First in vivo detection and characterization of hyaluronan‐coated extracellular vesicles in human synovial fluid

Mustonen, Anne‐Mari; Nieminen, Petteri; Joukainen, Antti; Jaroma, Antti; Kääriäinen, Tommi; Kröger, Heikki; Lázaro-Ibáñez, Elisa; Siljander, Pia; Kärjä, Vesa; Härkönen, Kai; Koistinen, Arto; Rilla, Kirsi

doi:10.1002/jor.23212

Cited by 30 publications

(26 citation statements)

References 39 publications

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“…For SF, differential centrifugation steps did not separate EVs based on morphology, nor on size, although size distribution of spherical EVs did show a tendency for EVs >100 nm to be recovered at 10,000 g (compared to 100/200,000 g ). The finding that 92–98% of EVs in our study had diameters between 20 and 200 nm is considerably different from the larger particles (up to 400 nm) detected in SF from patients with inflammatory joint disease (6, 9) (10, 19). These differences could be caused by differences in the methodology used to isolate EVs, for example, centrifugation protocols and density gradients.…”

Section: Discussioncontrasting

confidence: 99%

“…For developing a tailor‐made protocol for EV isolation from SF, previously published reports were first evaluated to determine which protocols to date have been used for EV isolation from SF and to survey which important technical steps should be considered for a standardized protocol. Of 18 articles published between 1995 and 2016 reporting EV isolation from SF (Table I) (2–19), 17 involved samples from human patients with joint disease. Only one study investigated healthy SF (of rat, canine, and equine origin) (14).…”

Section: Discussionmentioning

confidence: 99%

“…Although the recovery of total EVs (PKH67+) was not significantly affected when omitting HYase treatment, significantly less CD44+ EVs were recovered. Very recently the presence of HA‐coated EVs was detected in SF (19). One possible explanation for the lower detection of CD44+ EVs in the absence of HYase treatment in our study could be masking of antibody detection due to HA binding.…”

Section: Discussionmentioning

confidence: 99%

“…1. The protocol was based on earlier protocols used for EV isolation from SF as summarized in Table I, (2–19) but was more comprehensive than most of these, as it was made to meet the recently defined minimal requirements for functional EV studies (31). The technical specifications of ultracentrifugation steps (centrifugation speeds, rotor types, etc.)…”

Section: Methodsmentioning

confidence: 99%

“…In this context, EVs derived from synovial fluid (SF) are currently being investigated to unravel their role in these processes and their biomarker potential for joint disease. Several studies have examined EVs isolated from SF (2–19). In most of these studies EVs have only been isolated from human SF obtained from inflamed joints.…”

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confidence: 99%

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Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles

Boere

Lest

Libregts

et al. 2016

J of Extracellular Vesicle

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Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) – a prominent extracellular matrix component – it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20–200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery.

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Section: Discussioncontrasting

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles

Boere

Lest

Libregts

et al. 2016

J of Extracellular Vesicle

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Adequate enrichment of extracellular vesicles in laboratory medicine

Zhang

Gan

et al. 2023

Interdisciplinary Medicine

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Extracellular vesicles (EVs) and their clinical applications have been recognized as one of the most fast‐growing and promising fields. Numerous groups including us have expressed their concerns on the idea of isolating “pure” EVs for different purposes, as there are no specific markers to date that can examine the exact EV purity. Typically, major instrument need and EV yielding loss are primary compensations of getting EVs with higher purity. As such, especially in biomarker‐based clinical tests, ceaseless efforts for obtaining more pure EV are worth of discussion. We here propose alternative philosophy to tone down the importance of purity, but to emphasize the significance in clinical laboratory medicine to measure EVs obtained by well‐controlled isolation methods. As a result, EVs are enriched to the adequate degree to meet certain clinical applications such as disease diagnosis and/or prognosis. In this review, we will justify the adequate enrichment rationale with advances of EV definitions, markers and contaminants, EV isolation methods, as well as the conclusions acquired from clinical research using less pure but ease‐to‐operate EV isolation methods.

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Hyaluronic acid: An overlooked extracellular vesicle contaminant

Goncalves,

Ghebosu,

Tan

et al. 2023

J of Extracellular Vesicle

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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.

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First in vivo detection and characterization of hyaluronan‐coated extracellular vesicles in human synovial fluid

Cited by 30 publications

References 39 publications

Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles

Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles

Adequate enrichment of extracellular vesicles in laboratory medicine

Hyaluronic acid: An overlooked extracellular vesicle contaminant

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