Importance of isolation method on characteristics and bioactivity of extracellular vesicles from tomatoes

Ziyu Huang; Søren Drud Heydary Nielsen; Bradley Whitehead; Peter Nejsum; Milena Corredig; Martin Krøyer Rasmussen

doi:10.1016/j.jfca.2024.106064

Importance of isolation method on characteristics and bioactivity of extracellular vesicles from tomatoes

Ziyu Huang, Søren Drud Heydary Nielsen, Bradley Whitehead, Peter Nejsum, Milena Corredig, Martin Krøyer Rasmussen^*

^*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

6 Citations (Scopus)

5 Downloads (Pure)

Abstract

Plant-derived extracellular vesicles (EVs), including exosomes, are usually isolated by ultracentrifugation, followed by density gradient purification, without critically assessing the obtained material. The lack of characterization makes it difficult to compare findings between studies or ascribe effects to specific compounds. Thus, the study aimed to make a comprehensive characterization of the different subpopulations of EVs isolated using a range of ultracentrifugation forces and subsequent glucose gradient purification. Results showed that EVs obtained using 100,000 × g had a bilayer membrane structure, the highest abundance of proteins and microRNAs, and characteristics resembling exosomes. Additional gradient purification resulted in three distinct bands of EVs. EVs from the middle band had most characteristics resembling exosomes, such as the bilayer structure and high abundance of proteins and microRNAs. Furthermore, proteomics identified most proteins to be common between the different fractions, while band-specific proteins were also observed. In addition, the bioactive properties of the EVs also differed between bands, as assessed by evaluating the viability and proliferation of intestinal cells (Caco-2). In conclusion, the present findings clearly demonstrate that the characteristics and bioactive properties of plant EVs are highly dependable on isolation methods, which advocate for the development of standardized methods.

Original language	English
Article number	106064
Journal	Journal of Food Composition and Analysis
Volume	129
ISSN	0889-1575
DOIs	https://doi.org/10.1016/j.jfca.2024.106064
Publication status	Published - May 2024

Keywords

Exosome-like nanovesicle
Non-coding RNA
Proliferation
Sucrose gradient
Tomato fruits
Ultra-centrifugal force
Viability

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title = "Importance of isolation method on characteristics and bioactivity of extracellular vesicles from tomatoes",

abstract = "Plant-derived extracellular vesicles (EVs), including exosomes, are usually isolated by ultracentrifugation, followed by density gradient purification, without critically assessing the obtained material. The lack of characterization makes it difficult to compare findings between studies or ascribe effects to specific compounds. Thus, the study aimed to make a comprehensive characterization of the different subpopulations of EVs isolated using a range of ultracentrifugation forces and subsequent glucose gradient purification. Results showed that EVs obtained using 100,000 × g had a bilayer membrane structure, the highest abundance of proteins and microRNAs, and characteristics resembling exosomes. Additional gradient purification resulted in three distinct bands of EVs. EVs from the middle band had most characteristics resembling exosomes, such as the bilayer structure and high abundance of proteins and microRNAs. Furthermore, proteomics identified most proteins to be common between the different fractions, while band-specific proteins were also observed. In addition, the bioactive properties of the EVs also differed between bands, as assessed by evaluating the viability and proliferation of intestinal cells (Caco-2). In conclusion, the present findings clearly demonstrate that the characteristics and bioactive properties of plant EVs are highly dependable on isolation methods, which advocate for the development of standardized methods.",

keywords = "Exosome-like nanovesicle, Non-coding RNA, Proliferation, Sucrose gradient, Tomato fruits, Ultra-centrifugal force, Viability",

author = "Ziyu Huang and Nielsen, {S{\o}ren Drud Heydary} and Bradley Whitehead and Peter Nejsum and Milena Corredig and Rasmussen, {Martin Kr{\o}yer}",

note = "Publisher Copyright: {\textcopyright} 2024 The Authors",

year = "2024",

month = may,

doi = "10.1016/j.jfca.2024.106064",

language = "English",

volume = "129",

journal = "Journal of Food Composition and Analysis",

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Importance of isolation method on characteristics and bioactivity of extracellular vesicles from tomatoes. / Huang, Ziyu; Nielsen, Søren Drud Heydary; Whitehead, Bradley et al.
In: Journal of Food Composition and Analysis, Vol. 129, 106064, 05.2024.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

TY - JOUR

T1 - Importance of isolation method on characteristics and bioactivity of extracellular vesicles from tomatoes

AU - Huang, Ziyu

AU - Nielsen, Søren Drud Heydary

AU - Whitehead, Bradley

AU - Nejsum, Peter

AU - Corredig, Milena

AU - Rasmussen, Martin Krøyer

PY - 2024/5

Y1 - 2024/5

N2 - Plant-derived extracellular vesicles (EVs), including exosomes, are usually isolated by ultracentrifugation, followed by density gradient purification, without critically assessing the obtained material. The lack of characterization makes it difficult to compare findings between studies or ascribe effects to specific compounds. Thus, the study aimed to make a comprehensive characterization of the different subpopulations of EVs isolated using a range of ultracentrifugation forces and subsequent glucose gradient purification. Results showed that EVs obtained using 100,000 × g had a bilayer membrane structure, the highest abundance of proteins and microRNAs, and characteristics resembling exosomes. Additional gradient purification resulted in three distinct bands of EVs. EVs from the middle band had most characteristics resembling exosomes, such as the bilayer structure and high abundance of proteins and microRNAs. Furthermore, proteomics identified most proteins to be common between the different fractions, while band-specific proteins were also observed. In addition, the bioactive properties of the EVs also differed between bands, as assessed by evaluating the viability and proliferation of intestinal cells (Caco-2). In conclusion, the present findings clearly demonstrate that the characteristics and bioactive properties of plant EVs are highly dependable on isolation methods, which advocate for the development of standardized methods.

AB - Plant-derived extracellular vesicles (EVs), including exosomes, are usually isolated by ultracentrifugation, followed by density gradient purification, without critically assessing the obtained material. The lack of characterization makes it difficult to compare findings between studies or ascribe effects to specific compounds. Thus, the study aimed to make a comprehensive characterization of the different subpopulations of EVs isolated using a range of ultracentrifugation forces and subsequent glucose gradient purification. Results showed that EVs obtained using 100,000 × g had a bilayer membrane structure, the highest abundance of proteins and microRNAs, and characteristics resembling exosomes. Additional gradient purification resulted in three distinct bands of EVs. EVs from the middle band had most characteristics resembling exosomes, such as the bilayer structure and high abundance of proteins and microRNAs. Furthermore, proteomics identified most proteins to be common between the different fractions, while band-specific proteins were also observed. In addition, the bioactive properties of the EVs also differed between bands, as assessed by evaluating the viability and proliferation of intestinal cells (Caco-2). In conclusion, the present findings clearly demonstrate that the characteristics and bioactive properties of plant EVs are highly dependable on isolation methods, which advocate for the development of standardized methods.

KW - Exosome-like nanovesicle

KW - Non-coding RNA

KW - Proliferation

KW - Sucrose gradient

KW - Tomato fruits

KW - Ultra-centrifugal force

KW - Viability

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JO - Journal of Food Composition and Analysis

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Importance of isolation method on characteristics and bioactivity of extracellular vesicles from tomatoes

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