Study Of Extracellular Vesicles Research Articles

Efficient EVs separation and detection by an alumina-nanochannel-array-membrane integrated microfluidic chip and an antibody barcode biochip

BackgroundSmall endosome-derived lipid nanovesicles (30–200 nm) are actively secreted by living cells and serve as pivotal biomarkers for early cancer diagnosis. However, the study of extracellular vesicles (EVs) requires isolation and purification from various body fluids. Although traditional EVs isolation and detection technologies are mature, they usually require large amount of sample, consumes long-time, and have relatively low-throughput. How to efficiently isolate, purify and detect these structurally specific EVs from body fluids with high-throughput remains a great challenge in in vitro diagnostics and clinical research. ResultsHerein, we suggest a nanosized microfluidic device for efficient and economical EVs filtration based on an alumina nanochannel array membrane. We evaluated the filtration device performance of alumina membranes with different diameters and found that an optimized chamber array with a hydrophilic-treated channel diameter of 90 nm could realize a filtration efficiency of up to 82% without any assistance from chemical or physical separation methods. Importantly, by integrating meticulously designed multichannel microfluidic biochips, EVs can be captured in-situ and monitored by antibody barcode biochip. The proposed filtration chip together with the high-throughput detection chip were capable of filtration of a few tens of μL samples and recognition of different phonotypes. The practical filtration and detection of EVs from clinical samples demonstrated the high performance of the device. SignificantOverall, this work provides a cost-effective, highly efficient and automated EVs filtration chip and detection dual-function integrated chip platform, which can directly separate EVs from serum or cerebrospinal fluid with an efficiency of 82% and conduct in-situ detection. This small fluidic device can provide a powerful tool for highly efficient identifying and analyzing EVs, presenting great application potential in clinical detection.

Isolation of Hepatic and Adipose-Tissue-Derived Extracellular Vesicles Using Density Gradient Separation and Size Exclusion Chromatography.

In recent years, the study of extracellular vesicles (EVs) in the context of various diseases has dramatically increased due to their diagnostic and therapeutic potential. Typically, EVs are isolated in vitro from the cell culture of primary cells or cell lines or from bodily fluids. However, these cell culture methods do not represent the whole complexity of an in vivo microenvironment, and bodily fluids contain a high heterogeneous population of vesicles since they originate from different tissues. This highlights the need to develop new methods to isolate EVs directly from tissue samples. In the present study, we established a protocol for isolating EVs from hepatic and adipose tissue of mice, using a combination of ultracentrifugation and iodixanol-sucrose density gradient separation. EV isolation was confirmed with EV protein marker enrichment in Western blot assays, total protein quantification, and transmission electron microscopy. Regarding the liver tissue, we additionally implemented size exclusion chromatography (SEC) to further increase the purity grade of the EVs. The successful isolation of EVs from tissue samples will allow us to uncover a more precise molecular composition and functions, as well as their role in intercellular communication in an in vivo microenvironment.

Extracellular vesicles and nanoparticles: emerging complexities.

The study of extracellular vesicles (EVs) and nanoparticles (NPs) is rapidly expanding because recent discoveries have revealed a much greater complexity and diversity than was appreciated only a few years ago. New types of EVs and NPs have recently been described. Proteins and nucleic acids previously thought to be packaged in exosomes appear to be more enriched in different types of EVs and in two recently identified amembranous NPs, exomeres and supermeres. Thus, our understanding of the cell biology and intercellular communication facilitated by the release of EVs and NPs is in a state of flux. In this review, we describe the different types of EVs and NPs, highlight recent advances, and present major outstanding questions.

Application of annotation-agnostic RNA sequencing data analysis tools for biomarker discovery in liquid biopsy.

RNA sequencing analysis is an important field in the study of extracellular vesicles (EVs), as these particles contain a variety of RNA species that may have diagnostic, prognostic and predictive value. Many of the bioinformatics tools currently used to analyze EV cargo rely on third-party annotations. Recently, analysis of unannotated expressed RNAs has become of interest, since these may provide complementary information to traditional annotated biomarkers or may help refine biological signatures used in machine learning by including unknown regions. Here we perform a comparative analysis of annotation-free and classical read-summarization tools for the analysis of RNA sequencing data generated for EVs isolated from persons with amyotrophic lateral sclerosis (ALS) and healthy donors. Differential expression analysis and digital-droplet PCR validation of unannotated RNAs also confirmed their existence and demonstrates the usefulness of including such potential biomarkers in transcriptome analysis. We show that find-then-annotate methods perform similarly to standard tools for the analysis of known features, and can also identify unannotated expressed RNAs, two of which were validated as overexpressed in ALS samples. We demonstrate that these tools can therefore be used for a stand-alone analysis or easily integrated into current workflows and may be useful for re-analysis as annotations can be integrated post hoc.

Extracellular Vesicles as "Very Important Particles" (VIPs) in Aging.

In recent decades, extracellular vesicles have been recognized as "very important particles" (VIPs) associated with aging and age-related disease. During the 1980s, researchers discovered that these vesicle particles released by cells were not debris but signaling molecules carrying cargoes that play key roles in physiological processes and physiopathological modulation. Following the International Society for Extracellular Vesicles (ISEV) recommendation, different vesicle particles (e.g., exosomes, microvesicles, oncosomes) have been named globally extracellular vesicles. These vesicles are essential to maintain body homeostasis owing to their essential and evolutionarily conserved role in cellular communication and interaction with different tissues. Furthermore, recent studies have shown the role of extracellular vesicles in aging and age-associated diseases. This review summarizes the advances in the study of extracellular vesicles, mainly focusing on recently refined methods for their isolation and characterization. In addition, the role of extracellular vesicles in cell signaling and maintenance of homeostasis, as well as their usefulness as new biomarkers and therapeutic agents in aging and age-associated diseases, has also been highlighted.

Abstract IA027: Recent advances in the study of extracellular vesicles and nanoparticles in colorectal cancer

Abstract Extracellular vesicles (EVs) and nanoparticles (NPs) are increasingly recognized as important mediators of intercellular communication with implications for the diagnosis, monitoring and possibly treatment of colorectal cancer. We recently reviewed this topic in Gastroenterology. EVs are lipid bilayer-enclosed vesicles that can be classified as small (s) some of which are tetraspanin-containing, endosomally-derived exosomes (5-120 nm) and large that include plasma membrane-budding microvescles (150-800 nm). Nanoparticles are more recently discovered smaller amembranous nanoparticles that include exomeres (30-50 nm) and supermeres (25-35 nm). We have performed a comprehensive proteomic and RNA analysis of sEVs, exomeres and supermeres. Highlights from this analysis will be presented with a focus on their clinical relevance and the merits of parsing versus pooling biofluid fractions. Citation Format: Robert J. Coffey. Recent advances in the study of extracellular vesicles and nanoparticles in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr IA027.

Tackling the effects of extracellular vesicles in fibrosis

Fibrosis is a physiological process of tissue repair that turns into pathological when becomes chronic, damaging the functional structure of the tissue. In this review we outline the current status of extracellular vesicles as modulators of the fibrotic process at different levels. In adipose tissue, extracellular vesicles mediate the intercellular communication not only between adipocytes, but also between adipocytes and other cells of the stromal vascular fraction. Thus, they could be altering essential processes for the functionality of adipose tissue, such as adipocyte hypertrophy/hyperplasia, tissue plasticity, adipogenesis and/or inflammation, and ultimately trigger fibrosis. This process is particularly important in obesity, and may eventually, influence the development of obesity-associated alterations. In this regard, obesity is now recognized as an independent risk factor for the development of chronic kidney disease, although the role of extracellular vesicles in this connection has not been explored so far. Nonetheless, the role of extracellular vesicles in the onset and progression of renal fibrosis has been highlighted due to the critical role of fibrosis as a common feature of kidney diseases. In fact, the content of extracellular vesicles disturbs cellular signaling cascades involved in fibrosis in virtually all types of renal cells. What is certain is that the study of extracellular vesicles is complex, as their isolation and manipulation is still difficult to reproduce, which complicates the overview of their physiopathological effects. Nevertheless, new strategies have been developed to exploit the potential of extracellular vesicles and their cargo, both as biomarkers and as therapeutic tools to prevent the progression of fibrosis towards an irreversible event.

The Importance of Small Extracellular Vesicles in the Cerebral Metastatic Process.

Brain metastases represent more than 50% of all cerebral tumors encountered in clinical practice. Recently, there has been increased interest in the study of extracellular vesicles, and the knowledge about exosomes is constantly expanding. Exosomes are drivers for organotropic metastatic spread, playing important roles in the brain metastatic process by increasing the permeability of the blood–brain barrier and preparing the premetastatic niche. The promising results of the latest experimental studies raise the possibility of one day using exosomes for liquid biopsies or as drug carriers, contributing to early diagnosis and improving the efficacy of chemotherapy in patients with brain metastases. In this review, we attempted to summarize the latest knowledge about the role of exosomes in the brain metastatic process and future research directions for the use of exosomes in patients suffering from brain metastatic disease.

Challenges and opportunities in the study of extracellular vesicles: Global institutional context and national state of the art

ResumenEn la última década se ha incrementado el número de estudios y publicaciones sobre las vesículas extracelulares y los exosomas. En Colombia, ha habido interés y avances en su estudio, lo que se evidencia en el aumento de publicaciones y proyectos de investigación. Sin embargo, este es un campo de investigación aún en desarrollo, con desafíos analíticos y limitaciones técnicas, por lo cual, en el planteamiento de los proyectos de investigación y desarrollo, es necesario considerar cuál es el estado del campo científico a nivel mundial en cuanto a la nomenclatura y la clasificación de las vesículas extracelulares, las técnicas, recursos, requisitos y especificaciones de calidad y las instituciones que regulan el campo. La respuesta a esta pregunta permitirá desarrollar estudios que cumplan con los estándares internacionales, y las exigencias y recomendaciones institucionales. Sin embargo, la información científica disponible se encuentra dispersa y no todos los aspectos son tratados a cabalidad.En este actualización se condensa la información disponible y se presentan los términos oficiales para denominar las vesículas extracelulares y la nomenclatura aceptada actualmente, así como la evolución del campo, la homogenización de los parámetros experimentales, el establecimiento de autoridades científicas, instituciones y recursos, y las recomendaciones que se han generado a nivel mundial para el desarrollo de investigaciones en vesículas extracelulares, incluidos su aislamiento, caracterización y estudio funcional. Por último, se analiza el contexto nacional de una forma crítica, teniendo en cuenta las fortalezas institucionales, los errores usualmente cometidos, y las técnicas y tecnologías analíticas disponibles.

Circulating extracellular vesicles: friends and foes in neurodegeneration.

Extracellular vesicles have been identified as pivotal mediators of intercellular communication with critical roles in physiological and pathological conditions. Via this route, several molecules (e.g., nucleic acids, proteins, metabolites) can be transferred to proximal and distant targets to convey specific information. Extracellular vesicle-associated cargo molecules have been proposed as markers of several disease conditions for their potential of tracking down the generating cell. Indeed, circulating extracellular vesicles may represent biomarkers of dysfunctional cellular quality control systems especially in conditions characterized by the accrual of intracellular misfolded proteins. Furthermore, the identification of extracellular vesicles as tools for the delivery of nucleic acids or other cargo molecules to diseased tissues makes these circulating shuttles possible targets for therapeutic development. The increasing interest in the study of extracellular vesicles as biomarkers resides mainly in the fact that the identification of peripheral levels of extracellular vesicle-associated proteins might reflect molecular events occurring in hardly accessible tissues, such as the brain, thereby serving as a “brain liquid biopsy”. The exploitation of extracellular vesicles for diagnostic and therapeutic purposed might offer unprecedented opportunities to develop personalized approaches. Here, we discuss the bright and dark sides of extracellular vesicles in the setting of two main neurodegenerative diseases (i.e., Parkinson’s and Alzheimer’s diseases). A special focus will be placed on the possibility of using extracellular vesicles as biomarkers for the two conditions to enable disease tracking and treatment monitoring.

Extracellular Vesicles and Pancreatic Cancer: Insights on the Roles of miRNA, lncRNA, and Protein Cargos in Cancer Progression.

Pancreatic cancer (PC) is among the most devastating digestive tract cancers worldwide. This cancer is characterized by poor diagnostic detection, lack of therapy, and difficulty in predicting tumorigenesis progression. Although mutations of key oncogenes and oncosuppressor involved in tumor growth and in immunosurveillance escape are known, the underlying mechanisms that orchestrate PC initiation and progression are poorly understood or still under debate. In recent years, the attention of many researchers has been concentrated on the role of extracellular vesicles and of a particular subset of extracellular vesicles, known as exosomes. Literature data report that these nanovesicles are able to deliver their cargos to recipient cells playing key roles in the pathogenesis and progression of many pancreatic precancerous conditions. In this review, we have summarized and discussed principal cargos of extracellular vesicles characterized in PC, such as miRNAs, lncRNAs, and several proteins, to offer a systematic overview of their function in PC progression. The study of extracellular vesicles is allowing to understand that investigation of their secretion and analysis of their content might represent a new and potential diagnostic and prognostic tools for PC.

Exploiting Microfluidics for Extracellular Vesicle Isolation and Characterization: Potential Use for Standardized Embryo Quality Assessment.

Recent decades have seen a growing interest in the study of extracellular vesicles (EVs), driven by their role in cellular communication, and potential as biomarkers of health and disease. Although it is known that embryos secrete EVs, studies on the importance of embryonic EVs are still very limited. This limitation is due mainly to small sample volumes, with low EV concentrations available for analysis, and to laborious, costly and time-consuming procedures for isolating and evaluating EVs. In this respect, microfluidics technologies represent a promising avenue for optimizing the isolation and characterization of embryonic EVs. Despite significant improvements in microfluidics for EV isolation and characterization, the use of EVs as markers of embryo quality has been held back by two key challenges: (1) the lack of specific biomarkers of embryo quality, and (2) the limited number of studies evaluating the content of embryonic EVs across embryos with varying developmental competence. Our core aim in this review is to identify the critical challenges of EV isolation and to provide seeds for future studies to implement the profiling of embryonic EVs as a diagnostic test for embryo selection. We first summarize the conventional methods for isolating EVs and contrast these with the most promising microfluidics methods. We then discuss current knowledge of embryonic EVs and their potential role as biomarkers of embryo quality. Finally, we identify key ways in which microfluidics technologies could allow researchers to overcome the challenges of embryonic EV isolation and be used as a fast, user-friendly tool for non-invasive embryo selection.

Extracellular vesicles and pancreatitis: mechanisms, status and perspectives.

Comprehensive reviews and large population-based cohort studies have played an important role in the diagnosis and treatment of pancreatitis and its sequelae. The incidence and mortality of pancreatitis have been reduced significantly due to substantial advancements in the pathophysiological mechanisms and clinically effective treatments. The study of extracellular vesicles (EVs) has the potential to identify cell-to-cell communication in diseases such as pancreatitis. Exosomes are a subset of EVs with an average diameter of 50~150 nm. Their diverse and unique constituents include nucleic acids, proteins, and lipids, which can be transferred to trigger phenotypic changes of recipient cells. In recent years, many reports have indicated the role of EVs in pancreatitis, including acute pancreatitis, chronic pancreatitis and autoimmune pancreatitis, suggesting their potential influence on the development and progression of pancreatitis. Plasma exosomes of acute pancreatitis can effectively reach the alveolar cavity and activate alveolar macrophages to cause acute lung injury. Furthermore, upregulated exosomal miRNAs can be used as biomarkers for acute pancreatitis. Here, we summarized the current understanding of EVs in pancreatitis with an emphasis on their biological roles and their potential use as diagnostic biomarkers and therapeutic agents for this disease.

LncRNA Quantification from Extracellular Vesicles Isolated from Blood Plasma or Conditioned Media.

During the last years, the study of extracellular vesicles (EVs) and its cargo has gained interest in the scientific media. EVs have been found in all biofluids and it is postulated that all cells are capable to secrete a wide variety of these vesicles, which play a key role in different cell-to-cell communication processes as well as in the microenvironment modulation. In the EV cargo, DNA, protein, and RNA molecules can be found, including long noncoding RNAs (lncRNAs). Several authors consider the study of EV lncRNAs an ideal source of biomarkers due to the easy sampling of EVs in different biofluids and the high specificity of the lncRNA expression pattern.In the present chapter, a detailed explanation of the EV isolation workflow followed by RNA isolation and lncRNA gene expression study is provided for two sample sources: blood plasma and cell culture conditioned media. EVs from both plasma samples and cell cultured media are isolated using sequential ultracentrifugation method (UC), which has been reported as one of the best methods available to date in terms of purity. UC is followed by RNA extraction based on the combination of phenol/guanidine-based lysis of samples with silica-membrane-based purification of total RNA. LncRNA quantification is performed by qRT-PCR. This chapter includes detailed discussion on lncRNA quantification using hydrolysis probes, recommended housekeeping genes and evaluation of methods for comparing lncRNA levels between EVs and its parental cells. In summary, we describe here the main steps for a successful isolation of the EVs-lncRNA cargo, paying attention to how overcome the different challenges found in the experimental procedure and in the data analysis of lncRNA expression from this source.

DNA in extracellular vesicles: biological and clinical aspects.

The study of extracellular vesicles (EVs), especially in the liquid biopsy field, has rapidly evolved in recent years. However, most EV studies have focused on RNA or protein content and DNA in EVs (EV-DNA) has largely been unnoticed. In this review, we compile current evidence regarding EV-DNA and provide an extensive discussion on EV-DNA biology. We look into EV-DNA biogenesis and mechanisms of DNA loading into EVs, as well as describe the particularly significant function of DNA-carrying EVs in the maintenance of cellular homeostasis, intracellular communication, and immune response modulation. We also examine the current role of EV-DNA in the clinical setting, specifically in cancer, infections, pregnancy, and prenatal diagnosis.

Platelet removal by single-step centrifugation

The study of extracellular vesicles (EVs) in plasma requires removal of cells including platelets. At present, a two-step centrifugation protocol is recommended and commonly used. A simpler protocol that is less operator dependent is likely to improve the quality of plasma samples collected for EV research. The objective of this study is to develop an easy, fast and clinically applicable centrifugation protocol to produce essentially platelet-free plasma with a high yield for EV research. We compared the two-step centrifugation protocol to a single-step protocol at 5,000 g for 20 minutes. The removal of platelets was computationally predicted and experimentally validated. Flow cytometry was used to detect residual platelets and platelet-derived (CD61+) EVs. The single-step protocol at 5,000 g (i) is less laborious and approximately ten minutes faster, (ii) removes platelets as effective as the two-step centrifugation protocol, and (iii) has a ~ 10% higher plasma yield, whereas (iv) the recovery of platelet-derived EVs is comparable. For future research on plasma EVs we recommend the newly developed, easy and fast single-step protocol for preparation of platelet-free plasma for research on plasma biomarkers including EVs.

Exosomal Long NonCoding Rnas as Cancer Biomarkers and Therapeutic Targets

Extensive study of extracellular vesicles began about ten years ago. Exosomes are extracellular membrane vesicles 30–100 nm in diameter secreted by various types of cells and present in most biological fluids. For a long time they were considered non-functional cellular components. However, it has been proven that they serve as a means of intercellular exchange of information. They can move bioactive molecules such as proteins, lipids, RNA, and DNA. Several studies have shown that their contents, including proteins and non-coding nucleic acids, may be of particular interest as biomarkers of diseases. The most promising of all these molecules are non-coding RNAs (ncRNAs), including microRNAs and long non-coding RNAs (lncRNAs). LncRNAs are a large group of non-coding RNAs (ncRNAs) longer than 200 nucleotides. As regulatory factors lncRNAs play an important role in complex cellular processes, such as apoptosis, growth, differentiation, proliferation, etc. Despite many advances in diagnosis and treatment (surgery, radiation therapy, chemotherapy), cancer remains one of the most important public healthcare problems worldwide. Every day brings a better understanding of the role of exosomes in the development of cancer and metastases. Liquid biopsy has been developed as a method for the detection of cancer at an early stage. This is a series of minimally invasive tests of bodily fluids offering the advantage of real-time tracking of the tumour development. In fact, circulating exosomal lncRNAs have been found to be closely linked to processes of oncogenesis, metastasis and treatment. In this paper we review current studies into the functional role of exosomal lncRNAs in cancer and discuss their potential clinical use as diagnostic biomarkers and therapeutic targets for cancer.

Potential Mechanistic Links between Cancer Treatment and Alzheimer’s Disease: An Insight from the Study of Extracellular Vesicles

In the present work, we designed a microfluidic electrochemical immunosensor with enough sensibility and precision to quantify epithermal growth factor receptor (EGFR) in plasma extracellular vesicles (EVs) of plasma from breast cancer patients. The sensor employs SiNPs coated with chitosan (SiNPs-CH) as reaction's platform, based on the covalently immobilization of monoclonal anti-EGFR on SiNPs-CH retained in the central channel (CC) of the microfluidic device. The synthetized SiNPs-CH were characterized by UV–visible spectroscopy (UV–visible), energy dispersive spectrometry (EDS), Nanoparticle Tracking Analysis (NTA) and transmission electron microscopy (TEM). EGFR was quantified by a direct sandwich immunoassay measuring through a horseradish peroxidase (HRP)-conjugated anti-EGFR. The enzymatic product (benzoquinone) was detected by reduction at − 100 mV on a sputtering gold electrode. The measured current was directly proportional to the level of EGFR in human serum samples. The linear range was from 0 ng mL−1 to 50 ng mL−1. The detection limit was 1.37 pg mL−1, and the within- and between-assay coefficients of variation were below 6.25%. Finally, plasma samples from 30 early breast cancer patients and 20 healthy donor were analyzed by the novel method. EGFR levels in EVs (EVs-EGFR) were significantly higher than in the healthy control group (p = 0.002) and also, more sensitivity and specificity than normal serum markers like CEA and CA15.3 has been observed. EVs-EGFR concentration correlates with EGFR tumor status (p = 0.0003) as well as it correlate with the tumor size and pathological grade. To conclude, plasma EVs are suitable for proteomic characterization of cancer disease, as long as the employed method has sufficient sensitivity, like the case of immune-electrochemical nanosensors with incremented reaction surface.

Modern Techniques for the Isolation of Extracellular Vesicles and Viruses.

Extracellular signaling is pivotal to maintain organismal homeostasis. A quickly emerging field of interest within extracellular signaling is the study of extracellular vesicles (EV), which act as messaging vehicles for nucleic acids, proteins, metabolites, lipids, etc. from donor cells to recipient cells. This transfer of biologically active material within a vesicular body is similar to the infection of a cell through a virus particle, which transfers genetic material from one cell to another to preserve an infection state, and viruses are known to modulate EV. Although considerable heterogeneity exists within EV and viruses, this review focuses on those that are small (< 200nm in diameter) and of relatively low density (< 1.3g/mL). A multitude of isolation methods for EV and virus particles exist. In this review, we present an update on methods for their isolation, purification, and phenotypic characterization. We hope that the information we provide will be of use to basic science and clinical investigators, as well as biotechnologists in this emerging field. Graphical Abstract.

FCMPASS Software Aids Extracellular Vesicle Light Scatter Standardization.

The study of extracellular vesicles (EVs) is a rapidly growing field due to their great potential in many areas of clinical medicine including diagnostics, prognostics, theranostics, and therapeutics. Flow cytometry is currently one of the most popular methods of analyzing EVs due to it being a high-throughput, multiparametric technique, that is readily available in the majority of research labs. Despite its wide use, few commercial flow cytometers are designed specifically for the detection of EVs. Many flow cytometers used for EV analysis are working at their detection limits and are unable to detect the majority of EVs. Currently, very little standardization exists for EV flow cytometry, which is an issue because flow cytometers vary considerably in the way they collect scattered or fluorescent light from particles being interrogated. This makes published research hard to interpret, compare, and in some cases, impossible to reproduce. Here we demonstrate a method of flow cytometer light scatter standardization, utilizing flow cytometer postacquisition analysis software (FCMPASS ). FCMPASS is built upon Mie theory and enables the approximation of flow cytometer geometric parameters either by analyzing beads of known diameter and refractive index or by inputting the collection angle if known. The software is then able to create a scatter-diameter curve and scatter-refractive index curve that enables researchers to convert scattering data and instrument sensitivity into standardized units. Furthermore, with the correct controls, light scatter data can be converted to diameter distributions or refractive index distributions. FCMPASS therefore offers a freely available and ergonomic method of standardizing and further extending EV characterization using flow cytometry.