Journal of extracellular biology最新文献

{"title":"Systematic Evaluation of Isolation Techniques and Freeze-Thaw Effects on Plasma Extracellular Vesicle Heterogeneity and Subpopulation Profiling","authors":"Xueqi Li,&nbsp;Xiangyu Li,&nbsp;Lingjun Tong,&nbsp;Liqiao Hu,&nbsp;Yanfen Hong,&nbsp;Ruoyu Zhou,&nbsp;Zonghong Li,&nbsp;Ming Dong,&nbsp;Junjie Hou,&nbsp;Tao Xu,&nbsp;Wen Zhong","doi":"10.1002/jex2.70058","DOIUrl":"10.1002/jex2.70058","url":null,"abstract":"<p>Extracellular vesicles (EVs) are increasingly recognized as promising disease biomarkers and therapeutic carriers. However, standardizing blood-derived EV isolation remains challenging due to the heterogeneity of EV populations and variability among isolation techniques. In this study, we systematically evaluated three distinct EV isolation methods, including asymmetrical flow field-flow fractionation (AF4), size-exclusion chromatography (SEC) and automated centrifugal microfluidic disc system combined with functionalized membranes (Exo-CMDS), to compare their efficiency in isolating EVs from both freshly frozen and freeze-thawed plasma samples. We utilized an integrative approach combining Proximity-dependent Barcoding Assay (PBA) for single-EV surface protein profiling, Liquid Chromatography–Mass Spectrometry (LC-MS/MS) for bulk proteomic analysis, along with transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) to assess EV yield, morphology, surface protein expression and subpopulation diversity. Our results revealed significant differences in three EV isolation methods. AF4 is particularly enriched for EV subpopulations expressing high levels of classical tetraspanins (e.g., CD81, CD9 and CD151), and single-pass membrane proteins (e.g., ITGA4 and ITAGB1). Exo-CMDS demonstrated the highest reproducibility across samples, isolating specific EV subpopulations enriched in markers like CD5. SEC provided the highest yield but co-isolated significant amounts of non-vesicular particles, including lipoproteins. The findings contribute valuable insights toward standardized and reliable EV isolation practices for research and clinical applications.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"4 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jex2.70058","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144482285","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"iMSC-Derived Extracellular Vesicles Improve Atopic Dermatitis by Augmenting Skin Barrier Integrity and Inhibiting Inflammation, Pruritus and Th2 Immune Responses","authors":"Soo Kim,&nbsp;Jimin Kim,&nbsp;Ran Kim,&nbsp;Hongduk Kim,&nbsp;Seul Ki Lee,&nbsp;Seon-Yeong Jeong,&nbsp;Haedeun You,&nbsp;Somi Park,&nbsp;Tae Min Kim","doi":"10.1002/jex2.70067","DOIUrl":"10.1002/jex2.70067","url":null,"abstract":"<p>Atopic dermatitis (AD) is a chronic inflammatory disease characterized by severe itching and eczematous lesions. Despite various treatments, AD patients experience side effects and fail to achieve full remission. This study investigated the therapeutic potential of extracellular vesicles (EVs) derived from IFN-γ-primed induced mesenchymal stem cells (IFN-γ-iMSC-EVs) in a 2,4-dinitrochlorobenzene (DNCB)-induced AD mouse model. We also examined whether IFN-γ-iMSC-EVs could suppress IL-4/13-induced Th2 responses in keratinocytes. The therapeutic outcome of IFN-γ-iMSC-EVs was comparable to or more effective than baricitinib or clobetasol. While severe weight loss was observed in mice treated with clobetasol, no significant weight reduction occurred in those receiving IFN-γ-iMSC-EVs. Histological analysis demonstrated reduced skin thickness, decreased infiltration of mast cells and inflammatory cells, and suppression of the Th2 immune response, as evidenced by decreased signalling of IL-4, IL-13, and IL-31. IFN-γ-iMSC-EVs also led to a greater reduction in inflammation and pruritus compared to baricitinib and clobetasol. Additionally, skin barrier integrity and epidermal protein expression were improved in IFN-γ-iMSC-EVs. In IL-4/13-stimulated keratinocytes, the decrease in JAK1/2 gene expression and the increase in Keratin 1 gene expression were more prominent in IFN-γ-iMSC-EVs than in baricitinib. The results suggest that IFN-γ-iMSC-EVs have the potential to inhibit AD progression and represent a novel therapeutic option for AD.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"4 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jex2.70067","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144339648","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Serum Extracellular Vesicles Reveal Metabolic Responses to Time-Restricted Feeding in High-Fat Diet-Induced Obesity in Male Mice","authors":"Theresa Bushman,&nbsp;Te-Yueh Lin,&nbsp;Xuenan Jin,&nbsp;Qin Fu,&nbsp;Sheng Zhang,&nbsp;Xiaoli Chen","doi":"10.1002/jex2.70062","DOIUrl":"10.1002/jex2.70062","url":null,"abstract":"<p>Extracellular vesicle (EV) secretion and cargo composition are dysregulated in metabolic diseases. This study aimed to investigate how changes in serum EV concentration and protein composition reflect the metabolic effects of a high-fat diet (HFD) and time-restricted feeding (TRF), with a particular focus on adipocyte-derived EVs (Ad-EVs) in circulation. Mice were fed an HFD for 18 weeks prior to being placed either ad libitum or on a TRF for an additional 10 weeks. Mice on a normal chow ad libitum served as the control. The TRF group had food available for 10 h and fasted for 14 h per day. The serum EV size profile and amount displayed sex- and age-dependent changes in HFD-induced obesity, with age reducing EV amounts. HFD decreased small EV populations and increased larger EV populations, while TRF reversed these changes. Quantitative proteomic analysis showed that the abundance and composition of EV proteins changed in response to both acute stimulation with lipopolysaccharides (LPS) and HFD. Gene ontology analysis identified specific sets of EV proteins and their involved biological processes, reflecting the effect of LPS and HFD, as well as the reversal effect of TRF on metabolic and inflammatory pathways. EV proteins altered by HFD and those reversed by TRF had low protein overlap but significant functional overlap in biological processes. TRF activated the PPAR signalling pathway and the AKT-mTOR signalling pathway. The most significant impacts of HFD and TRF were observed on lipoprotein and carbohydrate metabolism, the complement system, and neutrophil degranulation. Additionally, we showed that serum Ad-EVs respond dynamically to HFD and TRF. Our findings suggest that EVs play a role in diet-induced metabolic and inflammatory responses, with changes in circulating EVs, particularly Ad-EVs, reflecting metabolic adaptations to dietary exposures and interventions.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"4 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jex2.70062","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144339646","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Extracellular Vesicle Lipids and Their Role in Delivery","authors":"Austin Brent,&nbsp;Paniz Shirmast,&nbsp;Nigel A. J. McMillan","doi":"10.1002/jex2.70064","DOIUrl":"10.1002/jex2.70064","url":null,"abstract":"<p>Small extracellular vesicles (sEVs) possess many advantageous characteristics which highlight their potential as nanocarriers for biomedical applications, including the ability to cross the blood brain barrier, improved biocompatibility and exhibit tissue tropism. Despite this potential, the clinical translation of sEVs has been hindered by a variety of factors and lipid nanoparticles (LNPs) remain as the gold standard for nanocarriers, indicating a knowledge gap which could unlock the potential of sEVs. A growing body of research suggests that the lipid profile, rather than the proteome, of sEVs may be contributing to these beneficial characteristics much more than previously thought. This review highlights and discusses the current state of the field in terms of lipid composition between sEVs originating from various cell sources and the roles which the different lipids play in the function of sEVs as natural nanocarriers within the body. We also discuss the potential of various EV-mimetics and synthetic EVs (synEVs) in terms of clinical translation which may provide a means to allow wider therapeutic adoption of EVs.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"4 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jex2.70064","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144339647","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Monitoring Pseudoprogression Using Circulating Small Extracellular Vesicles Expressing PD-L1 in a Melanoma Patient Treated With Immune Checkpoint Inhibitors","authors":"Charlée Nardin,&nbsp;Valentin Vautrot,&nbsp;Isen Naiken,&nbsp;Alexandre Doussot,&nbsp;Eve Puzenat,&nbsp;Célia De Girval,&nbsp;Carmen Garrido,&nbsp;François Aubin,&nbsp;Jessica Gobbo","doi":"10.1002/jex2.70066","DOIUrl":"10.1002/jex2.70066","url":null,"abstract":"<p>Immune checkpoint inhibitors (ICI) have revolutionized the treatment of metastatic malignancy. However, unique immune response patterns can occur, such as pseudoprogression, which corresponds to new lesion development or temporary tumour growth followed by regression. Misidentifying pseudoprogression may halt ICI therapy, due to the absence of biomarkers to distinguish progression from pseudoprogression. In 2020, our team proposed small extracellular vesicles expressing PD-L1 (sEV-PD-L1) as a predictor of melanoma treatment response. We report a case of pseudoprogression in a patient treated with nivolumab and ipilimumab for metastatic melanoma, and showing reduced circulating sEV-PD-L1. To our knowledge, this is the first report of PD-L1 monitoring in circulating sEV during pseudoprogression under ICI. A decrease in PD-L1 in circulating sEV might be an early sign of disease response to ICI, and may help to diagnose pseudoprogression. This case supports further evaluation of sEV-PD-L1 to identify responder patients to ICI, especially in case of pseudoprogression.</p><p>Trial Registration: EXOMEL1 P/2018/40 1 AC-2015-2496/DC-2014-2086. NCT05744076.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"4 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jex2.70066","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144339237","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Detection and Isolation of Tissue-Specific Extracellular Vesicles From the Blood","authors":"Lauren Newman,&nbsp;Andrew Rowland","doi":"10.1002/jex2.70059","DOIUrl":"10.1002/jex2.70059","url":null,"abstract":"<p>Extracellular vesicles (EVs) are nanosized, membrane-bound particles released by virtually all cell types, serving as messengers within tissues and across organs via the bloodstream. EVs encapsulate diverse molecular cargo that reflects the phenotypic state of their originating cells, making them promising candidates for liquid biopsy applications. However, the heterogeneity of circulating EVs, comprising particles from various cell types and non-vesicular entities like lipoproteins, poses significant challenges for isolating tissue-specific EV populations. This review examines current methodologies for detecting and isolating tissue-specific EVs from blood, focusing on immunoaffinity capture (IAC) strategies that leverage surface marker expression for specificity. Key considerations, including the selection and validation of markers, are discussed alongside advances in EV subtyping and isolation protocols. Challenges such as marker cross-reactivity, EV biogenesis and transport dynamics are highlighted to underscore the complexity of achieving clinical utility. By providing an overview of validated tissue-specific markers and isolation techniques, this review aims to facilitate the development of EV-based biomarkers with enhanced specificity and sensitivity, enabling minimally invasive monitoring of organ function and disease.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"4 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jex2.70059","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144339238","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Proteomics of Urinary Extracellular Vesicles Highlight the Involvement of Vitronectin and the Fibrinolytic and TNF Pathways as Mechanisms Underlying Renal Fibrosis in Kidney Transplant Patients","authors":"Marta Clos-Sansalvador,&nbsp;Sergio G. Garcia,&nbsp;Paula Rodríguez-Martínez,&nbsp;Marta Sanroque-Muñoz,&nbsp;Miriam Font-Morón,&nbsp;Cristina Grange,&nbsp;Benedetta Bussolati,&nbsp;Marcella Franquesa,&nbsp;Javier Juega,&nbsp;Francesc E. Borràs","doi":"10.1002/jex2.70056","DOIUrl":"10.1002/jex2.70056","url":null,"abstract":"<p>Vitronectin (VTN) is a potential non-invasive biomarker for renal fibrosis, originally described in urinary extracellular vesicles (uEV) from kidney transplant patients (KTx). However, VTN's specific role in renal fibrosis is unclear, as it is involved in various physiological processes. This study aims to identify other uEV-associated proteins linked to renal fibrosis to clarify which pathways involve VTN. uEV were isolated from 33 KTx patients and five healthy controls. uEV proteins were analysed using proximity extension assay (PEA), and data were normalized and compared using Welch's two-sided <i>t</i>-test to identify differentially expressed proteins between fibrotic (<i>n</i> = 31) and non-fibrotic patients (<i>n</i> = 7). Urinary VTN levels and monocyte chemoattractant protein-1 (MCP-1) were measured by ELISA. PEA analysis identified 33 proteins overexpressed in the fibrotic group. These proteins clustered in STRING analysis, primarily associating with coagulation, fibrinolysis and TNF-inflammation involving macrophages. ELISA detection of MCP-1 further validated the results. High levels of VTN in the fibrotic group were accompanied by the upregulation of fibrinolytic pathway components (PAI-1, tPA and uPAR), which are well-known to interact with VTN. This study highlights TNF-induced inflammation involving macrophages and fibrinolysis as key mechanisms underlying renal fibrosis with direct implications of VTN, which support VTN's potential as a biomarker for this pathological process.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"4 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jex2.70056","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144191005","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enolase and 16.5-kDa Tegument-Associated Protein in Fasciola hepatica Extracellular Vesicles: Clues to Their Role in Pathogenesis","authors":"Aránzazu González-Arce,&nbsp;Christian M. Sánchez-López,&nbsp;Liz F. Sánchez-Palencia,&nbsp;Antonio Marcilla,&nbsp;Dolores Bernal","doi":"10.1002/jex2.70055","DOIUrl":"10.1002/jex2.70055","url":null,"abstract":"<p>Fasciolosis, caused by <i>Fasciola hepatica</i>, is a parasitic zoonosis that induces liver fibrosis in infected hosts, including ruminants and humans. Extracellular vesicles secreted by <i>F. hepatica</i> (<i>Fh</i>EVs) play a crucial role in modulating host immune responses and promoting tissue re-modelling. This work explores the effects of two proteins found in <i>Fh</i>EVs, enolase (<i>Fh</i>enolase), enriched in the vesicular lumen, as well as the 16.5-kDa tegument-associated protein (<i>Fh</i>16.5TP), highly abundant in the EV membrane, on hepatic and liver-associated immune cells. Recombinant proteins (r-<i>Fh</i>enolase and r-<i>Fh</i>16.5TP) were produced to evaluate their impact on cell viability, inflammatory responses, proteomic profiles and EV secretion in THP1-XBlue CD14 macrophages, HepG2 hepatocytes and LX-2 hepatic stellate cells (HSCs). Interestingly, r-<i>Fh</i>enolase, but not r-<i>Fh</i>16.5TP, showed anti-inflammatory properties in lipopolysaccharide (LPS)–activated macrophages, by reducing NF-κB activation and inducing significant changes in the protein cargo of macrophage-derived EVs, which contained lower levels of the pro-inflammatory cytokines IL-1β, TNF-α and IL-6. Proteomic analysis of cells treated with r-<i>Fh</i>enolase revealed distinct alterations in proteins related to fibrotic and inflammatory pathways, including a reduction in extracellular matrix (ECM) proteins and suggesting a potential role in mitigating liver fibrosis. Furthermore, r-<i>Fh</i>enolase reduced EV production and fibrotic markers in hepatic cells, but not in macrophages. In contrast, r-<i>Fh</i>16.5TP increased pro-fibrotic proteins in both, cells and EVs, and increased EV production specifically in LX-2 cells, indicating its possible contribution to fibrosis progression in fasciolosis. These findings represent a first approach to analyse EV-associated proteins and study their potential role in the molecular mechanisms of <i>F. hepatica</i>–host interactions.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"4 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jex2.70055","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144191152","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enhanced Anti-Tumour Efficacy of iRGD-Modified Cell-Bound Membrane Vesicles (iRGD-CBMVs) as a Novel Drug Carrier","authors":"Haonan Zhao,&nbsp;Zhendong Huang,&nbsp;Qinghua Sheng,&nbsp;Wenxiang Shao,&nbsp;Min Zeng,&nbsp;Kun Wang,&nbsp;Yang Zhang,&nbsp;Ying Qin,&nbsp;Zhihao Xiong,&nbsp;Lizhen Chen,&nbsp;Huaying Wang,&nbsp;Tong Rong,&nbsp;Zhitao Qiu,&nbsp;Hongda Zhuang,&nbsp;Zhiwen Wu,&nbsp;Yuan Zhang,&nbsp;Wendiao Zhang,&nbsp;Yong Chen","doi":"10.1002/jex2.70052","DOIUrl":"10.1002/jex2.70052","url":null,"abstract":"<p>Cancer continues to be the foremost cause of mortality in humans. Persistent challenges in cancer treatment include inadequate drug targeting, severe toxicological side effects and uncontrolled drug distribution. The bioinspired membrane vesicle drug delivery systems have been emerging as promising therapeutic strategies. This study characterises unique cell-bound membrane vesicles (CBMVs), which are impervious to standard cleaning agents and effectively loaded with doxorubicin (DOX). For the first time, we used iRGD peptide to modify the CBMVs to enhance the CBMVs' targeting capabilities for cancer cells. Laser confocal microscopy and ¹H Nuclear Magnetic Resonance Spectra (¹H NMR) have confirmed the CBMVs' iRGD modification and effective encapsulation with DOX (iRGD-CBMVs-DOX). Then, we used the iRGD-CBMVs-DOX to treat tumour cell lines and tumour-bearing mouse models. Our research identified that iRGD-CBMVs-DOX proves effective in inhibiting cell growth and migration for tumour cell lines, significant anti-tumour ability, reduced organ toxicity and continuous drug administration were revealed in tumour-bearing mouse models. Additionally, the iRGD-CBMVs-DOX demonstrated sustained drug release, indicating their potential for prolonged circulation. These findings are pivotal in enhancing cancer treatment through novel nanomedicine strategies, and highlight the potential of iRGD-modified vesicles (e.g., iRGD-CBMVs) as efficient drug carriers, contributing to targeted and biocompatible drug delivery advancements for cancer treatment.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"4 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jex2.70052","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144191154","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Response to ‘Purification of Mesenchymal Stromal Cell-Derived Small Extracellular Vesicles Using Ultrafiltration’","authors":"Anders Toftegaard Boysen PhD","doi":"10.1002/jex2.70057","DOIUrl":"10.1002/jex2.70057","url":null,"abstract":"&lt;p&gt;Dear Editor,&lt;/p&gt;&lt;p&gt;I read the recent article ‘&lt;i&gt;Purification of mesenchymal stromal cell-derived small extracellular vesicles using ultrafiltration&lt;/i&gt;’ by Lei et al. (&lt;span&gt;2025&lt;/span&gt;) with great interest, as they have thoroughly characterised an ultrafiltration method also used by me (Boysen et al. &lt;span&gt;2024&lt;/span&gt;). I would like to weigh in with some of my own experiences and findings on their conclusions. The extracellular vesicle (EV) characterisation techniques (TEM and NTA) used by the authors have size and concentration determination limitations. While TEM does a great job of identifying EVs in their cup-shaped appearance, it is not a great tool in size and concentration determination, as the amount of EVs present is few and they have been dried, thereby having lost their true size and morphology. Similarly, NTA has drawbacks as EVs have a low refractive index, the limit of detection is regarded to be around 50 nm (Dragovic et al. &lt;span&gt;2011&lt;/span&gt;), leading to a potentially skewed size profile and reduced particle concentration. The study of Lei et al. is therefore at risk of not showing a potential loss of sub-50 nm EVs using ultrafiltration and their specified molecular weight cut-off (MWCO) values. While they argue that the pore size of the membranes used is 6.13, 8.84 and 10.48 nm for 100, 300 and 500 kDa MWCO, respectively, this is only true from a theoretical perspective based on the hydrodynamic radius of molecules. The pore size of commercially available filters is &lt;span&gt;not&lt;/span&gt; based on theoretical values but trial and error and the &lt;span&gt;average&lt;/span&gt; pore size of the selected filters by Lei et al. are 10, 35 and 55 nm for 100, 300 and 500 kDa MWCO respectively as seen in Figure 1 (determined using cryo-EM, personal correspondence with Merck Millipore and as reported by Pall corporation (Pall Corporation &lt;span&gt;2022&lt;/span&gt;)). We have translated a copious amount of the knowledge from virology to EV research, as EVs and viruses have many biophysical similarities, and we could also learn from virology in this case. MS2, a icosahedral ∼27 nm bacteriophage, is not retained on a 300 kDa MWCO filter, and can be found in the permeate (Wick and McCubbin &lt;span&gt;1999&lt;/span&gt;). While fouling, particle deposition, and potential loss of EVs can be reduced by adding shear force to the membrane, this force will not be homogeneous, and loss will occur (Hwang et al. &lt;span&gt;2016&lt;/span&gt;; Zhao et al. &lt;span&gt;2023&lt;/span&gt;). Cryo-EM, in contrast to TEM and NTA, can show sub-50 nm particles in their native state (Yuana et al. &lt;span&gt;2013&lt;/span&gt;). As a true size distribution of an EV sample, I have made individual measurements of 130 EVs derived from human embryonic kidney cells (HEK293T) cultured in chemically defined media (HEK VIP NX, Satorius) and enriched using size exclusion chromatography (35 nm, Izon) on a 100 kDa MWCO spin filter (Thermo Fisher) as shown below.\u0000\u0000 &lt;/p&gt;&lt;p&gt;Cryo-EM tends to exclude some of the larger EVs (Welsh et al.","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"4 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jex2.70057","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144191153","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}