TY - JOUR
T1 - Hyaluronic acid
T2 - An overlooked extracellular vesicle contaminant
AU - Goncalves, Jenifer P.
AU - Ghebosu, Raluca E.
AU - Tan, Xuan Ning Sharon
AU - Iannotta, Dalila
AU - Koifman, Na'ama
AU - Wolfram, Joy
N1 - Funding Information:
The cryogenic transmission electron microscopy work was performed at The University of Queensland Center of Microscopy and Microanalysis. This work used the Queensland node of the National Collaborative Research Infrastructure Strategy (NCRIS)‐enabled Australian National Fabrication Facility (ANFF). This work was partially funded by The University of Queensland, Australia (J.W.) and the Medical Research Future Fund, Australia under award number MRF2019485 (J.W.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the organizations and funding agencies. Figures 1 and 2 were partially made in ©BioRender—biorender.com.
Publisher Copyright:
© 2023 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.
PY - 2023/9
Y1 - 2023/9
N2 - The variable presence of contaminants in extracellular vesicle (EV) samples is one of the major contributors to a lack of inter-study reproducibility in the field. Well-known contaminants include protein aggregates, RNA-protein complexes and lipoproteins, which resemble EVs in shape, size and/or density. On the contrary, polysaccharides, such as hyaluronic acid (HA), have been overlooked as EV contaminants. Here, it is shown that low and medium molecular weight HA polymers are unexpectedly retained to some extent in EV fractions using two common isolation methods known for high purity: size-exclusion chromatography and tangential flow filtration. Although these isolation techniques are capable of efficient removal of non-EV-associated proteins, this is not the case for HA polymers, which are partially retained in a molecular weight-dependent manner, especially with size-exclusion chromatography. The supramolecular structure and hydrodynamic size of HA are likely to contribute to isolation in EV fractions of filtration-based approaches. Conversely, HA polymers were not retained with ultracentrifugation and polymer-based precipitation methods, which are known for co-isolating other types of contaminants. HA has a broad range of immunomodulatory effects, similar to those ascribed to various sources of EVs. Therefore, HA contaminants should be considered in future studies to avoid potential inaccurate attributions of functional effects to EVs.
AB - The variable presence of contaminants in extracellular vesicle (EV) samples is one of the major contributors to a lack of inter-study reproducibility in the field. Well-known contaminants include protein aggregates, RNA-protein complexes and lipoproteins, which resemble EVs in shape, size and/or density. On the contrary, polysaccharides, such as hyaluronic acid (HA), have been overlooked as EV contaminants. Here, it is shown that low and medium molecular weight HA polymers are unexpectedly retained to some extent in EV fractions using two common isolation methods known for high purity: size-exclusion chromatography and tangential flow filtration. Although these isolation techniques are capable of efficient removal of non-EV-associated proteins, this is not the case for HA polymers, which are partially retained in a molecular weight-dependent manner, especially with size-exclusion chromatography. The supramolecular structure and hydrodynamic size of HA are likely to contribute to isolation in EV fractions of filtration-based approaches. Conversely, HA polymers were not retained with ultracentrifugation and polymer-based precipitation methods, which are known for co-isolating other types of contaminants. HA has a broad range of immunomodulatory effects, similar to those ascribed to various sources of EVs. Therefore, HA contaminants should be considered in future studies to avoid potential inaccurate attributions of functional effects to EVs.
KW - extracellular vesicle contaminant
KW - hyaluronan
KW - size-exclusion chromatography
KW - tangential flow filtration
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U2 - 10.1002/jev2.12362
DO - 10.1002/jev2.12362
M3 - Letter
C2 - 37712345
AN - SCOPUS:85171358934
SN - 2001-3078
VL - 12
JO - Journal of Extracellular Vesicles
JF - Journal of Extracellular Vesicles
IS - 9
M1 - 12362
ER -