PT  - JOURNAL ARTICLE
AU  - Thanh Huyen Phan
AU  - Shiva Kamini Divakarla
AU  - Jia Hao Yeo
AU  - Qingyu Lei
AU  - Priyanka Tharkar
AU  - Taisa Nogueira Pansani
AU  - Kathryn G. Leslie
AU  - Maggie Tong
AU  - Victoria A. Coleman
AU  - Åsa Jämting
AU  - Mar-Dean Du Plessis
AU  - Elizabeth J. New
AU  - Bill Kalionis
AU  - Philip Demokritou
AU  - Hyun-Kyung Woo
AU  - Yoon-Kyoung Cho
AU  - Wojciech Chrzanowski
TI  - New multiscale characterisation methodology for effective determination of isolation-structure-function relationship of extracellular vesicles
AID  - 10.1101/2021.02.09.430523
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.02.09.430523
4099  - http://biorxiv.org/content/early/2021/02/18/2021.02.09.430523.short
4100  - http://biorxiv.org/content/early/2021/02/18/2021.02.09.430523.full
AB  - 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 new multiscale characterisation 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.Competing Interest StatementThe authors have declared no competing interest.AFMAtomic Force Microscopy;AFM-IRAtomic Force Microscope InfraredSpectroscopy;CMSC29chorionic mesenchymal stromal/stem cell line;CEVsextracellular vesicles derived from chorionic mesenchymal stromal/stem cell;DMSC23decidual mesenchymal stromal/stem cell line;DEVsextracellular vesicles derived from decidual mesenchymal stromal/stem cell;EVsextracellular vesicles;HBSS(-)hanks’ balanced salt solution;LPSlipopolysaccharide;MSCmesenchymal stromal/stem cell;PTAParticle Tracking Analysis;nFCMNano-flow Cytometry;RMMResonant Mass Measurement;TRPSTunable Resistive Pulse Sensing;TFFtangential flow filtration.