Hyaluronic acid: An overlooked extracellular vesicle contaminant

Abstract

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.

Inorganic Phosphate-Induced Extracellular Vesicles from Vascular Smooth Muscle Cells Contain Elevated Levels of Hyaluronic Acid, Which Enhance Their Interaction with Very Small Superparamagnetic Iron Oxide Particles

“…A previous study reported possible contaminations of EV fractions with a high-molecular-weight HA fragment of 289 kDa, which could influence our measurements in this case [ 43 ]. However, the targeted up- and downregulation of HA in cells and EV by iloprost and siRNAs, respectively, and the according changes in the interaction of cells and EV with VSOP underline the validity of our conclusions.…”

Extracellular Vesicle and Lipoprotein Interactions

“…Results from EV studies are highly impacted by the EV isolation technique used, which should also be considered in the study of the lipoprotein–EV interface. For example, it is known that the levels of co-isolated contaminants vary substantially based on the EV isolation method. , It is also critical to note that EV isolation methods may induce non-physiologically relevant interactions between EVs and lipoproteins. Ultracentrifugation, a common EV isolation technique that employs high centrifugal forces, can induce EV aggregation and alter EV morphology. , Therefore, ultracentrifugation may also cause complexation between EVs and lipoproteins.…”