Proteomics, Genomics & Microarrays
Exosomes: A Promising New Tool for Diagnostic and Clinical Applications Dr Maja Petkovic (
majap@ansbio.com) and Ben Smith (
bens@amsbio.com) - AMSBIO.
Exosomes and extracellular vesicles (EV) are currently the subject of a lot of scientifi c interest; implicated in intracellular communication and with their ability to be used as a tool in biomarker discovery, they have the potential to enable advances in clinical settings. EVs are nanosized lipid membrane vesicles that are released from a vast number of different cell types into the extracellular space [1]. They are categorised into 2 broad areas of exosomes and ectosomes [2]. Exosomes are vesicles ranging in size of ~40-160nm in diameter and form through the endocytic pathway to be released from the cell by fusion of endosomal multivesicular bodies (MVBs) with the plasma membrane [2]. Ectosomes differ by directly budding from the surface of cells and include a number of subspecies; microvesicles, migrasomes, exophers, apoptotic bodies, and large oncosomes with a size range of ~50nm-5um [3].
UC is the benchmark for EV isolation techniques as it provides great purity and yield, as well as having established protocols for EVs studies. However, UC does have huge drawbacks in the field. Standard isolation protocols can take a full lab day for a set of isolations, it requires large sample starting volumes, and above all, UC needs an experienced EV isolation researcher to create isolation reproducibility. This method also has huge challenges in scalability which is why it is NOT suggested for clinical applications.
The increasing popularity of SEC has arisen from its ability to provide a simple and reproducible isolation protocol, the fact it is faster than UC, and because it can effi ciently isolate out contaminants such as soluble proteins, lipoproteins, and nano sized non vesicular extracellular particles. Drawbacks to the SEC method are the tedious fractioning of isolated samples, volume constraints, and isolating based on size.
TFF, traditionally used on large biomanufacturing facilities, has gained a lot of interest in its use for EV isolation. This method uses ultrafi ltration membranes with pores in a column that provides a tangential fl ow to avoid fi lter clogging and cake formation. This creates the ability to be scaled for different clinical applications. Drawbacks to this method includes co-isolating non EVs that have comparable size profi le to EVs.
Figure 1. Illustration of cells secreting exosomes.
EVs are involved in various physiological and pathological processes, such as intercellular communication, immune regulation, and tumour progression. They contain various bioactive molecules, including proteins, lipids, nucleic acids, and metabolites, that can be transferred to recipient cells and modulate their functions. There is an abundant amount of evidence showing EVs are distributed widely in bio fl uids including blood, urine, saliva, breast milk, cerebral spinal fl uid, synovial fl uid, and more [4]. There is also increasing evidence to demonstrate that the surface of an EV and its luminal content correspond with different pathophysiological states of the body [5]. The upshot is that EVs could potentially be a great source for potential biomarkers in the diagnosis and prognosis of a number of disease states. EVs also hold promising therapeutic applications for new drug discovery and drug delivery tools from the increasing evidence of their high biocompatibility, limited immunogenicity, cargo diversity, stability, and ability to cross the blood-brain barrier [6].
With their inherent properties of cell-to-cell signalling, EVs could offer considerable advantages within the medical and scientifi c fi elds. While this newly researched fi eld is promising, it holds many questions and areas that need to be explored further before the clinical applications of EVs can be maximised. This includes research in mechanisms of EV biogenesis, cargo loading, and uptake, as well as their safety and effi cacy in clinical settings.
Isolation Techniques
Working with EVs holds many challenges due to their broad heterogeneity and issues with contaminations from soluble proteins, lipoproteins, and other non-EV contaminants during EV isolation. This is why understanding the limits and advantages of different EV isolation techniques are important to determine the best EV isolation tool to match the research goals of the study. Different approaches to isolating EVs include ultracentrifugation (UC), size-exclusion chromatography (SEC), Tangential Flow Filtration (TFF), immunoaffi nity, and precipitation methods.
A recent study [7] reviewed various isolation methods of EVs and found that UC is considered the gold standard for EV isolation, with newer techniques such as SEC and precipitation methods are becoming more popular.
Immunoaffi nity provides a different principle of isolation based on surface markers present on EVs. The key benefi t of this method is the extreme purity of EVs that can be extracted with the ability to subtype EV populations based on their surface markers. Drawbacks to this method include the low yield from isolating a subpopulation of EVs, and the inability to get the targeting motif removed from the EV surface marker.
Lastly, the precipitation method provides a high yield of EVs by using polyethylene glycol (PEG) to decrease the solubility of the mixture to isolate EVs out of the solution. This method does have an extremely high yield of EVs, is cost effective, and has a simple protocol. However, in the isolation process impurities such as lipoproteins and other soluble proteins will contaminate isolated EV samples. The precipitation method cannot do large volumes of isolation, and so this method is most impactful for early- stage screening of EV populations.
Companies such as AMSBIO offer a variety of exosome isolation kits that are based on ultracentrifugation, size-exclusion chromatography and precipitation methods, that can help researchers to select the best method based on the parameters of their specifi c research or project.
EV Standards
Whilst standardised EV isolation methods can and will produce usable EVs for use in studies, by nature EVs are also inherently heterogeneous, due both to the vast number of cells producing EVs and the physiological conditions that these EVs are expressed from. This means that for the best results for diagnostic applications or when developing exosome-based therapies it can be better to use an externally produced set of EV standards. These standards can include purifi ed exosomes from a specifi c cell type or a mixture of exosomes from multiple cell types. They can also include synthetic exosomes that have been engineered to contain specifi c biomarkers. The use of consistent, well-characterised exosome standards can facilitate the development of new diagnostic tests and therapies. A report in the Journal of Extracellular Vesicles [8] described the importance of using exosome standards to ensure the quality control of exosome-based diagnostics and therapeutics. AMSBIO offers a wide range of exosome standards that can be used in clinical settings, including purifi ed exosomes from a specifi c cell type, exosome standards for diagnostic applications and synthetic exosomes that have been engineered to contain specifi c biomarkers.
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