Patricia C.Neves, RicardoCorreia, Joana Simõesinfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-03-02T09:30:52Zoai:estudogeral.uc.pt:10316/105481Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T21:22:02.801574Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Development of an optimized and scalable method for isolation of umbilical cord blood-derived small extracellular vesicles for future clinical use |
title |
Development of an optimized and scalable method for isolation of umbilical cord blood-derived small extracellular vesicles for future clinical use |
spellingShingle |
Development of an optimized and scalable method for isolation of umbilical cord blood-derived small extracellular vesicles for future clinical use Development of an optimized and scalable method for isolation of umbilical cord blood-derived small extracellular vesicles for future clinical use Cardoso, Renato M. S. angiogenesis cell signaling cellular therapy clinical translation microRNA stem cells tissue regeneration umbilical cord blood Animals Biomarkers Mice MicroRNAs Exosomes Extracellular Vesicles Fetal Blood Cardoso, Renato M. S. angiogenesis cell signaling cellular therapy clinical translation microRNA stem cells tissue regeneration umbilical cord blood Animals Biomarkers Mice MicroRNAs Exosomes Extracellular Vesicles Fetal Blood |
title_short |
Development of an optimized and scalable method for isolation of umbilical cord blood-derived small extracellular vesicles for future clinical use |
title_full |
Development of an optimized and scalable method for isolation of umbilical cord blood-derived small extracellular vesicles for future clinical use |
title_fullStr |
Development of an optimized and scalable method for isolation of umbilical cord blood-derived small extracellular vesicles for future clinical use Development of an optimized and scalable method for isolation of umbilical cord blood-derived small extracellular vesicles for future clinical use |
title_full_unstemmed |
Development of an optimized and scalable method for isolation of umbilical cord blood-derived small extracellular vesicles for future clinical use Development of an optimized and scalable method for isolation of umbilical cord blood-derived small extracellular vesicles for future clinical use |
title_sort |
Development of an optimized and scalable method for isolation of umbilical cord blood-derived small extracellular vesicles for future clinical use |
author |
Cardoso, Renato M. S. |
author_facet |
Cardoso, Renato M. S. Cardoso, Renato M. S. Rodrigues, Sílvia C. Gomes, Claudia F. Duarte, Filipe V. Romao, Maryse Leal, Ermelindo C. Freire, Patricia C. Neves, Ricardo Correia, Joana Simões Rodrigues, Sílvia C. Gomes, Claudia F. Duarte, Filipe V. Romao, Maryse Leal, Ermelindo C. Freire, Patricia C. Neves, Ricardo Correia, Joana Simões |
author_role |
author |
author2 |
Rodrigues, Sílvia C. Gomes, Claudia F. Duarte, Filipe V. Romao, Maryse Leal, Ermelindo C. Freire, Patricia C. Neves, Ricardo Correia, Joana Simões |
author2_role |
author author author author author author author author |
dc.contributor.author.fl_str_mv |
Cardoso, Renato M. S. Rodrigues, Sílvia C. Gomes, Claudia F. Duarte, Filipe V. Romao, Maryse Leal, Ermelindo C. Freire, Patricia C. Neves, Ricardo Correia, Joana Simões |
dc.subject.por.fl_str_mv |
angiogenesis cell signaling cellular therapy clinical translation microRNA stem cells tissue regeneration umbilical cord blood Animals Biomarkers Mice MicroRNAs Exosomes Extracellular Vesicles Fetal Blood |
topic |
angiogenesis cell signaling cellular therapy clinical translation microRNA stem cells tissue regeneration umbilical cord blood Animals Biomarkers Mice MicroRNAs Exosomes Extracellular Vesicles Fetal Blood |
description |
Extracellular vesicles (EV) are a promising therapeutic tool in regenerative medicine. These particles were shown to accelerate wound healing, through delivery of regenerative mediators, such as microRNAs. Herein we describe an optimized and upscalable process for the isolation of EV smaller than 200 nm (sEV), secreted by umbilical cord blood mononuclear cells (UCB-MNC) under ischemic conditions and propose quality control thresholds for the isolated vesicles, based on the thorough characterization of their protein, lipid and RNA content. Ultrafiltration and size exclusion chromatography (UF/SEC) optimized methodology proved superior to traditional ultracentrifugation (UC), regarding production time, standardization, scalability, and vesicle yield. Using UF/SEC, we were able to recover approximately 400 times more sEV per mL of media than with UC, and upscaling this process further increases EV yield by about 3-fold. UF/SEC-isolated sEV display many of the sEV/exosomes classical markers and are enriched in molecules with anti-inflammatory and regenerative capacity, such as hemopexin and miR-150. Accordingly, treatment with sEV promotes angiogenesis and extracellular matrix remodeling, in vitro. In vivo, UCB-MNC-sEV significantly accelerate skin regeneration in a mouse model of delayed wound healing. The proposed isolation protocol constitutes a significant improvement compared to UC, the gold-standard in the field. Isolated sEV maintain their regenerative properties, whereas downstream contaminants are minimized. The use of UF/SEC allows for the standardization and upscalability required for mass production of sEV to be used in a clinical setting. |
publishDate |
2021 |
dc.date.none.fl_str_mv |
2021-06 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10316/105481 http://hdl.handle.net/10316/105481 https://doi.org/10.1002/sctm.20-0376 |
url |
http://hdl.handle.net/10316/105481 https://doi.org/10.1002/sctm.20-0376 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
2157-6564 2157-6580 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.publisher.none.fl_str_mv |
Oxford University Press |
publisher.none.fl_str_mv |
Oxford University Press |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
instname_str |
Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
instacron_str |
RCAAP |
institution |
RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
collection |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository.name.fl_str_mv |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
repository.mail.fl_str_mv |
|
_version_ |
1822183344302981120 |
dc.identifier.doi.none.fl_str_mv |
10.1002/sctm.20-0376 |