Isolation and characterization of exosomes from cell culture supernatants and biological fluids.
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.
- SourceAvailable from: Guoqiang Sun[Show abstract] [Hide abstract]
ABSTRACT: Reprogrammed glucose metabolism as a result of increased glycolysis and glucose uptake is a hallmark of cancer. Here we show that cancer cells can suppress glucose uptake by non-tumour cells in the premetastatic niche, by secreting vesicles that carry high levels of the miR-122 microRNA. High miR-122 levels in the circulation have been associated with metastasis in breast cancer patients, and we show that cancer-cell-secreted miR-122 facilitates metastasis by increasing nutrient availability in the premetastatic niche. Mechanistically, cancer-cell-derived miR-122 suppresses glucose uptake by niche cells in vitro and in vivo by downregulating the glycolytic enzyme pyruvate kinase. In vivo inhibition of miR-122 restores glucose uptake in distant organs, including brain and lungs, and decreases the incidence of metastasis. These results demonstrate that, by modifying glucose utilization by recipient premetastatic niche cells, cancer-derived extracellular miR-122 is able to reprogram systemic energy metabolism to facilitate disease progression.Nature cell biology. 01/2015;
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ABSTRACT: Objective Whether circulating microvesicles convey bioactive signals in neurodegenerative diseases remains currently unknown. In this study, we investigated the biochemical composition and biological function of exosomes isolated from sera of patients with Parkinson's disease (PD).Methods Proteomic analysis was performed on microvesicle preparations from grouped samples of patients with genetic and sporadic forms of PD, amyotrophic lateral sclerosis, and healthy subjects. Nanoparticle-tracking analysis was used to assess the number and size of exosomes between patient groups. To interrogate their biological effect, microvesicles were added to primary rat cortical neurons subjected to either nutrient deprivation or sodium arsenite.ResultsAmong 1033 proteins identified, 23 exosome-associated proteins were differentially abundant in PD, including the regulator of exosome biogenesis syntenin 1. These protein changes were detected despite similar exosome numbers across groups suggesting that they may reflect exosome subpopulations with distinct functions. Accordingly, we showed in models of neuronal stress that Parkinson's-derived microvesicles have a protective effect.InterpretationCollectively, these data suggest for the first time that immunophenotyping of circulating exosome subpopulations in PD may lead to a better understanding of the systemic response to neurodegeneration and the development of novel therapeutics.Annals of Clinical and Translational Neurology. 02/2015;
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ABSTRACT: Background Radiation Therapy is one of the standard treatments for early stage cancer. It is used together with chemotherapy for localised tumour treatment. However, the increase in radioresistance is reducing the effectiveness of the treatment (Baumann 2008, Hiraoka 1984, Weichselbaum 1985). The mechanisms of radioresistance are poorly understood, although, collective evidence is supporting MicroRNA’s as a biological and functional converter (Thery Clayton 2006, Bussolati 2011). It is considered that individual Radioresistant cells act like stem cells, reviving the population, and in turn resulting in a resistant population. Methods The model OE33 Radioresistant and Parental lines generated by Lynam-Lennons et al (Lynam-Lennons 2011) were being studied in this project for characterisation of exosome release and uptake. The experiment involved isolating and quantifying exosome release between the two cell lines, while additionally measuring uptake of genetic information through exosomes. Results The conclusions of this study show that it is possible to isolate and quantify exosomes in two ways, additionally that the Nanodrop is the easier of these methods. Secondly, in confluent cells, there is a significant difference between Radioresistant and Parental exosome release. The Radioresistant cells expelled measurably larger quantities of exosomes than the Parental cell lines. Thirdly, the study showed that it might be possible to insert plasmids into isolated exosomes, although this has not been confirmed. Additionally, it is still unclear whether the exosomes can be taken up by the lines tested. Conclusions An unexpected result directed the conclusion that, in less confluent cells, the level of exosomes released was larger, thus a negative correlation can be observed. Limitations The limitations were the number of performed repeats, with populations of high confluency. I believe repeating further tests would reinforce my conclusions. A further study would involve examining the uptake of genetic material in both exosomes and cells05/2013, Degree: BSc (Hons) Biology, Supervisor: Stephen Maher