Isolation and characterization of extracellular vesicles from human milk for potential use as a dietary supplement in clinical research with preterm infants

Background/objectives: Human milk (HM) is the gold standard for neonatal nutrition, providing essential macronutrients and bioactive compounds that promote immune and gastrointestinal development. Among these components, HM-derived extracellular vesicles (HMEVs) are emerging as key mediators of inte...

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Autores: Moreno-Casillas, JL, Ripoll-Seguer, L, Rumba-Matano, E, Parra-Llorca, A, Cernada, M, Vaya, MJ, Briz, EL, Padilla-López, AM, Brusola, AG, Lendl, B, Quintás, G, Gormaz, M, Kuligowski, J
Formato: artículo
Estado:Versión publicada
Fecha de publicación:2026
País:España
Recursos:Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunitat Valenciana (FISABIO)
Repositorio:r-FISABIO. Repositorio Institucional de Producción Científica
OAI Identifier:oai:dnet:r-fisabio___::afcd99d6090c50c43e2ac3324138d65c
Acesso em linha:https://fisabio.portalinvestigacion.com/publicaciones/20822
Access Level:acceso abierto
Palavra-chave:donor human milk (DHM) supplementation
human milk-derived extracellular vesicles (HMEVs)
necrotizing enterocolitis (NEC)
neonatal intestinal protection
preterm infant nutrition
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spelling Isolation and characterization of extracellular vesicles from human milk for potential use as a dietary supplement in clinical research with preterm infantsMoreno-Casillas, JLRipoll-Seguer, LRumba-Matano, EParra-Llorca, ACernada, MVaya, MJBriz, ELPadilla-López, AMBrusola, AGLendl, BQuintás, GGormaz, MKuligowski, Jdonor human milk (DHM) supplementationhuman milk-derived extracellular vesicles (HMEVs)necrotizing enterocolitis (NEC)neonatal intestinal protectionpreterm infant nutritionBackground/objectives: Human milk (HM) is the gold standard for neonatal nutrition, providing essential macronutrients and bioactive compounds that promote immune and gastrointestinal development. Among these components, HM-derived extracellular vesicles (HMEVs) are emerging as key mediators of intestinal maturation and protection against necrotizing enterocolitis (NEC). HMEVs carry miRNAs, proteins, and bioactive lipids that resist digestion and modulate critical signaling pathways in the immature gut, making them particularly relevant for preterm infants. Methods: This study describes the development and adaptation of a robust, scalable workflow for isolating HMEVs from donor HM for potential use as a nutritional supplement. An initial laboratory-scale isolation protocol was successfully scaled up to a sterile, clinically compatible process. Key modifications included increasing ultracentrifugation speed, eliminating filtration, and replacing phosphate-buffered saline (PBS) with a resuspension medium suitable for nutritional applications. Results: The scaled procedure increased the processed milk volume from 25 mL to 63 mL while reducing the HMEVs isolation time from 14 hs to 7.7 h. Tunable Resistive Pulse Sensing (TRPS) and ExoView((R)) analysis showed that these modifications led to an approximate 2-fold increase in EV recovery following centrifugation speed optimization. In addition, omission of filtration yielded a 4.3-fold increase in HMEV recovery. The isolated HMEVs exhibited high purity, reaching up to 3.98 & times; 10(13) particles/g protein. The final product was resuspended in 5% glucose solution, chosen for its physiological compatibility, favorable osmolarity (< 380 mOsmol/kg), and ability to preserve EV stability for up to 30 days at -80 degrees C. The HMEV preparation was produced under sterile conditions, and endotoxin testing returned negative results. Conclusions: This optimized and scalable method enables the safe and efficient isolation of HMEVs for use in neonatal nutritional supplements. These findings establish methodological groundwork for future translational studies of milk-derived EVs aimed at supporting intestinal development and immune protection, and potentially reducing the risk of NEC in preterm infants.FRONTIERS MEDIA SA2026info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttps://fisabio.portalinvestigacion.com/publicaciones/20822Frontiers in NutritionISSN: 2296861Xreponame:r-FISABIO. Repositorio Institucional de Producción Científicainstname:Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunitat Valenciana (FISABIO)Inglésinfo:eu-repo/semantics/openAccessoai:dnet:r-fisabio___::afcd99d6090c50c43e2ac3324138d65c2026-06-11T12:45:17Z
dc.title.none.fl_str_mv Isolation and characterization of extracellular vesicles from human milk for potential use as a dietary supplement in clinical research with preterm infants
title Isolation and characterization of extracellular vesicles from human milk for potential use as a dietary supplement in clinical research with preterm infants
spellingShingle Isolation and characterization of extracellular vesicles from human milk for potential use as a dietary supplement in clinical research with preterm infants
Moreno-Casillas, JL
donor human milk (DHM) supplementation
human milk-derived extracellular vesicles (HMEVs)
necrotizing enterocolitis (NEC)
neonatal intestinal protection
preterm infant nutrition
title_short Isolation and characterization of extracellular vesicles from human milk for potential use as a dietary supplement in clinical research with preterm infants
title_full Isolation and characterization of extracellular vesicles from human milk for potential use as a dietary supplement in clinical research with preterm infants
title_fullStr Isolation and characterization of extracellular vesicles from human milk for potential use as a dietary supplement in clinical research with preterm infants
title_full_unstemmed Isolation and characterization of extracellular vesicles from human milk for potential use as a dietary supplement in clinical research with preterm infants
title_sort Isolation and characterization of extracellular vesicles from human milk for potential use as a dietary supplement in clinical research with preterm infants
dc.creator.none.fl_str_mv Moreno-Casillas, JL
Ripoll-Seguer, L
Rumba-Matano, E
Parra-Llorca, A
Cernada, M
Vaya, MJ
Briz, EL
Padilla-López, AM
Brusola, AG
Lendl, B
Quintás, G
Gormaz, M
Kuligowski, J
author Moreno-Casillas, JL
author_facet Moreno-Casillas, JL
Ripoll-Seguer, L
Rumba-Matano, E
Parra-Llorca, A
Cernada, M
Vaya, MJ
Briz, EL
Padilla-López, AM
Brusola, AG
Lendl, B
Quintás, G
Gormaz, M
Kuligowski, J
author_role author
author2 Ripoll-Seguer, L
Rumba-Matano, E
Parra-Llorca, A
Cernada, M
Vaya, MJ
Briz, EL
Padilla-López, AM
Brusola, AG
Lendl, B
Quintás, G
Gormaz, M
Kuligowski, J
author2_role author
author
author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv donor human milk (DHM) supplementation
human milk-derived extracellular vesicles (HMEVs)
necrotizing enterocolitis (NEC)
neonatal intestinal protection
preterm infant nutrition
topic donor human milk (DHM) supplementation
human milk-derived extracellular vesicles (HMEVs)
necrotizing enterocolitis (NEC)
neonatal intestinal protection
preterm infant nutrition
description Background/objectives: Human milk (HM) is the gold standard for neonatal nutrition, providing essential macronutrients and bioactive compounds that promote immune and gastrointestinal development. Among these components, HM-derived extracellular vesicles (HMEVs) are emerging as key mediators of intestinal maturation and protection against necrotizing enterocolitis (NEC). HMEVs carry miRNAs, proteins, and bioactive lipids that resist digestion and modulate critical signaling pathways in the immature gut, making them particularly relevant for preterm infants. Methods: This study describes the development and adaptation of a robust, scalable workflow for isolating HMEVs from donor HM for potential use as a nutritional supplement. An initial laboratory-scale isolation protocol was successfully scaled up to a sterile, clinically compatible process. Key modifications included increasing ultracentrifugation speed, eliminating filtration, and replacing phosphate-buffered saline (PBS) with a resuspension medium suitable for nutritional applications. Results: The scaled procedure increased the processed milk volume from 25 mL to 63 mL while reducing the HMEVs isolation time from 14 hs to 7.7 h. Tunable Resistive Pulse Sensing (TRPS) and ExoView((R)) analysis showed that these modifications led to an approximate 2-fold increase in EV recovery following centrifugation speed optimization. In addition, omission of filtration yielded a 4.3-fold increase in HMEV recovery. The isolated HMEVs exhibited high purity, reaching up to 3.98 & times; 10(13) particles/g protein. The final product was resuspended in 5% glucose solution, chosen for its physiological compatibility, favorable osmolarity (< 380 mOsmol/kg), and ability to preserve EV stability for up to 30 days at -80 degrees C. The HMEV preparation was produced under sterile conditions, and endotoxin testing returned negative results. Conclusions: This optimized and scalable method enables the safe and efficient isolation of HMEVs for use in neonatal nutritional supplements. These findings establish methodological groundwork for future translational studies of milk-derived EVs aimed at supporting intestinal development and immune protection, and potentially reducing the risk of NEC in preterm infants.
publishDate 2026
dc.date.none.fl_str_mv 2026
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv https://fisabio.portalinvestigacion.com/publicaciones/20822
url https://fisabio.portalinvestigacion.com/publicaciones/20822
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv FRONTIERS MEDIA SA
publisher.none.fl_str_mv FRONTIERS MEDIA SA
dc.source.none.fl_str_mv Frontiers in Nutrition
ISSN: 2296861X
reponame:r-FISABIO. Repositorio Institucional de Producción Científica
instname:Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunitat Valenciana (FISABIO)
instname_str Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunitat Valenciana (FISABIO)
reponame_str r-FISABIO. Repositorio Institucional de Producción Científica
collection r-FISABIO. Repositorio Institucional de Producción Científica
repository.name.fl_str_mv
repository.mail.fl_str_mv
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