Global trend in exosome isolation and application: an update concept in management of diseases.

Extracellular vesicles (EVs) secreted by various cells offer great potential for use in the diagnosis and treatment of disease. EVs are heterogeneous membranous vesicles. Exosomes are a subtype of EVs, 40-150 nm spherical vesicles with a lipid layer derived from endosomes. Exosomes, which are involved in signal transduction and maintain homeostasis, are released from almost all cells, tissues, and body fluids. Although several methods exist to isolate and characterize EVs and exosomes, each technique has significant drawbacks and limitations that prevent progress in the field. New approaches in the biology of EVs show great potential for isolating and characterizing EVs, which will help us better understand their biological function. The strengths and limitations of conventional strategies and novel methods (microfluidic) for EV isolation are outlined in this review. We also present various exosome isolation techniques and kits that are commercially available and assess the global market demand for exosome assays.

Molecular mechanisms and therapeutic application of extracellular vesicles from plants.

Small extracellular vesicles (sEVs) isolated from animal sources are among the most investigated types of cell-free therapeutic tools to cure different diseases. sEVs have been isolated from a variety of sources, ranging from prokaryotes to animals and plants. Human-derived sEVs have many uses in pre- and clinical studies in medicine and drug delivery, while plant-derived EVs, also known as plant-derived nanovesicles (PDNVs), have not been widely investigated until the second decade of the 21st century. For the past five years, there has been a rapid rise in the use of plant EVs as a therapeutic tool due to the ease of massive production with high efficacy and yield of preparation. Plant EVs contain various active biomolecules such as proteins, regulatory RNAs, and secondary metabolites and play a key role in inter-kingdom communications. Many studies have already investigated the potential application of plant EVs in preventing and treating cancer, inflammation, infectious diseases, and tissue regeneration with no sign of toxicity and are therefore considered safe. However, due to a lack of universal markers, the properties of plant EVs have not been extensively studied. Concerns regarding the safety and therapeutic function of plant EVs derived from genetically modified plants have been raised. In this paper, we review the physiological role of EVs in plants. Moreover, we focus on molecular and cellular mechanisms involved in the therapeutic effects of plant EVs on various human diseases. We also provide detailed information on the methodological aspects of plant EV isolation and analysis, which could pave the way for future clinical translation.

Exploring capabilities of elemental mass spectrometry for determination of metal and biomolecules in extracellular vesicles.

Extracellular vesicles (EVs) are increasingly recognized as crucial components influencing various pathophysiological processes, such as cellular homeostasis, cancer progression, and neurological disease. However, the lack of standardized methods for EV isolation and classification, coupled with ambiguity in biochemical markers associated with EV subtypes, remains a major challenge. This Trends article highlights the most common approaches for EV isolation and characterization, along with recent applications of elemental mass spectrometry (MS) to analyse metals and biomolecules in EVs obtained from biofluids or in vitro cellular models. Considering the promising capabilities of elemental MS, the article also looks ahead to the potential analysis of EVs at the single-vesicle and single-cell levels using ICP-MS. These approaches may offer valuable insights into individual characteristics of EVs and their functions, contributing to a deeper understanding of their role in various biological processes.

Size-Exclusion Chromatography: A Path to Higher Yield and Reproducibility Compared to Sucrose Cushion Ultracentrifugation for Extracellular Vesicle Isolation in Multiple Myeloma.

Small extracellular vesicles (EVs) play a pivotal role in intercellular communication across various physiological and pathological contexts. Despite their growing significance as disease biomarkers and therapeutic targets in biomedical research, the lack of reliable isolation techniques remains challenging. This study characterizes vesicles that were isolated from conditioned culture media (CCM) sourced from three myeloma cell lines (MM.1S, ANBL-6, and ALMC-1), and from the plasma of healthy donors and multiple myeloma patients. We compared the efficacy, reproducibility, and specificity of isolating small EVs using sucrose cushion ultracentrifugation (sUC) vs. ultrafiltration combined with size-exclusion chromatography (UF-SEC). Our results demonstrate that UF-SEC emerges as a more practical, efficient, and consistent method for EV isolation, outperforming sUC in the yield of EV recovery and exhibiting lower variability. Additionally, the comparison of EV characteristics among the three myeloma cell lines revealed distinct biomarker profiles. Finally, our results suggest that HBS associated with Tween 20 improves EV recovery and preservation over PBS. Standardization of small EV isolation methods is imperative, and our comparative evaluation represents a significant step toward achieving this goal.

Comparative Characteristic of the Methods of Isolation of Oocytes and Spermatozoa in Laboratory Animals (Review).

Over the past decade, there has been an increasing trend in the use of assisted reproductive technologies, which have significantly expanded the opportunities to overcome the problem of infertility. However, the problem of increasing the effectiveness of in vitro fertilization remains open. Isolation of germ cells from animals is a necessary process for various experimental studies. Animal germ cells can be used in experiments to study physical, chemical, genetic, immunological, and microbiological factors affecting reproduction efficiency and for the development of techniques that increase the effectiveness of in vitro fertilization. All of the above determines the relevance of studying existing methods of oocyte and sperm isolation for experimental in vitro studies. Here we discuss the existing methods of sperm and oocyte isolation from animals and their advantages and disadvantages, and also substantiate priority methods for use.

Biological, functional and nutritional properties of caseinomacropeptide from sweet whey.

Bioactive peptides derived from bovine milk proteins have gained much attention due to their health promoting functions. All over the world, cheese industry generates high volumes of sweet whey that could be used as an alternative source of bioactive peptide in nutraceuticals and food industry. Caseinomacropeptide (CMP) is a bioactive peptide derived from κ-casein by the action of chymosin during cheese manufacturing. CMP consist of two forms which are glycosylated (gCMP) and non-glycosylated (aCMP). The predominant carbohydrate in gCMP is N-acetylneuraminic (sialic acid) which gives functional and biological properties to gCMP. Due to its unique composition and technological characteristics such as wide pH range solubility, emulsifying, gelling, and foaming ability, CMP has received special attention. Therefore, there is an increased interest in researches for isolation and concentration of CMP. However, the isolation and purification methods are not cost-effective. It would be easier to optimize the conditions for isolation, purification, and utilization of CMP in nutraceuticals and food industry through deeper understanding of the effective factors. In this review, the structure of CMP, biological activities, isolation, and purification methods, the factors affecting functional properties and application areas of CMP in food industry are discussed.

A review on comparative studies addressing exosome isolation methods from body fluids.

Exosomes emerged as valuable sources of disease biomarkers and new therapeutic tools. However, extracellular vesicles isolation with exosome-like characteristics from certain biofluids is still challenging which can limit their potential use in clinical settings. While ultracentrifugation-based procedures are the gold standard for exosome isolation from cell cultures, no unique and standardized method for exosome isolation from distinct body fluids exists. The complexity, specific composition, and physical properties of each biofluid constitute a technical barrier to obtain reproducible and pure exosome preparations, demanding a detailed characterization of both exosome isolation and characterization methods. Moreover, some isolation procedures can affect downstream proteomic or RNA profiling analysis. This review compiles and discussed a set of comparative studies addressing distinct exosome isolation methods from human biofluids, including cerebrospinal fluid, plasma, serum, saliva, and urine, also focusing on body fluid specific challenges, physical properties, and other potential variation sources. This summarized information will facilitate the choice of exosome isolation methods, based on the type of biological samples available, and hopefully encourage the use of exosomes in translational and clinical research.

Comparative analysis of extracellular vesicles isolated from human mesenchymal stem cells by different isolation methods and visualisation of their uptake.

Various types of cells secrete extracellular vesicle (EVs) which contain proteins, lipids and nucleic acids and play important roles in inter-cellular signalling and pathological processes to impact the recipient cells. EVs have demonstrated their potential as biomarkers for disease and as therapeutic agents in regenerative medicine. In recent times, EVs derived from mesenchymal stem cells (MSCs), which are widely used as a promising medicinal product in many clinical applications, are being tested in many preclinical trials. However, the lack of standardization of MSC-derived EV isolation and analysis methods, restricts the utility of MSC-derived EVs in the clinical setting. Here, we focused on optimising the isolation method for EVs derived from MSCs. Four samples of EVs were isolated from human adipose derived MSC culture medium by differential ultracentrifugation with three different ultracentrifuge durations to investigate the influence of ultracentrifuge time on quality and quantity of MSC-derived EVs. Additionally, we used a commercial kit to extract EVs from MSC cultured medium and compared it with the ultracentrifugation method. The EV samples were then characterised for particle concentration, protein concentration, size distribution and the presence of known EV protein markers, by western blot and flow cytometry. A comparison of these results for the five samples demonstrated that 1 h of differential ultracentrifugation was optimal to isolate high quality and quantity of MSC-derived EVs from MSC cultured medium. Additionally, fluorescence imaging of the freshly isolated vs frozen EVs showed that freshly isolated EVs are taken up by cells more efficiently than frozen EVs. These finding establish a simple and reliable method of EV isolation from MSCs.

Effect of Ecotype and Starch Isolation Methods on the Physicochemical, Functional, and Structural Properties of Ethiopian Potato (Plectranthus edulis) Starch.

The Ethiopian potato (Plectranthus edulis) is an annual tuber crop indigenous to Ethiopia. The crop is underutilized and not much studied despite its high yield of starch, which has a good potential to contribute to the effort in meeting the quickly growing demand for starch. In this study, the effects of the ecotype and isolation methods on the physicochemical, functional, structural, and crystalline properties of starches were evaluated. Starches were isolated from two Ethiopian potato ecotypes (Loffo and Chanqua) using distilled water (DW), 0.01% sodium metabisulphite (SMS), and 1M sodium chloride (NaCl) in the isolation media. The results showed that the lowest starch yield was obtained from Chanqua using DW (97.4%), while the maximum was from Loffo using SMS (99.3%). The L* (lightness) and whiteness values of the starches obtained from Loffo were higher than those of Chanqua starches, with NaCl and SMS extractants yielding the highest values. The bulk density, water activity (aw), pH, proximate composition (moisture content, protein, ash, fat, crude fiber, and carbohydrate contents), and techno-functional properties were established. The majority of these parameters varied depending on both the isolation method and the ecotype. The crystallinity pattern of all starches showed B-type diffraction, with differences in diffraction peak intensities between all starches. FTIR tests showed structural changes as a function of the ecotype and isolation procedure used. The Loffo ecotype exhibited considerably better results, and the SMS isolation method was found to be the most effective way to acquire the highest starch quality in most of the characteristics evaluated.

A Concise Profile of Gallic Acid-From Its Natural Sources through Biological Properties and Chemical Methods of Determination.

Nature is a valuable source of anti-oxidants that have a health-promoting effect by inhibiting various undesirable changes leading to cell degradation and, consequently, potential disease ailments. One of them is gallic acid which has been used as a healing agent since ancient times. Currently, due to various beneficial properties, this compound is considered to be one of the main phenolic acids of great importance in numerous industries. It is commonly used as a substance protecting against the harmful effects of UV radiation, an astringent in cosmetic preparations, and a preservative in food products. Therefore, gallic acid is now deemed essential for both human health and industry. Increasingly better methods of its isolation and analysis are being developed, and new solutions are being sought to increase its production. This review, presenting a concise characterization of gallic acid, updates the knowledge about its various biological activities and methods used for its isolation and determination, including chromatographic and non-chromatographic methods.

Comparison of Methods for MicroRNA Isolation from Extracellular Vesicles Obtained from Ascitic Fluids.

Secreted extracellular vesicles (EVs) contain active biomolecules, including miRNAs, composition of which reflects epigenetic changes occurring in cells during pathological processes, in particular, malignant transformation. The accumulated pool of data on the role of EVs in carcinogenesis has stimulated investigations of the EV-derived cancer markers. The most important factor limiting development of this scientific direction is lack of "gold standards" both for methods of EV isolation from biological fluids and for analyzing their molecular content, including composition of miRNAs. Here we first examined efficacy of various methods for small RNA isolation from EVs contained in ascitic fluid for subsequent miRNA analysis. Comparison of different commercial kits showed advantages of the methods based on phenol-chloroform extraction: Total Exosome RNA & Protein Isolation Kit and miRNeasy Serum/Plasma Kit. Analysis of the small RNA transcriptome showed presence of various classes of molecules in the EVs, among which proportion of miRNAs averaged 6% and reaching 10% with the Total Exosome RNA & Protein Isolation Kit. The PureLink miRNA Isolation Kit demonstrated the lowest efficiency. The miRNeasy Advanced Serum/Plasma Kit showed the highest concentration of the small RNA fraction, miRNA proportion of which, however, did not exceed that obtained with the miRNeasy Serum/Plasma Kit and Total Exosome RNA & Protein Isolation Kit. Moreover, RT-PCR analysis of the individual molecules showed lower levels of each of investigated miRNAs (miR-1246, miR-200b-5p, miR-200c-3p, and miR-23a-3p) when using the miRNeasy Advanced Serum/Plasma Kit. In conclusion, Total Exosome RNA & Protein Isolation Kit and miRNeasy Serum/Plasma Kit can be considered as optimal kits in terms of performance based on combination of the studied characteristics, including small RNA concentration, percentage of microRNA according to bioanalyzer and sequencing results, and levels of individual miRNAs detected by RT-PCR.

Beyond the Extracellular Vesicles: Technical Hurdles, Achieved Goals and Current Challenges When Working on Adipose Cells.

Adipose tissue and its crosstalk with other organs plays an essential role in the metabolic homeostasis of the entire body. Alteration of this communication (i.e., due to obesity) is related to the development of several comorbidities including type 2 diabetes, cardiovascular diseases, or cancer. Within the adipose depot, adipocytes are the main cell type and thus the main source of secreted molecules, which exert modulating effects not only at a local but also at a systemic level. Extracellular vesicles (EVs) have recently emerged as important mediators in cell-cell communication and account for part of the cellular secretome. In recent years, there has been a growing body of research on adipocyte-derived extracellular vesicles (Ad-EVs). However, there is still a lack of standardized methodological approaches, especially regarding primary adipocytes. In this review, we will provide an outline of crucial aspects when working on adipose-derived material, with a special focus on primary adipocytes. In parallel, we will point out current methodological challenges in the EV field and how they impact the transcriptomic, proteomic and functional evaluations of Ad-EVs.

Extracellular Vesicles as a Therapeutic Tool for Kidney Disease: Current Advances and Perspectives.

Extracellular vesicles (EVs) have been described as important mediators of cell communication, regulating several physiological processes, including tissue recovery and regeneration. In the kidneys, EVs derived from stem cells have been shown to support tissue recovery in diverse disease models and have been considered an interesting alternative to cell therapy. For this purpose, however, several challenges remain to be overcome, such as the requirement of a high number of EVs for human therapy and the need for optimization of techniques for their isolation and characterization. Moreover, the kidney's complexity and the pathological process to be treated require that EVs present a heterogeneous group of molecules to be delivered. In this review, we discuss the recent advances in the use of EVs as a therapeutic tool for kidney diseases. Moreover, we give an overview of the new technologies applied to improve EVs' efficacy, such as novel methods of EV production and isolation by means of bioreactors and microfluidics, bioengineering the EV content and the use of alternative cell sources, including kidney organoids, to support their transfer to clinical applications.

Single Extracellular Vesicle Analysis Performed by Imaging Flow Cytometry and Nanoparticle Tracking Analysis Evaluate the Accuracy of Urinary Extracellular Vesicle Preparation Techniques Differently.

Small extracellular vesicles isolated from urine (uEVs) are increasingly recognized as potential biomarkers. Meanwhile, different uEV preparation strategies exist. Conventionally, the performance of EV preparation methods is evaluated by single particle quantification, Western blot, and electron microscopy. Recently, we introduced imaging flow cytometry (IFCM) as a next-generation single EV analysis technology. Here, we analyzed uEV samples obtained with different preparation procedures using nanoparticle tracking analysis (NTA), semiquantitative Western blot, and IFCM. IFCM analyses demonstrated that urine contains a predominant CD9+ sEV population, which exceeds CD63+ and CD81+ sEV populations. Furthermore, we demonstrated that the storage temperature of urine samples negatively affects the recovery of CD9+ sEVs. Although overall reduced, the highest CD9+ sEV recovery was obtained from urine samples stored at -80 °C and the lowest from those stored at -20 °C. Upon comparing the yield of the different uEV preparations, incongruencies between NTA and IFCM data became apparent. Results obtained by both NTA and IFCM were consistent with Western blot analyses for EV marker proteins; however, NTA results correlated with the amount of the impurity marker uromodulin. Despite demonstrating that the combination of ultrafiltration and size exclusion chromatography appears as a reliable uEV preparation technique, our data challenge the soundness of traditional NTA for the evaluation of different EV preparation methods.

Influencing Factors on the Physicochemical Characteristics of Tea Polysaccharides.

Tea polysaccharides (TPSs) are one of the main bioactive constituents of tea with various biological activities such as hypoglycemic effect, antioxidant, antitumor, and immunomodulatory. The bioactivities of TPSs are directly associated with their structures such as chemical composition, molecular weight, glycosidic linkages, and conformation among others. To study the relationship between the structures of TPSs and their bioactivities, it is essential to elucidate the structure of TPSs, particularly the fine structures. Due to the vast variation nature of monosaccharide units and their connections, the structure of TPSs is extremely complex, which is also affected by several major factors including tea species, processing technologies of tea and isolation methods of TPSs. As a result of the complexity, there are few studies on their fine structures and chain conformation. In the present review, we aim to provide a detailed summary of the multiple factors influencing the characteristics of TPS chemical structures such as variations of tea species, degree of fermentation, and preparation methods among others as well as their applications. The main aspects of understanding the structural difference of TPSs and influencing factors are to assist the study of the structure and bioactivity relationship and ultimately, to control the production of the targeted TPSs with the most desired biological activity.

Effective methods for isolation and purification of extracellular vesicles from plants.

Plant extracellular vesicles (EVs) play critical roles in the cross-kingdom trafficking of molecules from hosts to interacting microbes, most notably in plant defense responses. However, the isolation of pure, intact EVs from plants remains challenging. A variety of methods have been utilized to isolate plant EVs from apoplastic washing fluid (AWF). Here, we compare published plant EV isolation methods, and provide our recommended method for the isolation and purification of plant EVs. This method includes a detailed protocol for clean AWF collection from Arabidopsis thaliana leaves, followed by EV isolation via differential centrifugation. To further separate and purify specific subclasses of EVs from heterogeneous vesicle populations, density gradient ultracentrifugation and immunoaffinity capture are then utilized. We found that immunoaffinity capture is the most precise method for specific EV subclass isolation when suitable specific EV biomarkers and their corresponding antibodies are available. Overall, this study provides a guide for the selection and optimization of EV isolation methods for desired downstream applications.

[Research strategies for endophytes in medicinal plants based on high-throughput sequencing and traditional culture and isolation methods].

The research on endophytes of medicinal plants mainly relies on the traditional culture and isolation methods. Because of their functions such as promoting host growth, improving stress resistance, promoting the accumulation of medicinal active ingredients or directly producing medicinal active ingredients, the endophytes of medicinal plants have gradually attracted wide attention. However, it was found that the strains isolated by traditional methods were not the true dominant endophytes of medicinal plants by comparing the results of traditional culture isolation with high-throughput sequencing. The blind and random nature of traditional methods leads to the lack of standards in terms of medium selection, culture time and interaction between species. On the contrary, high-throughput sequencing technology is an emerging molecular biology technology developed in recent decades. Due to its high resolution level and indepen-dent culture, it can be used for thorough analysis of the community structure and diversity of environmental microorganisms. Therefore, we proposed the strategy of using high-throughput sequencing technology to guide the traditional culture and isolation of endophytes from medicinal plants. Firstly, the endophytic structure and diversity of medicinal plants were completely clear by high-throughput sequencing technology, and the dominant endophytes of the host were unequivocal. Then according to the characteristics of each dominant endophytes design or query suitable medium for its growth to culture and isolation. Finally, the function of the isolates was studied. This method can prevent researchers from missing out on the important functional strains of the host, expand the research scope of endophytes of medicinal plants, and facilitate the in-depth excavation and utilization of endophytes of medicinal plants.

Extracellular vesicle isolation methods: rising impact of size-exclusion chromatography.

Extracellular vesicles (EVs) include a variety of nanosized vesicles released to the extracellular microenvironment by the vast majority of cells transferring bioactive lipids, proteins, mRNA, miRNA or non-coding RNA, as means of intercellular communication. Remarkably, among other fields of research, their use has become promising for immunomodulation, tissue repair and as source for novel disease-specific molecular signatures or biomarkers. However, a major challenge is to define accurate, reliable and easily implemented techniques for EV isolation due to their nanoscale size and high heterogeneity. In this context, differential ultracentrifugation (dUC) has been the most widely used laboratory methodology, but alternative procedures have emerged to allow purer EV preparations with easy implementation. Here, we present and discuss the most used of the different EV isolation methods, focusing on the increasing impact of size exclusion chromatography (SEC) on the resulting EV preparations from in vitro cultured cells-conditioned medium and biological fluids. Comparatively, low protein content and cryo-electron microscopy analysis show that SEC removes most of the overabundant soluble plasma proteins, which are not discarded using dUC or precipitating agents, while being more user friendly and less time-consuming than gradient-based EV isolation. Also, SEC highly maintains the major EVs' characteristics, including vesicular structure and content, which guarantee forthcoming applications. In sum, together with scaling-up possibilities to increase EV recovery and manufacturing following high-quality standards, SEC could be easily adapted to most laboratories to assist EV-associated biomarker discovery and to deliver innovative cell-free immunomodulatory and pro-regenerative therapies.

Impact of Extracellular Vesicle Isolation Methods on Downstream Mirna Analysis in Semen: A Comparative Study.

Seminal plasma (SP) contains a unique concentration of miRNA, mostly contained in small extracellular vesicles (sEVs) such as exosomes, some of which could be clinically useful for diagnosis and/or prognosis of urogenital diseases such as prostate cancer (PCa). We optimized several exosome-EV isolation technologies for their use in semen, evaluating EV purifying effectiveness and impact on the downstream analysis of miRNAs against results from the standard ultracentrifugation (UC) method to implement the use of SP sEV_miRNAs as noninvasive biomarkers for PCa. Our results evidenced that commercial kits designed to isolate exosomes/EVs from blood or urine are mostly applicable to SP, but showed quantitative and qualitative variability between them. ExoGAG 3500× g and the miRCURY Cell/Urine/CSF 1500× g methods resulted as equivalent alternative procedures to UC for isolating exosomes/sEVs from semen for nanoparticle characteristics and quality of RNA contained in vesicles. Additionally, the expression profile of the altered semen sEV-miRNAs in PCa varies depending on the EV isolation method applied. This is possibly due to different extraction techniques yielding different proportions of sEV subtypes. This is evidence that the exosome-EV isolation method has a significant impact on the analysis of the miRNAs contained within, with important consequences for their use as clinical biomarkers. Therefore, miRNA analysis results for EVs cannot be directly extrapolated between different EV isolation methods until clear markers for delineation between microvesicles and exosomes are established. However, EV extraction methodology affects combined models (semen exosome miRNA signatures plus blood Prostate specific antigen (PSA) concentration for PCa diagnosis) less; specifically our previously described (miR-142-3p + miR-142-5p + miR-223-3p + PSA) model functions as molecular marker from EVs from any of the three isolation methods, potentially improving the efficiency of PSA PCa diagnosis.

An Update on Isolation Methods for Proteomic Studies of Extracellular Vesicles in Biofluids.

Extracellular vesicles (EVs) are lipid bilayer enclosed particles which present in almost all types of biofluids and contain specific proteins, lipids, and RNA. Increasing evidence has demonstrated the tremendous clinical potential of EVs as diagnostic and therapeutic tools, especially in biofluids, since they can be detected without invasive surgery. With the advanced mass spectrometry (MS), it is possible to decipher the protein content of EVs under different physiological and pathological conditions. Therefore, MS-based EV proteomic studies have grown rapidly in the past decade for biomarker discovery. This review focuses on the studies that isolate EVs from different biofluids and contain MS-based proteomic analysis. Literature published in the past decade (2009.1-2019.7) were selected and summarized with emphasis on isolation methods of EVs and MS analysis strategies, with the aim to give an overview of MS-based EV proteomic studies and provide a reference for future research.