Comparison of isolation methods of exosomes and exosomal RNA from cell culture medium and serum.
01 Sep 2017-International Journal of Molecular Medicine (Spandidos Publications)-Vol. 40, Iss: 3, pp 834-844
TL;DR: It was found that combinations of the TEI and TER methods resulted in high extraction efficiency and purity of small RNA obtained using CCM, and different methods may introduce variations in the concentration, purity and size of exosomes and exoRNA.
Abstract: Exosomes are cell-derived vesicles and are abundant in biological fluids; they contain RNA molecules which may serve as potential diagnostic biomarkers in 'precision medicine'. To promote the clinical application of exosomal RNA (exoRNA), many isolation methods must be compared and validated. Exosomes in cell culture medium (CCM) and serum may be isolated using ultracentrifugation (UC), ExoQuick or Total Exosome Isolation Reagent (TEI), and exoRNA may be extracted using TRIzol-LS, SeraMir, Total Exosome RNA Isolation (TER), HiPure Liquid RNA/miRNA kit (HLR), miRNeasy or exoRNeasy. ExoRNA was assessed using NanoDrop, Bioanalyzer 2100, quantitative polymerase chain reaction and high-throughput sequencing. UC showed the lowest recovery of particles, but the highest protein purity for exosome isolation. For isolation of exoRNA, we found that combinations of the TEI and TER methods resulted in high extraction efficiency and purity of small RNA obtained using CCM. High yield and a narrow size distribution pattern of small RNA were shown in exoRNA isolated by exoRNeasy from serum. In RNA profile analysis, the small RNA constituent ratio, miRNA content and amount varied as a result of methodological differences. This study showed that different methods may introduce variations in the concentration, purity and size of exosomes and exoRNA. Herein we discuss the advantages and disadvantages of each method and their application to different materials, therefore providing a reference according to research design.
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TL;DR: The droplet-based single-exosome-counting enzyme-linked immunoassay (droplet digital ExoELISA) approach enables absolute counting of cancer-specific exosomes to achieve unprecedented accuracy and may have the potential for early diagnosis of cancer and accelerate the discovery of cancer exosomal biomarkers for clinical diagnosis.
Abstract: Exosomes shed by tumor cells have been recognized as promising biomarkers for cancer diagnostics due to their unique composition and functions. Quantification of low concentrations of specific exosomes present in very small volumes of clinical samples may be used for noninvasive cancer diagnosis and prognosis. We developed an immunosorbent assay for digital qualification of target exosomes using droplet microfluidics. The exosomes were immobilized on magnetic microbeads through sandwich ELISA complexes tagged with an enzymatic reporter that produces a fluorescent signal. The constructed beads were further isolated and encapsulated into a sufficient number of droplets to ensure only a single bead was encapsulated in a droplet. Our droplet-based single-exosome-counting enzyme-linked immunoassay (droplet digital ExoELISA) approach enables absolute counting of cancer-specific exosomes to achieve unprecedented accuracy. We were able to achieve a limit of detection (LOD) down to 10 enzyme-labeled exosome comple...
266 citations
TL;DR: Focusing on product development and technological improvements will enable acoustofluidic separation to find real-world applications, the researchers conclude.
Abstract: Acoustofluidics, the integration of acoustics and microfluidics, is a rapidly growing research field that is addressing challenges in biology, medicine, chemistry, engineering, and physics. In particular, acoustofluidic separation of biological targets from complex fluids has proven to be a powerful tool due to the label-free, biocompatible, and contact-free nature of the technology. By carefully designing and tuning the applied acoustic field, cells and other bioparticles can be isolated with high yield, purity, and biocompatibility. Recent advances in acoustofluidics, such as the development of automated, point-of-care devices for isolating sub-micron bioparticles, address many of the limitations of conventional separation tools. More importantly, advances in the research lab are quickly being adopted to solve clinical problems. In this review article, we discuss working principles of acoustofluidic separation, compare different approaches of acoustofluidic separation, and provide a synopsis of how it is being applied in both traditional applications, such as blood component separation, cell washing, and fluorescence activated cell sorting, as well as emerging applications, including circulating tumor cell and exosome isolation.
240 citations
TL;DR: Focusing on advantages and limitations of methods for exosome isolation and characterization, approaches are proposed to facilitate further progress in the development of exosomes as biomarkers in human disease.
Abstract: A growing body of evidence emphasizes the important role exosomes in different physiological and pathological conditions. Exosomes, virus-size extracellular vesicles (EVs), carry a complex molecular cargo, which is actively processed in the endocytic compartment of parental cells. Exosomes carry and deliver this cargo to recipient cells, serving as an intercellular communication system. The methods for recovery of exosomes from supernatants of cell lines or body fluids are not uniformly established. Yet, studies of the quality and quantity of exosome cargos underlie the concept of “liquid biopsy.” Exosomes are emerging as a potentially useful diagnostic tool and a predictor of disease progression, response to therapy and overall survival. Although many novel approaches to exosome isolation and analysis of their cargos have been introduced, the role of exosomes as diagnostic or prognostic biomarkers of disease remains unconfirmed. This review considers existing challenges to exosome validation as disease biomarkers. Focusing on advantages and limitations of methods for exosome isolation and characterization, approaches are proposed to facilitate further progress in the development of exosomes as biomarkers in human disease.
220 citations
Cites background from "Comparison of isolation methods of ..."
...Although 588 common miRNAs were found in all three exosome samples, each sample isolated by the different method contained approximately 200 miRNAs, which were unique for each exosome preparation [18]....
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TL;DR: Comparatively, low protein content and cryo-electron microscopy analysis show that SEC removes most of the overabundant soluble plasma proteins, while being more user friendly and less time-consuming than gradient-based EV isolation.
Abstract: 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.
195 citations
University of California, San Diego1, Harvard University2, Universiti Putra Malaysia3, University of California, San Francisco4, University of Texas MD Anderson Cancer Center5, Thomas Jefferson University6, University of Michigan7, Brigham and Women's Hospital8, Mayo Clinic9, Oregon Health & Science University10, Pacific Northwest Diabetes Research Institute11
TL;DR: An interactive web-based application (miRDaR) was developed to help investigators select the optimal exRNA isolation method for their studies and returns a ranked list of ex RNA isolation methods prioritized by complexity, expression level, and reproducibility.
189 citations
Cites methods from "Comparison of isolation methods of ..."
...Although standard RNA-seq methods perform poorly on exRNA samples, small RNA-seq has been successfully used to profile exRNA isolated using several methods from different biofluids (Burgos et al., 2013; Cheng et al., 2014; Freedman et al., 2016; Li et al., 2015, 2018; Shah et al., 2017; Tang et al., 2017; Williams et al., 2013; Yeri et al., 2017)....
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References
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TL;DR: This unit describes different approaches for exosome purification from various sources, and discusses methods to evaluate the purity and homogeneity of the purified exosomes preparations.
Abstract: Exosomes are small membrane vesicles found in cell culture supernatants and in different biological fluids. Exosomes form in a particular population of endosomes, called multivesicular bodies (MVBs), by inward budding into the lumen of the compartment. Upon fusion of MVBs with the plasma membrane, these internal vesicles are secreted. Exosomes possess a defined set of membrane and cytosolic proteins. The physiological function of exosomes is still a matter of debate, but increasing results in various experimental systems suggest their involvement in multiple biological processes. Because both cell-culture supernatants and biological fluids contain different types of lipid membranes, it is critical to perform high-quality exosome purification. This unit describes different approaches for exosome purification from various sources, and discusses methods to evaluate the purity and homogeneity of the purified exosome preparations.
4,492 citations
"Comparison of isolation methods of ..." refers methods in this paper
...The UC method was used as previously described (5)....
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...The commonly used protocol for isolation of exosomes is ultracentrifugation (UC); the final step of which is centrifugation at 100,000 x g at least for 70 min to pellet the small vesicles that correspond to exosomes (5)....
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TL;DR: GPC1+ crExos may serve as a potential non-invasive diagnostic and screening tool to detect early stages of pancreatic cancer to facilitate possible curative surgical therapy.
Abstract: Exosomes are lipid-bilayer-enclosed extracellular vesicles that contain proteins and nucleic acids. They are secreted by all cells and circulate in the blood. Specific detection and isolation of cancer-cell-derived exosomes in the circulation is currently lacking. Using mass spectrometry analyses, we identify a cell surface proteoglycan, glypican-1 (GPC1), specifically enriched on cancer-cell-derived exosomes. GPC1(+) circulating exosomes (crExos) were monitored and isolated using flow cytometry from the serum of patients and mice with cancer. GPC1(+) crExos were detected in the serum of patients with pancreatic cancer with absolute specificity and sensitivity, distinguishing healthy subjects and patients with a benign pancreatic disease from patients with early- and late-stage pancreatic cancer. Levels of GPC1(+) crExos correlate with tumour burden and the survival of pre- and post-surgical patients. GPC1(+) crExos from patients and from mice with spontaneous pancreatic tumours carry specific KRAS mutations, and reliably detect pancreatic intraepithelial lesions in mice despite negative signals by magnetic resonance imaging. GPC1(+) crExos may serve as a potential non-invasive diagnostic and screening tool to detect early stages of pancreatic cancer to facilitate possible curative surgical therapy.
2,102 citations
"Comparison of isolation methods of ..." refers background in this paper
...They may therefore serve as biomarkers for the development of superior, sensitive and minimally invasive diagnostic alternatives in ‘precision medicine’ (2,3)....
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University of Gothenburg1, University of Melbourne2, University of California, San Diego3, Semmelweis University4, Cedars-Sinai Medical Center5, University of Oxford6, Pohang University of Science and Technology7, Agency for Science, Technology and Research8, La Trobe University9, Brown University10, Icahn School of Medicine at Mount Sinai11, Hiroshima University12, Utrecht University13, Johns Hopkins University14, French Institute of Health and Medical Research15
TL;DR: The International Society for Extracellular Vesicles (ISEV) provides researchers with a minimal set of biochemical, biophysical and functional standards that should be used to attribute any specific biological cargo or functions to EVs.
Abstract: Secreted membrane-enclosed vesicles, collectively called extracellular vesicles (EVs), which include exosomes, ectosomes, microvesicles, microparticles, apoptotic bodies and other EV subsets, encompass a very rapidly growing scientific field in biology and medicine. Importantly, it is currently technically challenging to obtain a totally pure EV fraction free from non-vesicular components for functional studies, and therefore there is a need to establish guidelines for analyses of these vesicles and reporting of scientific studies on EV biology. Here, the International Society for Extracellular Vesicles (ISEV) provides researchers with a minimal set of biochemical, biophysical and functional standards that should be used to attribute any specific biological cargo or functions to EVs.
2,028 citations
"Comparison of isolation methods of ..." refers background in this paper
...The relative proportions of different proteins (including exosome markers) seem to vary in the different types of EV (21)....
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...As Lötvall et al recommend (21), the composition of EVs should ideally be compared with that of the secreting cells....
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...to the minimal requirements for EV studies suggested by Lötvall et al (21), investigators should report the amount of several (three or more) ‘exosome-enriched’ and non-EV protein markers in any EV preparation....
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TL;DR: The novel role of exosomes highlights a new perspective into intercellular mediation of tissue injury and repair, and engenders novel approaches to the development of biologics for tissue repair.
1,816 citations
"Comparison of isolation methods of ..." refers methods in this paper
...In addition, sucrose density gradients, ultrafiltration (6), high performance liquid chromatography-based protocols (7) and immunoaffinitycapture methods (8), singly or combined with the application of UC, can provide high enrichment and purity of exosomes (9)....
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TL;DR: The small vesicles shed from the surface of many cells upon stimulation, considered for a long time to be artefacts, are now recognized as specific structures that are distinct from the exosomes released upon exocytosis of multivesicular bodies.
1,673 citations
"Comparison of isolation methods of ..." refers background in this paper
...Different types of vesicles have been identified, such as exosomes, microvesicles, apoptotic bodies, secreted proteins and retrovirus-like vesicles (1,4)....
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