New Multiscale Characterization Methodology for Effective Determination of Isolation-Structure-Function Relationship of Extracellular Vesicles

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

doi:10.3389/fbioe.2021.669537

New Multiscale Characterization Methodology for Effective Determination of Isolation-Structure-Function Relationship of Extracellular Vesicles

Front Bioeng Biotechnol. 2021 Jun 7:9:669537. doi: 10.3389/fbioe.2021.669537. eCollection 2021.

Authors

Thanh Huyen Phan¹, Shiva Kamini Divakarla¹, Jia Hao Yeo², Qingyu Lei¹, Priyanka Tharkar¹, Taisa Nogueira Pansani³, Kathryn G Leslie², Maggie Tong², Victoria A Coleman⁴, Åsa Jämting⁴, Mar-Dean Du Plessis⁴, Elizabeth J New^{2

5}, Bill Kalionis⁶, Philip Demokritou⁷, Hyun-Kyung Woo^{8

9}, Yoon-Kyoung Cho^{8

9}, Wojciech Chrzanowski¹

Affiliations

¹ Sydney School of Pharmacy, Faculty of Medicine and Health, Sydney Nano Institute, The University of Sydney, Camperdown, NSW, Australia.
² School of Chemistry, The University of Sydney, Camperdown, NSW, Australia.
³ Department of Dental Materials and Prosthodontics, Araraquara School of Dentistry, UNESP-Universidade Estadual Paulista, Araraquara, Brazil.
⁴ Nanometrology Section, National Measurement Institute Australia, Lindfield, NSW, Australia.
⁵ School of Chemistry, Faculty of Science, Sydney Nano Institute, The University of Sydney, Camperdown, NSW, Australia.
⁶ Maternal-Fetal Medicine Pregnancy Research Centre, The Royal Women's Hospital, and Department of Obstetrics and Gynaecology, The University of Melbourne, Parkville, VIC, Australia.
⁷ Department of Environmental Health, Center for Nanotechnology and Nanotoxicology, Harvard T.H. Chan School of Public Health, Boston, MA, United States.
⁸ Center for Soft and Living Matter, Institute for Basic Science (IBS), Ulsan, South Korea.
⁹ Department of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, South Korea.

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 characterize them effectively. Technical limitations in comprehensive characterization of EVs lead 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 likely to contribute to the biological and functional effects of EVs on single cell and cell cultures. We anticipate that our new multiscale characterization approach, which goes beyond traditional experimental methodology, 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.

Keywords: exosomes; extracellular vesicles; isolation methods; mesenchymal stromal/stem cell; nanodosimetry; single vesicle analysis; tangential flow filtration; ultracentrifugation.