Isolation and Purification of Exosomes and Microvesicles Using the NTA Technique

Introduction

Exosomes and microvesicles (MVs) are extracellular vesicular bodies that are found in a wide range of eukaryotic and prokaryotic organisms and play a major role in many pathological and physiological processes. The structure, function, cellular origin and characterization of these cell-derived vesicular bodies have been widely studied, although they are still topics of much debate. Previously, there has been a lack of suitable techniques for detecting, analyzing, phenotyping and enumerating these structures and this article shows how the NTA technique can be used to address these issues.

Definitions and Nomenclature

With respect to the definitions and nomenclature of exosomes and MVs, Gyorgy et al. (2011) described them as being 50 to 100 nm and 100 to 1000 nm in diameter, respectively. However, the definition and nomenclature of exosomes and MVs, as yet, still varies. According to Simpson et al. (2009), exosomes are vesicles of endocytic origin that are released by cells following fusion of multivesicular bodies with the plasma membrane. The terminology of extracellular organelles can be quite confusing and in order to distinguish the functional and biochemical activities of these structures, it is important to clarify the nomenclature and also to enhance purification strategies.

According to Lee (2011), since MVs are so heterogenous, many different names have been used to describe them under different experimental conditions. Some of the most common terms used are exosomes, MVs, ectosomes, microparticles, shed vesicles, exosome-like vesicles and oncosomes. Platelet-derived microparticles or PMP are heterogeneous vesicles that measure less than 1μm in diameter. They are produced from the plasma membrane following activation of platelets by different stimuli. This discrete population of vesicles differ somewhat from the exosomes derived from the intracellular multivesicular bodies and from the microparticles derived from megakaryocytes.

It can therefore be concluded that the variation in definition and nomenclature of microvesicular bodies can be attributed to the fact that they originate from a broad range of cellular origins and serve many different functions, all of which still need to be clarified. Herring et al. (2013) explored the function of cellular exocytic vesiculation in disease, health and transfusion medicine and found that microparticles (MPs), which are derived from many cell types, are released into the circulation as a result of complement activation, shear stress, agonist interaction with cell surface receptors, cellular damage or proapoptotic stimulation.

Origin, Occurrence and Role

The following mechanisms are involved in the origination of MVs:

  • Breakdown of dying cells into apoptotic bodies
  • Blebbing of the cellular plasma membrane (ectosomes)
  • Endosomal processing and discharge of plasma membrane material in the form of exosomes

Exosomes are present in many types of bodily fluids including blood, saliva, breast milk, amniotic fluid and urine, amongst others. Given the varied molecular structures associated with their construction, exosomes are thought to play a number of different roles. Since exosomes derived from breast milk carry immunorelevant structures, they are thought to play a role in directing the immune response and in the development of the immune system in infants.
In cell signaling processes, exosomes are believed to play an important role and to have a strong association with disease progression. Since MVs are capable of transferring bioactive molecular content to recipient cells, they are recognized as mediators of intercellular communication. These processes may take place at both the local and systemic level, contributing to the formation of microenvironmental niches and fields. Examples of the bioactive content of MVs include proteases, growth factors and their receptors, signaling molecules, adhesion molecules, mRNA, microRNA and DNA sequences.

Although there is much debate with respect to the physiological function of exosomes, increasing amounts of evidence from different experimental systems indicate their role in many biological processes.

Preparation and Detection Protocol Development

As mentioned above, the methods used to separate and prepare microvesicles and exosomes vary significantly, which can have a significant effect on the results obtained. Yuana et al. (2012) have considered this lack of visibility regarding the true nanoparticulate nature of a specimen in their evaluation of pre-analytical and analytical problems in blood microparticle analysis. They concluded that although the results of plasma microparticle measurements recorded in the literature are widely varied, this can be attributed not only to poor standardization of MP assays, but to differences in pre-analytical conditions.

Yuana et al. (2011) also stressed the need to obtain platelet-free plasma samples and warned against the insufficient calibration of traditional flow cytometric analysis. On comparing the NTA and DLS techniques, they found that DLS had a lower sensitivity in polydisperse samples, as demonstrated by cell-derived microparticles. In contrast, the NTA technique is capable of precisely sizing particles in a sample, although particles of a larger size decrease the number of small particles identified by the software. The NTA technique was not considered to be as easy to operate as the DLS technique and required some skill form the user. However, Yuana et al. (2010) had previously observed that NTA validates the size and number concentration of microparticles detected through atomic force microscopy.

Similarly, Giebel and Ludwig in 2011 used both the EM and NTA techniques to size their sample solutions enriched with exosomes, which revealed that they mostly included particles that ranged between 80 and 160 nm, while the same sample appeared to be significantly smaller when EM-based technologies were used.

In a similar study, exosomes derived from three different types of human cells were characterized by Sokolova et al. (2011) using SEM and NTA techniques. The stability of these exosomes were investigated when stored at -20°C, 4ºC, and 37 ºC. This revealed that the size of the exosomes reduced at 4°C and 37 °C, suggesting degradation or a structural change. On the other hand, the size of the exosomes remained unaffected by multiple ultracentrifugation and multiple freezing and thawing. It was therefore concluded that the NTA technique is suitable for studying exosomes.

According to Gabriel and Giordano (2010), NTA provides a number of benefits in the characterization of particle size distribution. They said that the technique provides speed, precision, and ease of operation and can be used to easily establish the particle’s hydrodynamic volume, size and surface characteristics, as well as the interaction of its surface with specific ligands. Additionally, the technique can be extended to allow for the real-time observation of surface reactions and without reporter group conjugation to the reactant. The NTA technique also makes it possible to assess the kinetics of binding and binding constants without any chemical alternation, providing real benefits in terms of clinical diagnostics, therapeutic monitoring, and product development. The method was also used to study myristoylated alanine-rich C-kinase substrate (MARCKS) peptide as a probe to target microvesicles, as well as to validate a method for the profiling and quantification of exosomes in human plasma using a protein microarray based on biotin-labeled anti-tetraspanin antibodies.

According to Soo et al.(2012), NTA helped in establishing the relative concentration and size distribution of exosomes and microvesicles in biological fluids and cell-culture supernatants during their analysis of microvesicle release from human T lymphoblastoid cell lines.

In another study, Shiba et al.(2012) described their analysis of the interaction between solid materials and isolated exosomes using the NTA technique. According to Fang et al.(2012), NTA proved to be an ideal technique for the characterization and quantification of exosomes in their studies of the renal system.

Such studies prove useful to researchers involved in the separation, purification and storage of exosome samples.

Isolation and Purification Methodology

Since different types of lipid membranes are found in biological fluids and cell-culture supernatants, it is essential to carry out high-quality exosome purification. Théry et al. (2006) have suggested a number of methods for purifying exosomes from different sources.

Today’s isolation procedures for exosome isolation involve a two-step differential centrifugation step. By contrast, other preparation techniques involve the use of Annexin V-coated magnetic beads, sucrose gradient centrifugation, immunoisolation, ExoMir® filtration technologies and ExoQuick® precipitation technologies. Typically, the isolation and analysis approach may involve a combination of methods such as that described by Mathias et al. (2009), which used size filtration, followed by ultracentrifugation to isolate and purify the exosomes from LIM 1215, the colon carcinoma cell line.

However, problems still arise. In a recent analysis by Mathivanan et al. (2010) looking at the various methods of exosome purification, the researchers found that the transfer and propagation of infectious retroviruses and prions could be artefacts of the purification strategies. Quah and O’Neill (2007) also demonstrated that mycoplasma contaminants were present in exosome fractions of dendritic cells generated in cultures. This research emphasized the strong relationship between exosomes and infectious agents such as Mycoplasma and cautioned against the use of purification processes to prepare exosomes for immunity-related studies.

Researchers Sorokina et al. (2013) performed the quantitative and qualitative analysis of preservation methods on extracellular microvesicles in order to facilitate the use of EV for clinical application, recognizing how important it is that efficient methods for their long-term storage are developed that won’t compromise their function. Quantitative analysis of the EVs pre- and post-preservation (BCA and NTA protein assay) was carried out after EVs from lung were preserved in PBS at 4oC and -20oC for a period of 1 week, but no differences were observed. Moreover, both preserved and fresh EVs did not affect the viability of the whole bone marrow cells in co-culture.

In an international conference held in Boston, USA in 2013, Heusermann et al. and Kremenskoy et al. described rapid and efficient techniques for isolating exosomal-like vesicles from body fluids and cell culture medium and exosome isolation, fluorescence labeling, analysis and characterization of cell uptake. At the same conference Yuana et al (2013) reviewed the issue of standardizing the collection, detection and handling of extracellular vesicles and pointed out that although flow cytometry is mainly used for detecting EV, it only detects 1 to 2% of all the EV present and the results obtained from EV studies are not easy to compare between laboratories.

The researchers therefore strived to develop standardized collection and handling protocols and to carry out sensitive detection of EV using methods such as NTA and resistive pulse sensing (RPS). The researchers found that, compared to flow cytometry, both NTA and RPS detected between 1,000 and 10,000 times more particles in all of the EV preparations tested. However, generally, the EV concentration and particle size were more affected by the single freeze/thaw cycle than by centrifugation. The team concluded that the reconstitution solution, the type of EV and the detection limit of the methods used to determine EV are important factors in the standardization of protocols.

Zeringer et al. (2013) verified the identity and purity of exosomes by EM and NTA and attempted to develop new procedures for isolating exosomes from human blood serum and HeLa cell culture media and characterizing their RNA content. The researchers claimed that by using a Total exosome RNA and protein isolation kit, they were able to complete the isolation process within a fraction of the time taken when using current standard protocols. They were also able to recover fully intact exosomes in higher yields. This work was then extended to a following report, wherein NTA was used to show that their isolation protocol represented a set of reagents and a workflow that allowed quick and efficient exosome extraction, followed by isolation and analysis of RNA using qRT-PCR and other methods.

Nordin et al. (2013) compared spin filtration, ultracentrifugation, and spin filtration with sequential LC fractioning to isolate exosomes from cell culture media in order to try and find out whether spin filtration with size exclusion chromatography fractioning provides a more reliable and scalable approach than ultracentrifugation. Using RNA and protein content analysis, NTA, Western blotting and electron microscopy, the researchers demonstrated that by means of spin-filtration and sequential LC fractionation, it is possible to purify high yields of exosomes from large media volumes. However, more studies were needed for this approach to become the gold standard for exosome purification.

Chute et al. (2013) recommended an alternative method, which involved using a peptide with an affinity for canonical heat shock proteins, for capturing and enriching extracellular microvesicles. The researchers demonstrated that it is possible to purify and analyze these microvesicles by protein content. Here, NTA was similar to other standard methods used for isolation of extracellular microvesicles.

Conclusion

In recent years, the analysis of exosomes and microvesicles has become extremely important in research laboratories. However, a lack of suitable techniques for detection, analysis, and phenotyping of these vesicular bodies is proving to be a major hindrance. The above details demonstrate how the NTA technique has proved a reliable and scalable technique for both qualitative and quantitative analysis of extracellular microvesicles.

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