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Nanocharacterisation of extracellular vesicles uncovers the correlation between isolation methods, physicochemical composition and their biological function

View ORCID ProfileThanh Huyen Phan, Shiva Kamini Divakarla, Jia Hao Yeo, Qingyu Lei, Priyanka Tharkar, Taisa Nogueira Pansani, Kathryn G. Leslie, Maggie Tong, Victoria A. Coleman, Åsa Jämting, Mar-Dean Du Plessis, Elizabeth J. New, Bill Kalionis, Philip Demokritou, Hyun-Kyung Woo, Yoon-Kyoung Cho, View ORCID ProfileWojciech Chrzanowski
doi: https://doi.org/10.1101/2021.02.09.430523
Thanh Huyen Phan
1The University of Sydney, Sydney Nano Institute, Faculty of Medicine and Health, Sydney School of Pharmacy, Pharmacy and Bank Building A15, Camperdown, NSW 2006, Australia
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  • ORCID record for Thanh Huyen Phan
Shiva Kamini Divakarla
1The University of Sydney, Sydney Nano Institute, Faculty of Medicine and Health, Sydney School of Pharmacy, Pharmacy and Bank Building A15, Camperdown, NSW 2006, Australia
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Jia Hao Yeo
3The University of Sydney, School of Chemistry, Camperdown, NSW 2006, Australia
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Qingyu Lei
1The University of Sydney, Sydney Nano Institute, Faculty of Medicine and Health, Sydney School of Pharmacy, Pharmacy and Bank Building A15, Camperdown, NSW 2006, Australia
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Priyanka Tharkar
1The University of Sydney, Sydney Nano Institute, Faculty of Medicine and Health, Sydney School of Pharmacy, Pharmacy and Bank Building A15, Camperdown, NSW 2006, Australia
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Taisa Nogueira Pansani
4UNESP – Univ. Estadual Paulista, Araraquara School of Dentistry, Department of Dental Materials and Prosthodontics, Araraquara, Centro 14801-903, Brazil
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Kathryn G. Leslie
3The University of Sydney, School of Chemistry, Camperdown, NSW 2006, Australia
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Maggie Tong
3The University of Sydney, School of Chemistry, Camperdown, NSW 2006, Australia
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Victoria A. Coleman
5National Measurement Institute Australia, Nanometrology Section, Lindfield, NSW 2070, Australia
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Åsa Jämting
5National Measurement Institute Australia, Nanometrology Section, Lindfield, NSW 2070, Australia
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Mar-Dean Du Plessis
5National Measurement Institute Australia, Nanometrology Section, Lindfield, NSW 2070, Australia
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Elizabeth J. New
2The University of Sydney, Sydney Nano Institute, Faculty of Science, School of Chemistry, Camperdown, NSW 2006, Australia
3The University of Sydney, School of Chemistry, Camperdown, NSW 2006, Australia
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Bill Kalionis
6Department of Maternal-Fetal Medicine Pregnancy Research Centre and University of Melbourne Department of Obstetrics and Gynaecology, Royal Women’s Hospital, Parkville, VIC 3052, Australia
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Philip Demokritou
7Harvard T.H Chan School of Public Health, Center for Nanotechnology and Nanotoxicology, Department of Environmental Health, MA 02115, USA
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Hyun-Kyung Woo
8Center for Soft and Living Matter, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
9Department of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea
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Yoon-Kyoung Cho
8Center for Soft and Living Matter, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
9Department of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea
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Wojciech Chrzanowski
1The University of Sydney, Sydney Nano Institute, Faculty of Medicine and Health, Sydney School of Pharmacy, Pharmacy and Bank Building A15, Camperdown, NSW 2006, Australia
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  • For correspondence: wojciech.chrzanowski@sydney.edu.au
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Abstract

Extracellular vesicles (EVs) have been lauded as next generation medicines, but very few EV-based therapeutics have progressed to clinical use. Limited clinical translation is largely due to technical barriers that hamper our ability to mass-produce EVs, i.e. to isolate, purify and characterise them effectively. Technical limitations in comprehensive characterisation of EVs leads to unpredicted biological effects of EVs. Here, using a range of optical and non-optical techniques, we showed that the differences in molecular composition of EVs isolated using two isolation methods correlated with the differences in their biological function. Our results demonstrated that the isolation method determines the composition of isolated EVs at single and sub-population levels. Besides the composition, we measured for the first time the dry mass and predicted sedimentation of EVs. These parameters were shown to correlate well with the biological and functional effects of EVs on single cell and cell cultures. We anticipate that our multiscale characterisation approach will support fundamental understanding of EVs as well as elucidate the functional effects of EVs in in vitro and in vivo studies. Our findings and methodology will be pivotal for developing optimal isolation methods and establishing EVs as mainstream therapeutics and diagnostics. This innovative approach is applicable to a wide range of sectors including biopharma and biotechnology as well as to regulatory agencies.

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Competing Interest Statement

The authors have declared no competing interest.

  • List of Abbreviations

    AFM
    Atomic Force Microscopy
    AFM-IR
    Atomic Force Microscope Infrared-Spectroscopy
    CMSC29
    chorionic mesenchymal stromal/stem cell line
    CEVs
    extracellular vesicles derived from chorionic mesenchymal stromal/stem cell
    DMSC23
    decidual mesenchymal stromal/stem cell line
    DEVs
    extracellular vesicles derived from decidual mesenchymal stromal/stem cell
    EVs
    extracellular vesicles
    HBSS(-)
    hanks’ balanced salt solution
    LPS
    lipopolysaccharide
    MSC
    mesenchymal stromal/stem cell
    PTA
    Particle Tracking Analysis
    nFCM
    Nano-flow Cytometry
    RMM
    Resonant Mass Measurement
    TRPS
    Tunable Resistive Pulse Sensing
    TFF
    tangential flow filtration
  • Copyright 
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    Nanocharacterisation of extracellular vesicles uncovers the correlation between isolation methods, physicochemical composition and their biological function
    Thanh Huyen Phan, Shiva Kamini Divakarla, Jia Hao Yeo, Qingyu Lei, Priyanka Tharkar, Taisa Nogueira Pansani, Kathryn G. Leslie, Maggie Tong, Victoria A. Coleman, Åsa Jämting, Mar-Dean Du Plessis, Elizabeth J. New, Bill Kalionis, Philip Demokritou, Hyun-Kyung Woo, Yoon-Kyoung Cho, Wojciech Chrzanowski
    bioRxiv 2021.02.09.430523; doi: https://doi.org/10.1101/2021.02.09.430523
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    Nanocharacterisation of extracellular vesicles uncovers the correlation between isolation methods, physicochemical composition and their biological function
    Thanh Huyen Phan, Shiva Kamini Divakarla, Jia Hao Yeo, Qingyu Lei, Priyanka Tharkar, Taisa Nogueira Pansani, Kathryn G. Leslie, Maggie Tong, Victoria A. Coleman, Åsa Jämting, Mar-Dean Du Plessis, Elizabeth J. New, Bill Kalionis, Philip Demokritou, Hyun-Kyung Woo, Yoon-Kyoung Cho, Wojciech Chrzanowski
    bioRxiv 2021.02.09.430523; doi: https://doi.org/10.1101/2021.02.09.430523

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