Ion exchange chromatography as a simple and scalable method to isolate biologically active small extracellular vesicles from conditioned media

Autores
Malvicini, Ricardo; Santa Cruz, Diego Mario; Tolomeo, Anna Maria; Muraca, Maurizio; Yannarelli, Gustavo Gabriel; Pacienza, Natalia Alejandra
Año de publicación
2023
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
In the last few years, extracellular vesicles (EVs) have become of great interest due to their potential as biomarkers, drug delivery systems, and, in particular, therapeutic agents. However, there is no consensus on which is the best way to isolate these EVs. The choice of the isolation method depends on the starting material (i.e., conditioned culture media, urine, serum, etc.) and their downstream applications. Even though there are numerous methods to isolate EVs, few are compatible with clinical applications as they are not scalable. In the present work, we set up a protocol to isolate EVs from conditioned media by ion exchange chromatography, a simple, fast, and scalable method, suitable for clinical production. We performed the isolation using an anion exchange resin (Q sepharose) and eluted the EVs using 500 mM NaCl. We characterized the elution profile by measuring protein and lipid concentration, and CD63 by ELISA. Moreover, we immunophenotyped all the eluted fractions, assessed the presence of TSG101, calnexin, and cytochrome C by western blot, analyzed nanoparticle size and distribution by tRPS, and morphology by TEM. Finally, we evaluated the immunomodulatory activity in vitro. We found that most EVs are eluted and concentrated in a single peak fraction, with a mean particle size of <150nm and expression of CD9, CD63, CD81, and TSG101 markers. Moreover, sEVs in fraction 4 exerted an anti-inflammatory activity on LPS-stimulated macrophages. In summary, we set up a chromatographic, scalable, and clinically compatible method to isolate and concentrate small EVs from conditioned media, which preserves the EVs biological activity.
Fil: Malvicini, Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina. Università di Padova; Italia. Fondazione Istituto Di Ricerca Pediatrica Città Della Speranza; Italia. Consorzio Per la Ricerca Sanitaria; Italia
Fil: Santa Cruz, Diego Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina
Fil: Tolomeo, Anna Maria. Fondazione Istituto Di Ricerca Pediatrica Città Della Speranza; Italia. Consorzio Per la Ricerca Sanitaria; Italia. Università di Padova; Italia
Fil: Muraca, Maurizio. Fondazione Istituto Di Ricerca Pediatrica Città Della Speranza; Italia. Consorzio Per la Ricerca Sanitaria; Italia. Università di Padova; Italia
Fil: Yannarelli, Gustavo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina
Fil: Pacienza, Natalia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina
Materia
Ion exchange chromatography
Extracellular vesicles
Isolation Method
Conditioned media
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/227882
Acceder
id CONICETDig_6e31f7cb36f606ea3a573ac68773f6a1
oai_identifier_str oai:ri.conicet.gov.ar:11336/227882
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Ion exchange chromatography as a simple and scalable method to isolate biologically active small extracellular vesicles from conditioned mediaMalvicini, RicardoSanta Cruz, Diego MarioTolomeo, Anna MariaMuraca, MaurizioYannarelli, Gustavo GabrielPacienza, Natalia AlejandraIon exchange chromatographyExtracellular vesiclesIsolation MethodConditioned mediahttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1In the last few years, extracellular vesicles (EVs) have become of great interest due to their potential as biomarkers, drug delivery systems, and, in particular, therapeutic agents. However, there is no consensus on which is the best way to isolate these EVs. The choice of the isolation method depends on the starting material (i.e., conditioned culture media, urine, serum, etc.) and their downstream applications. Even though there are numerous methods to isolate EVs, few are compatible with clinical applications as they are not scalable. In the present work, we set up a protocol to isolate EVs from conditioned media by ion exchange chromatography, a simple, fast, and scalable method, suitable for clinical production. We performed the isolation using an anion exchange resin (Q sepharose) and eluted the EVs using 500 mM NaCl. We characterized the elution profile by measuring protein and lipid concentration, and CD63 by ELISA. Moreover, we immunophenotyped all the eluted fractions, assessed the presence of TSG101, calnexin, and cytochrome C by western blot, analyzed nanoparticle size and distribution by tRPS, and morphology by TEM. Finally, we evaluated the immunomodulatory activity in vitro. We found that most EVs are eluted and concentrated in a single peak fraction, with a mean particle size of <150nm and expression of CD9, CD63, CD81, and TSG101 markers. Moreover, sEVs in fraction 4 exerted an anti-inflammatory activity on LPS-stimulated macrophages. In summary, we set up a chromatographic, scalable, and clinically compatible method to isolate and concentrate small EVs from conditioned media, which preserves the EVs biological activity.Fil: Malvicini, Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina. Università di Padova; Italia. Fondazione Istituto Di Ricerca Pediatrica Città Della Speranza; Italia. Consorzio Per la Ricerca Sanitaria; ItaliaFil: Santa Cruz, Diego Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; ArgentinaFil: Tolomeo, Anna Maria. Fondazione Istituto Di Ricerca Pediatrica Città Della Speranza; Italia. Consorzio Per la Ricerca Sanitaria; Italia. Università di Padova; ItaliaFil: Muraca, Maurizio. Fondazione Istituto Di Ricerca Pediatrica Città Della Speranza; Italia. Consorzio Per la Ricerca Sanitaria; Italia. Università di Padova; ItaliaFil: Yannarelli, Gustavo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; ArgentinaFil: Pacienza, Natalia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; ArgentinaPublic Library of Science2023-09info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/227882Malvicini, Ricardo; Santa Cruz, Diego Mario; Tolomeo, Anna Maria; Muraca, Maurizio; Yannarelli, Gustavo Gabriel; et al.; Ion exchange chromatography as a simple and scalable method to isolate biologically active small extracellular vesicles from conditioned media; Public Library of Science; Plos One; 18; 9; 9-2023; 1-111932-6203CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0291589info:eu-repo/semantics/altIdentifier/doi/10.1371/journal.pone.0291589info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-15T14:23:50Zoai:ri.conicet.gov.ar:11336/227882instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-15 14:23:51.158CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Ion exchange chromatography as a simple and scalable method to isolate biologically active small extracellular vesicles from conditioned media
title Ion exchange chromatography as a simple and scalable method to isolate biologically active small extracellular vesicles from conditioned media
spellingShingle Ion exchange chromatography as a simple and scalable method to isolate biologically active small extracellular vesicles from conditioned media
Malvicini, Ricardo
Ion exchange chromatography
Extracellular vesicles
Isolation Method
Conditioned media
title_short Ion exchange chromatography as a simple and scalable method to isolate biologically active small extracellular vesicles from conditioned media
title_full Ion exchange chromatography as a simple and scalable method to isolate biologically active small extracellular vesicles from conditioned media
title_fullStr Ion exchange chromatography as a simple and scalable method to isolate biologically active small extracellular vesicles from conditioned media
title_full_unstemmed Ion exchange chromatography as a simple and scalable method to isolate biologically active small extracellular vesicles from conditioned media
title_sort Ion exchange chromatography as a simple and scalable method to isolate biologically active small extracellular vesicles from conditioned media
dc.creator.none.fl_str_mv Malvicini, Ricardo
Santa Cruz, Diego Mario
Tolomeo, Anna Maria
Muraca, Maurizio
Yannarelli, Gustavo Gabriel
Pacienza, Natalia Alejandra
author Malvicini, Ricardo
author_facet Malvicini, Ricardo
Santa Cruz, Diego Mario
Tolomeo, Anna Maria
Muraca, Maurizio
Yannarelli, Gustavo Gabriel
Pacienza, Natalia Alejandra
author_role author
author2 Santa Cruz, Diego Mario
Tolomeo, Anna Maria
Muraca, Maurizio
Yannarelli, Gustavo Gabriel
Pacienza, Natalia Alejandra
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv Ion exchange chromatography
Extracellular vesicles
Isolation Method
Conditioned media
topic Ion exchange chromatography
Extracellular vesicles
Isolation Method
Conditioned media
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv In the last few years, extracellular vesicles (EVs) have become of great interest due to their potential as biomarkers, drug delivery systems, and, in particular, therapeutic agents. However, there is no consensus on which is the best way to isolate these EVs. The choice of the isolation method depends on the starting material (i.e., conditioned culture media, urine, serum, etc.) and their downstream applications. Even though there are numerous methods to isolate EVs, few are compatible with clinical applications as they are not scalable. In the present work, we set up a protocol to isolate EVs from conditioned media by ion exchange chromatography, a simple, fast, and scalable method, suitable for clinical production. We performed the isolation using an anion exchange resin (Q sepharose) and eluted the EVs using 500 mM NaCl. We characterized the elution profile by measuring protein and lipid concentration, and CD63 by ELISA. Moreover, we immunophenotyped all the eluted fractions, assessed the presence of TSG101, calnexin, and cytochrome C by western blot, analyzed nanoparticle size and distribution by tRPS, and morphology by TEM. Finally, we evaluated the immunomodulatory activity in vitro. We found that most EVs are eluted and concentrated in a single peak fraction, with a mean particle size of <150nm and expression of CD9, CD63, CD81, and TSG101 markers. Moreover, sEVs in fraction 4 exerted an anti-inflammatory activity on LPS-stimulated macrophages. In summary, we set up a chromatographic, scalable, and clinically compatible method to isolate and concentrate small EVs from conditioned media, which preserves the EVs biological activity.
Fil: Malvicini, Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina. Università di Padova; Italia. Fondazione Istituto Di Ricerca Pediatrica Città Della Speranza; Italia. Consorzio Per la Ricerca Sanitaria; Italia
Fil: Santa Cruz, Diego Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina
Fil: Tolomeo, Anna Maria. Fondazione Istituto Di Ricerca Pediatrica Città Della Speranza; Italia. Consorzio Per la Ricerca Sanitaria; Italia. Università di Padova; Italia
Fil: Muraca, Maurizio. Fondazione Istituto Di Ricerca Pediatrica Città Della Speranza; Italia. Consorzio Per la Ricerca Sanitaria; Italia. Università di Padova; Italia
Fil: Yannarelli, Gustavo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina
Fil: Pacienza, Natalia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina
description In the last few years, extracellular vesicles (EVs) have become of great interest due to their potential as biomarkers, drug delivery systems, and, in particular, therapeutic agents. However, there is no consensus on which is the best way to isolate these EVs. The choice of the isolation method depends on the starting material (i.e., conditioned culture media, urine, serum, etc.) and their downstream applications. Even though there are numerous methods to isolate EVs, few are compatible with clinical applications as they are not scalable. In the present work, we set up a protocol to isolate EVs from conditioned media by ion exchange chromatography, a simple, fast, and scalable method, suitable for clinical production. We performed the isolation using an anion exchange resin (Q sepharose) and eluted the EVs using 500 mM NaCl. We characterized the elution profile by measuring protein and lipid concentration, and CD63 by ELISA. Moreover, we immunophenotyped all the eluted fractions, assessed the presence of TSG101, calnexin, and cytochrome C by western blot, analyzed nanoparticle size and distribution by tRPS, and morphology by TEM. Finally, we evaluated the immunomodulatory activity in vitro. We found that most EVs are eluted and concentrated in a single peak fraction, with a mean particle size of <150nm and expression of CD9, CD63, CD81, and TSG101 markers. Moreover, sEVs in fraction 4 exerted an anti-inflammatory activity on LPS-stimulated macrophages. In summary, we set up a chromatographic, scalable, and clinically compatible method to isolate and concentrate small EVs from conditioned media, which preserves the EVs biological activity.
publishDate 2023
dc.date.none.fl_str_mv 2023-09
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/227882
Malvicini, Ricardo; Santa Cruz, Diego Mario; Tolomeo, Anna Maria; Muraca, Maurizio; Yannarelli, Gustavo Gabriel; et al.; Ion exchange chromatography as a simple and scalable method to isolate biologically active small extracellular vesicles from conditioned media; Public Library of Science; Plos One; 18; 9; 9-2023; 1-11
1932-6203
CONICET Digital
CONICET
url http://hdl.handle.net/11336/227882
identifier_str_mv Malvicini, Ricardo; Santa Cruz, Diego Mario; Tolomeo, Anna Maria; Muraca, Maurizio; Yannarelli, Gustavo Gabriel; et al.; Ion exchange chromatography as a simple and scalable method to isolate biologically active small extracellular vesicles from conditioned media; Public Library of Science; Plos One; 18; 9; 9-2023; 1-11
1932-6203
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0291589
info:eu-repo/semantics/altIdentifier/doi/10.1371/journal.pone.0291589
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Public Library of Science
publisher.none.fl_str_mv Public Library of Science
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
_version_ 1846082654293721088
score 13.216834

Publicaciones similares