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. 2023 Sep 15;12(9):12362. doi: 10.1002/jev2.12362

Hyaluronic acid: An overlooked extracellular vesicle contaminant

Jenifer P Goncalves 1, Raluca E Ghebosu 1, Xuan Ning Sharon Tan 1, Dalila Iannotta 2, Na'ama Koifman 3, Joy Wolfram 1,2,✉
PMCID: PMC10502654  PMID: 37712345

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.

Keywords: extracellular vesicle contaminant, hyaluronan, size‐exclusion chromatography, tangential flow filtration

1. INTRODUCTION

There has been an exponential increase in extracellular vesicle (EV) studies due to the critical role that these biological nanoparticles play in intercellular communication, paving the way for new diagnostic/therapeutic approaches (Beetler et al., 2022; Ghodasara et al., 2023) and an improved understanding of (patho)physiological mechanisms (van Niel et al., 2018). However, the field is plagued by a lack of standardization of procedures, ranging from the processing of source materials, EV isolation, sample characterization, tracking studies and functional analysis (Théry et al., 2018). One of the major contributors to a lack of inter‐study reproducibility is varying levels of contaminants in EV samples. Known EV contaminants, primarily protein aggregates and lipoproteins, have similar shapes and sizes as EVs and can be detected by commonly used techniques, such as immunoblotting of proteins and cryogenic transmission electron microscopy (Busatto et al., 2022; Théry et al., 2018; Tian et al., 2020). A common method of assessing the purity of EV samples is based on the protein‐to‐particle ratio (Théry et al., 2018). In addition to protein‐based contaminants, non‐EV‐associated RNA bound to larger structures, such as protein‐RNA complexes, RNA aggregates, lipid‐RNA complexes, is another important contaminant to consider (Auber et al., 2019; Lehrich et al., 2021; Urzi et al., 2022).

In terms of contaminants, as well as EV cargo, the major focus has been on RNA and proteins. It is only recently that carbohydrates have been given consideration as EV‐associated biomolecules (Goncalves et al., 2022; Yang et al., 2022). Nevertheless, polysaccharides have previously been overlooked as EV contaminants. For example, hyaluronic acid (HA) is released extracellularly by many types of vertebrate cells and can be present in a variety of size ranges (de Oliveira et al., 2016). HA, also known as hyaluronan and hyaluronate, is a glycosaminoglycan composed of repeating units of D‐glucuronic acid and N‐acetyl‐D‐glucosamine. HA is synthesized by membrane‐associated HA synthases in molecular weights ranging from 100 to 4000 kDa, and can be enzymatically broken down to even smaller polymers in the extracellular environment (Itano et al., 1999; Snetkov et al., 2020). Under physiological conditions, HA is usually present in high molecular weight forms, and lower molecular weight polymers have been associated with tissue damage and inflammation (Cowman et al., 2015; Marinho et al., 2021).

2. RESULTS

Although HA has been overlooked as a contaminant in EV samples, it is conceivable that high molecular weight polymers (e.g., 1000 kDa) would co‐isolate with EV samples. However, here it is shown that HA polymers that would be expected to be removed during EV isolation due to their low or medium molecular weights are unexpectedly retained using two common isolation methods: tangential flow filtration (Busatto et al., 2018) and commercial size‐exclusion chromatography columns (Walker et al., 2020) (Figure 1a,b). HA of medium molecular weight (289 kDa) was assessed, as it is less than half of the lower cut‐off size for tangential flow filtration (750 kDa used in this study), and would be expected to be removed. However, when a solution of commercial 289 kDa HA (concentration‐matched to that of conditioned medium from MDA‐MB‐231 human breast cancer cells) was processed through a tangential flow filtration‐based EV isolation protocol, the polymer was only partially eliminated through sequential filtration steps. Specifically, 65.5% of the initial amount of HA remained in the sample (permeate) after the first filtration step through a filter with a pore size of 650 nm (Figure 1a). In the second step of the tangential flow filtration protocol, separation and diafiltration (purification) through a filter with a pore size of 750 kDa are usually performed to collect the retentate (EV sample) and remove smaller contaminants (permeate), such as those in the 289 kDA range. However, this step could only remove a further 55.7% of the initial 289 kDa HA amount, resulting in 9.8% of the initial polymer remaining in the final purified sample (Figure 1a). The retained amount of HA is higher than what has been reported for protein contaminants using the tangential flow filtration protocol, which is capable of near complete removal of non‐EV‐associated proteins (Busatto et al., 2018). In addition, the initial HA concentration can be enriched after tangential flow filtration due to the concentration step.

FIGURE 1.

FIGURE 1

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Assessment of hyaluronic acid (HA) as a contaminant in extracellular vesicle (EV) isolation. (a‐d) Samples were processed by tangential flow filtration (a), size‐exclusion chromatography (b), ultracentrifugation (c) and a precipitation kit (d). Aliquots were collected from each step, and HA was quantified by an enzyme‐linked immunosorbent assay (ELISA). Graphs show the percentage of retained HA in various samples (300 ng/mL of 289 kDa or 37 kDa HA, human plasma and conditioned medium of MDA‐MB‐231 human breast cancer cells) after EV isolation. Data represent mean ± standard deviation (SD) of at least three replicates. Tangential flow filtration data of free HA solutions were obtained using at least three independent filters, while size‐exclusion chromatography data were obtained using at least two independent columns. ANOVA with Tukey's multiple comparisons test. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Size‐exclusion chromatography is a gel filtration‐based method that is known for high EV purity compared to other common isolation methods (Monguio‐Tortajada et al., 2019), and studies have shown that large proteins can be removed from EV samples using commercial size‐exclusion chromatography columns (Busatto et al., 2021). Therefore, this method was used in addition to tangential flow filtration to assess the removal of 289 kDa HA. Unexpectedly, 23.3% of HA was present in fractions that are usually used for EV collection (Figure 1b). On the contrary, there was minimal retention of the 289 kDa HA in EV fractions using non‐filtration‐based methods, namely ultracentrifugation and a precipitation kit (Figure 1c,d).

To assess whether the isolation of HA in EV fractions was dependent on polymer size, a low molecular weight HA (37 kDa) was also processed using the four previously mentioned isolation methods. Results showed that more HA (95.7%) is eliminated from EV fractions by tangential flow filtration (Figure 1a) and size‐exclusion chromatography (92%; Figure 1b) with the low molecular weight polymer compared to the medium molecular weight one. It is worth noting that the displayed results reflect the variability observed when repeating the isolation step several times using different lots of tangential flow filtration filters and size‐exclusion chromatography columns. Less variability in HA removal was observed when repeating the isolation steps several times using the same filter or column (data not shown), highlighting that filters and columns that pass quality control may still display slight variations in performance. Similar to the results obtained with 289 kDa HA, 37 kDa HA was eliminated with EV isolation protocols based on ultracentrifugation or a precipitation kit (Figure 1c,d). Taken together, the abundance of HA contaminants varies depending on the molecular weight of HA and the EV isolation method, with size‐exclusion chromatography resulting in the highest levels of HA retention in EV fractions.

The efficient removal of contaminants through filtration is affected by several different factors, including shape and hydration layer. While a globular shape (proteins) is likely to enable efficient filtration, HA can display a non‐spherical supramolecular structure and high hygroscopicity, causing a substantial hydration layer to form around the polymer (Cowman et al., 2015; de Oliveira et al., 2016). Notably, these characteristics of its structure could impact the filtration of HA. Nanoparticle tracking analysis revealed that 300 ng/mL and 2 μg/mL solutions of 289 kDa HA (corresponding to common concentrations of HA in MDA‐MB‐231 conditioned cell culture medium pre‐ and post‐tangential flow filtration, respectively) had particle levels similar to the control buffer (106/mL; sucrose buffer; data not shown). Additionally, the HA polymers could not be visualized by cryogenic transmission electron microscopy even at high concentrations (2000 μg/mL; Figure 2a). Although HA was undetectable by nanoparticle tracking analysis and cryogenic transmission electron microscopy, the supramolecular structure and hydrodynamic size of HA could be responsible for the co‐isolation of the HA polymers in EV fractions.

FIGURE 2.

FIGURE 2

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EV characterization and illustration of size‐dependent co‐isolation of HA with various isolation methods. (a) Cryogenic transmission electron microscopy images of water, a 2 mg/mL 289 kDa HA solution and MDA‐MB‐231 human breast cancer cell‐derived EVs isolated using four EV isolation methods. Scale bars = 50 nm. (b) HA amount (ELISA) per particle (nanoparticle tracking analysis) when subjecting conditioned medium from MDA‐MB‐231 cells to various EV isolation methods. Data represent mean ± SD of three replicates. ANOVA with Tukey's multiple comparisons test. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. (c) Schematic summarizing the size‐dependent co‐isolation of HA as a contaminant in EV samples with various isolation methods.

The issue of low and medium molecular weight HA being retained with some EV isolation protocols is further complicated by studies indicating that HA is a component of EVs obtained from synovial fluid (Mustonen et al., 2022, 2016), mesothelial cells (Rilla et al., 2013) and cancer cells (Paul et al., 2022; Pendiuk Goncalves et al., 2023; Rilla et al., 2013). Specifically, studies have reported that HA is associated with the EV surface (Arasu et al., 2017; Pirri et al., 2022; Rilla et al., 2014). Many EVs, such as those obtained from cancer cells, are known to express CD44, a membrane receptor for HA (Rontogianni et al., 2019). Therefore, a potential mechanism for HA binding to the EV surface may be through CD44. However, conclusive evidence that HA is bound to EVs is challenging to obtain, as the polymer is not visible by cryogenic transmission electron microscopy, which is capable of clearly distinguishing the characteristic EV phospholipid bilayer. (Broad et al., 2023) Therefore, the proportion of HA that is associated with EVs versus being an unassociated contaminant is challenging to assess. This study provides the first evidence that low‐ and medium‐molecular weight HA can be retained in EV fractions in the absence of EVs. To assess the retention of HA in biological samples containing EVs, cell culture medium and plasma were processed with various isolation methods. The presence of EVs following each isolation method was confirmed by cryogenic transmission electron microscopy (Figure 2a).

When processing conditioned medium from MDA‐MB‐231 human breast cancer cells by various EV isolation methods, the proportion of retained HA varied substantially. In the case of the conditioned medium, tangential flow filtration retained 42.9% (Figure 1a), and size‐exclusion chromatography retained 88.8% (Figure 1b), while ultracentrifugation and polymer‐based precipitation retained 0.6% and 1% of the initial HA amount, respectively (Figure 1c,d). Although the amount of HA per isolated particle was similar for tangential flow filtration and size‐exclusion chromatography, there was a substantial reduction with ultracentrifugation and a precipitation kit (Figure 2b). Processing of human plasma by size‐exclusion chromatography resulted in 32.4% of the initial HA amount being retained in the EV fraction (Figure 1b). Based on the retention results in the absence of EVs (Figure 1a,b), it is likely that HA contaminants constitute some of the HA detected in EV samples isolated by tangential flow filtration and size‐exclusion chromatography. On the other hand, the centrifugal forces of ultracentrifugation and the water‐binding properties of the polymer in the precipitation kit may strip HA from EV surfaces.

It is worth noting that HA contaminants could possibly contribute to EV‐attributed functional effects, such as immunomodulation, although evidence of this is lacking. In fact, HA has been shown to have pro‐inflammatory or anti‐inflammatory effects, which vary depending on the molecular weight (Cowman et al., 2015; Marinho et al., 2021). Therefore, it is possible that co‐isolated HA contaminants may contribute to immunomodulatory effects observed with cancer cell‐derived EVs (Marar et al., 2021). Alternatively, it could be speculated that anti‐inflammatory effects observed in numerous studies with mesenchymal stromal cell (MSC)‐derived EVs (Cruz et al., 2015; Seo et al., 2019; Tian et al., 2020; Wang et al., 2019, 2021) may be partially attributed to co‐isolated HA contaminants. For example, MSC‐derived EVs reduce inflammation in standard in vitro and in vivo assays, such as those based on macrophage responses to lipopolysaccharide (LPS) (Pacienza et al., 2019), a Toll‐like receptor 4 (TLR4) ligand. Notably, HA binds to TLR4 (Turley et al., 2002), and can have similar anti‐inflammatory effects in LPS‐based studies (Lee et al., 2021; Rayahin et al., 2015; You et al., 2021). Although the HA concentration in functional studies is higher than what was present in the MDA‐MB‐231 conditioned cell culture medium, the EV isolation process can concentrate HA levels. Additionally, other EV sources, such as synovium, have much higher initial levels of HA, which could potentially result in higher contaminant levels (Bruno et al., 2022).

3. DISCUSSION

HA represents a previously overlooked contaminant in EV samples that is likely to be broadly relevant, as this polymer is released into the extracellular space by a wide variety of vertebrate cell types (as well as some prokaryotes) and found in numerous biological fluids (Cowman et al., 2015; de Oliveira et al., 2016). HA with medium and low molecular weights was shown to be partially captured in the same fractions as EVs using two different filtration‐based isolation methods (Busatto et al., 2018; Walker et al., 2020). A previous study has also demonstrated that processing human plasma through commercial size‐exclusion chromatography columns results in the enrichment of glycan features that most likely correspond to chondroitin sulfate, dermatan sulfate and/or Type II keratan sulfate (Walker et al., 2020). It is possible that this enrichment is due to other glycosaminoglycan contaminants in EV samples. The presence of HA contaminants brings into question whether functional effects should be attributed to EVs, as HA has similar immunomodulatory properties as those ascribed to pathological and therapeutic EVs.

Taken together, the presence of EV‐bound and contaminant HA is dependent on the selected EV isolation technique, as levels varied greatly when the same samples were subjected to four isolation methods. Tangential flow filtration and size‐exclusion chromatography are likely to result in EV samples with polysaccharide contaminants, while ultracentrifugation and precipitation kits may remove EV‐bound polysaccharides. In addition to EV‐bound and contaminant polysaccharides, other advantages and disadvantages, should also be considered when selecting an isolation method. Filtration‐based methods are known to preserve EV morphology and display high purity in terms of protein‐based contaminants (Böing et al., 2014; Brennan et al., 2020; Busatto et al., 2018; Iannotta et al., 2021; Konoshenko et al., 2018). However, lipoproteins and other EV‐sized contaminants often co‐isolate with these methods (Brennan et al., 2020; Iannotta et al., 2021; Liangsupree et al., 2021). On the other hand, ultracentrifugation can result in less contamination in terms of some lipoprotein types, but lower EV yields, increased protein contamination and substantial EV damage due to the high centrifugal forces (Brennan et al., 2020; Busatto et al., 2018; Iannotta et al., 2021; Konoshenko et al., 2018). Precipitation methods have low purity and the polymer provided with the kit can contaminate EV samples (Konoshenko et al., 2018; Paolini et al., 2016). This article urges the EV community to consider the implications of EV‐bound and contaminant polysaccharides, such as HA.

4. MATERIALS AND METHODS

4.1. Sample preparation

HA solutions of medium (289 kDa, R&D Systems, GLR004) and low (37 kDa, R&D Systems, GLR001) molecular weight at 300 ng/mL were prepared in PBS (pH 7.4, Gibco/Thermo Fisher Scientific, 10010–031) from a 2000 μg/mL water‐based stock solution. Human MDA‐MB‐231 breast cancer cells (ATCC, HTB‐26) were seeded in 175 cm2 flasks and cultured in high glucose Dulbecco's Modified Eagle's Medium (DMEM, Sigma‐Aldrich/Merck, D5796) containing 100 U/mL of penicillin and 100 μg/mL of streptomycin (Gibco, 15140‐122), and 10% fetal bovine serum (FBS; Gibco, 26140‐079) at 37°C in 5% CO2 until reaching 90% confluency. Cells were then washed twice with PBS and cultured in DMEM without FBS for 24 h to produce EVs. The conditioned medium was collected and centrifuged at 800 × g and 4°C for 30 min to remove dead cells and large cellular debris. Human plasma obtained from the Australian Red Cross Lifeblood (under the University of Queensland's Human Research Ethics Approval number 2022/HE000652) was centrifuged at 2000 × g for 30 min at 4°C. The lipid‐rich top layer was gently removed using a pipette and discarded.

4.2. Tangential flow filtration

Samples were processed using a KrosFlo KR2i TFF System (Repligen), as previously described (Busatto et al., 2018, 2020, 2021; Tian et al., 2020; Wang et al., 2021). Briefly, samples (0.2–0.8 L of 300 ng/mL HA solution or conditioned cell culture medium) were filtered using sterile hollow fibre modified polyethersulfone membranes with 0.65 μm (Spectrum Labs, D02‐E65U‐07‐S) and 750 kDa (Spectrum Labs, D02‐E750‐05‐S) molecular weight cut‐off pores to remove any remaining cell debris and small biomolecules, respectively. Filters were washed with sterile PBS (3× volume of the filter) prior to processing the samples. The input flow rate was 130 mL/min for the first filter and 65 mL/min for the second filter to keep the shear force below 2000/s. Samples were concentrated and diafiltered six times in sterile cryoprotective sucrose buffer (5% sucrose, 50 mM Tris and 2 mM MgCl2 [Walker et al., 2022]) using a flow rate of 55 mL/min, concentrated to 9–11 mL, and stored at −80°C. EVs isolated by this method have been authenticated by nanoparticle tracking analysis, and Western blotting in previous publications (Busatto et al., 2018, 2020, 2021, 2022; Walker et al., 2020).

4.3. Size‐exclusion chromatography

Samples were processed using qEVoriginal 70 nm Gen 2 Columns (Izon, ICO‐70) according to the manufacturer's instructions. Briefly, either a 300 ng/mL HA solution, the conditioned cell culture medium, or pure human plasma were eluted in sucrose buffer, and the collected fractions of 0.5 mL were pooled into groups of 5–6 fractions and stored at −80°C.

4.4. Ultracentrifugation

Samples were processed using an OPTIMA XPN100 ultracentrifuge equipped with a type 50.2 Ti fixed‐angle rotor (k factor 209, Beckman Coulter), as previously described (Lobb et al., 2015). The conditioned cell culture medium and 300 ng/mL HA solution (50 mL of input volume) were centrifuged at 100,000 × g for 90 min at 4°C. The supernatants were discarded, the pellets resuspended in 25 mL of PBS, and the centrifugation step was repeated to obtain the EV pellets, which were resuspended in 400 μL of sucrose buffer, and stored at −80°C.

4.5. Precipitation kit

Samples were processed using miRCURY Exosome Cell/Urine/CSF Kit (Qiagen, 767443) according to the manufacturer's instructions. Briefly, the conditioned cell culture medium and 300 ng/mL HA solutions (6 mL of input volume) were incubated with the precipitation buffer for 60 min at 4°C, then centrifuged at 3200 × g for 30 min at 20°C. The supernatants were removed and the pellets resuspended in 180 μL of sucrose buffer for storage at −80°C.

4.6. HA quantification

HA was quantified in the samples using the Hyaluronan Quantikine ELISA Kit (R&D Systems, DHYAL0) according to the manufacturer's instructions. When required, samples were diluted in PBS before measurements.

4.7. Nanoparticle tracking analysis

A NanoSight NS300 (Software NTA 3.4 Build 3.4.4; Malvern Panalytical Ltd, Malvern) equipped with a 405 nm laser was used to assess particle size distribution profiles and concentration in HA solutions and MDA‐MB‐231 EVs prepared in ultrapure water (Invitrogen/Thermo Fisher Scientific, 10977‐015). Three one‐minute videos were recorded using a camera level of 10 and a detection threshold of five. Each replicate was measured under a continuous syringe pump flow rate of 40 μL/min.

4.8. Cryogenic transmission electron microscopy

Cryogenic transmission electron microscopy is uniquely capable of accurately identifying EV lipid bilayers, which is a key authentication feature of EVs (Broad et al., 2023). The EVs obtained from the conditioned medium of MDA‐MB‐231 cells through the four isolation methods were prepared using the Leica EM GP2 robotic vitrification system. Medium molecular weight HA (289 kDa) at the stock concentration (2000 μg/mL; in water) was also imaged and ultrapure water was used as a control. The preparation process took place under controlled temperature (22°C) and humidity (95%). A 2 μL dispersion of HA or EVs was applied onto a carbon film with circular holes (3.5 μm diameter) and a 1 μm interspace in both dimensions. This film was supported on a 200‐mesh copper grid. Any excess solution was automatically blotted for a period of 2–2.5 s and swiftly immersed in liquid ethane near its freezing point (−182.8°C). The grids were then promptly moved into liquid nitrogen for storage. The samples were imaged using a Jeol Cryo ARM 300 (JEM‐Z300FSC) transmission electron microscope (TEM) in a frozen hydrated state at −176°C. The microscope was equipped with a cold field emission gun (FEG) and an in‐column Omega energy filter. The images were captured with zero energy loss at an acceleration voltage of 300 kV and a filter setting of 20 eV. To ensure minimal exposure, the images were recorded under low‐dose conditions using the SerialEM software and a Gatan K3 direct detector camera (Mastronarde, 2005).

AUTHOR CONTRIBUTIONS

Jenifer P. Goncalves: Conceptualization; Investigation; Formal analysis; Validation; Visualization; Writing—original draft; Writing—review and editing. Raluca Ghebosu: Investigation; Formal analysis; Validation; Visualization; Writing—review and editing. Xuan Ning Sharon Tan: Investigation; Writing—review and editing. Dalila Iannotta: Investigation; Writing—original draft; Writing—review and editing. Na'ama Koifman: Investigation; Writing—review and editing. Joy Wolfram: Conceptualization; Funding acquisition; Project administration; Supervision; Writing—original draft; Writing—review and editing.

CONFLICT OF INTEREST STATEMENT

The authors report no conflicts of interest. Associate Professor Joy Wolfram is listed on an extracellular vesicle grant from Ionis Pharmaceuticals, but the topic of this article is not related to the grant.

ACKNOWLEDGMENTS

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.

Goncalves, J. P. , Ghebosu, R. E. , Tan, X. N. S. , Iannotta, D. , Koifman, N. , & Wolfram, J. (2023). Hyaluronic acid: an overlooked extracellular vesicle contaminant. Journal of Extracellular Vesicles, 12, e12362. 10.1002/jev2.12362

Jenifer P. Goncalves and Raluca E. Ghebosu contributed equally to this study.

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