**2. Techniques used for collection, measurement of concentration and preservation of EVs**

Because of the heterogenicity of EVs, a method of collecting a specific population of EVs of interest must be established. Moreover, the methods of efficient collection of EVs have been investigated in different studies. Previous reports have used two main methods: the ultracen‐ trifugation method [7, 8] and FACS methods [9] for collecting EVs. Many studies have compared the efficiency of these collection methods and evaluated the effect of *in vivo* biodistribution of EVs [10, 11]. These observations suggested that bulk EVs are heterogeneous populations and that there is a need to collect a specific population of interest. In the following section, we will compare different methods for isolating EVs and different techniques used for collection and preservation of EVs.

## **2.1. Isolation techniques**

**1. Introduction**

86 Tumor Metastasis

[3]).

nuclei and cytoplasmic organelles [3, 4] **Figure 1**.

Extracellular vesicles (EVs) are membrane-bound particles shed into the extracellular environ‐ ment by many types of cell under different circumstances ranging from normal physiological conditions to pathological conditions like cancer. There are several ways of classifying EVs, including size and mode of biogenesis. Some authors use the designation EVs interchangea‐ bly with other terms like exosomes and microvesicles (MVs). This has led to some confusion andinconsistencyastotheparticlesactuallybeingstudied.Therefore,wehaveincludedasection on the various isolation procedures to emphasise the importance of standardization. Indeed, in many studies, there is no or unconvincing characterization of preparations. In this chapter, we will use EVs as a broad term to encompass three categories: exosomes, microvesicles and apoptotic bodies [1]. Exosomes (30–100 nm diameter) are formed in multi-vesicular bodies (MVB) [2] and released upon MVB exocytosis [3]. They carry several kinds of cargo, depend‐ ing on the surrounding physiological conditions prevailing at the time of their formation and this will determine their effect upon recipient cells. MVs (100 nm–1 μm) are produced by the outward blebbing and fission of the plasma membrane and appear to have possibly more selectively sorted cargo. MVs express surface receptors that differ depending on the mem‐ brane composition of the donor cell [2]. Apoptotic bodies (1–5 μm) are usually released by tumour cells undergoing apoptosis, are packaged indiscriminately and are often fragmented

**Figure 1.** Schematic illustrating the relative sizes of the different classes of EVs (Adapted with permission from Ref.

When first discovered, the release of EVs from cells was thought to be a mechanism for removal of waste and harmful substances from the cell. Nowadays, they are viewed as mediators of intercellular communication through the transfer of biologically active molecules from donor to recipient cells where they can modulate the phenotype and function of those recipient cells [1]. EVs can interact through their surface proteins with receptors on the target cell, triggering

In general, the isolation of EVs from biological fluids and cell cultures requires a series of standard differential centrifugation steps [22]. These sequentially remove dead cells and large cellular debris and then larger intracellular organelles, prior to obtaining a pellet from the cleared cell supernatant. Several modifications/elaborations of the basic procedure have been introduced to purify EVs as well as fractionate them further into discrete size groups; these are illustrated in **Figure 2**.

Heat shock proteins (HSPs), usually associated with cytoprotective functions or as receptor chaperones, are often associated with the cell surface of cancer cells. This feature has been exploited for the separation of EVs from biological fluids and conditioned cell culture growth media. Synthetic peptides called venceremin (Vn), with specific affinity to HSPs, have been used to precipitate out EVs expressing these proteins, in a procedure [16] that has advantages of speed and simplicity over those methods using standard ultracentrifugation. Further refinement of such a strategy, utilizing targets with an exclusively cell surface localization, could be used to distinguish the intracellularly generated exosomes from particles that are formed from the plasma membrane. Other techniques for separating EVs include a salting out process using sodium acetate, and the exoquick technique marketed as a kit by several companies. The latter is also based on selective precipitation of EVs but uses a commercial agglutinating agent as well as two centrifugation steps. Exoquick has been claimed to produce the highest concentration of EVs when compared to differential centrifugation or salting out methods [14].

**Figure 2.** Summary of procedures for isolation of EVs from biological fluids or cell cultures.

Currently, there is no standard isolation protocol for clearly discriminating the different classes of EVs whether by size, density, morphology of the particles or molecular markers [2]. Various procedures have been described in an attempt to separate them. Among the different groups of EVs, the isolation of exosomes is the one most frequently reported in the literature [13]. Separation of exosomes from MVs usually involves a combination of low-speed differential centrifugation steps followed by sucrose gradient ultracentrifugation [21]. Apoptotic bodies can be collected at low-speed centrifugation of approximately 2,000 g. Microvesicles need a higher speed ranging from 10,000 to 20,000 g. Exosomes are pelleted by ultracentrifugation above 100,000 g for 1 hour or more [12]. Alternatively, immune selection of MVs can be performed instead of the differential centrifugation step. This involves the adherence of MVs to magnetic beads bearing antibodies against tumour-associated markers found on the surface of MVs. Ultracentrifugation would still be needed following this immunoselection in order to recover the exosomes in the eluate from the magnetic beads. Apoptotic bodies, on the other hand, can be separated from exosomes by flotation on a continuous sucrose gradient. Separa‐ tion of exosomes from biological fluids and other EVs through the steps mentioned above takes approximately 4–6 hours.

Another procedure devised to shorten the time for preparation is based on the use of a microfluidic device, which is said to allow extraction and purification of exosomal RNA from 100 to 400 μl serum samples in an hour. This device relies on immunoaffinity isolation of exosomes from cell-free supernatant or serum samples. The sample is allowed to flow inside a microchannel coated with IgG against CD63 (which is highly expressed in exosomes from all cell origins). Specificity was demonstrated by showing that fluorescence intensity was higher in the microchannel coated with anti-CD63 antibodies compared to that coated with (control) anti-CD4 antibodies. As opposed to magnetic bead-based systems, the microfluidic device extracts exosomes directly from the serum in a single step. It does not require incuba‐ tion, washing or centrifugation. This technique is not only faster compared to other methods of separation but is also cheaper, requires smaller volumes of samples, and fewer reagents [22].

Another method commonly used for isolation is size exclusion chromatography, which relies on size differences to separate EVs. Immunoaffinity chromatography is also an option for capturing exosomes with antibodies that recognize a marker on the surface of exosomes [13].
