1. Introduction
Extracellular vesicles (EVs) can be largely divided into three main subtypes: apoptotic bodies, microvesicles, and exosomes, and are classified based on their cellular origin, physiochemical, and biomolecular properties [
1] (
Figure 1). The largest of these EVs, apoptotic bodies arise from the outward blebbing of an apoptotic cell membrane, resulting in phosphatidylserine-rich vesicles 500–5000 nm in diameter. Microvesicles originate as particles shedding from the plasma membrane and are enriched with phosphatidylserine and cholesterol, and typically are 100–1000 nm in diameter. Exosomes are the smallest EVs (30–150 nm) and are formed by the exocytosis of multivesicular bodies (MVBs) liberating intraluminal vesicles upon fusion with the plasma membrane [
2]. The biogenesis of these intraluminal vesicles occurs through endosomal sorting complexes required for transport (ESCRT) or a soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor (SNARE)-based system. Exosomal membranes are characterized by the presence of specific lipid species: cholesterol, sphingomyelin, ceramide, and phosphatidylserine, some of which can be used to distinguish them from liposomes [
3,
4]. Exosomes represent an evolutionarily conserved mode of communication [
5,
6] and serve as critical mediators of intercellular communication and potentiate organ cross-talk.
Exosomes were first discovered in 1983 in two studies published within a week of each other, as endosomal vesicles containing transferrin receptors that were secreted from maturing reticulocytes [
7,
8]. The presence of exosomes was relatively ignored (classified as ‘cellular dust’) until interest was revived with discoveries demonstrating their capacity to serve as mediators of intercellular communication [
9], potential drug delivery vectors [
10], and bio-markers of various chronic and acute diseases [
11,
12,
13,
14,
15]. Exosomes are a snapshot of the parent cell that produces them, and upon uptake by the recipient cell, can modify cell function by virtue of their enclosed cargo. Exosomal cargo can include proteins, DNA species (mitochondrial DNA and nuclear DNA), RNA species (mRNA, microRNA, and lncRNA), lipids, and metabolites. Exosomal cargo can change depending on the cellular milieu, cell of origin, and even the exosome preparation technique [
16].
Exosome research is plagued with the use of inconsistent isolation methodologies, nomenclature, and a lack of standardized data acquisition and analysis strategies. This limits the interpretation of the research conducted on exosomes [
17]. Furthermore, due to the differential protein constitution of exosomes, using conventional immuno-marking methodologies to isolate them is challenging, as not all exosomes express the same classical protein markers, nor are all established markers exclusive to exosomes, as they have been found in other subtypes of EVs as well. Indeed, the International Society for Extracellular Vesicles (ISEV) endorsed use of a new standardized nomenclature unless researchers can reliably establish the endosomal origin of their exosomal preparations. The Minimal Information for Studies of Extracellular Vesicles (MISEV) 2018 guidelines recommend characterization of EVs according to size [small EVs (sEVs < 200 nm); medium/large EVs (m/lEV > 200 nm)], density [low, medium, high], biochemical composition [e.g., CD81
+/CD63
+ EVs], or cell of origin [e.g., oncosomes, hypoxic EVs, etc.] [
18]. Irrespective of nomenclature, exosomes and other EVs have been isolated by a number of techniques such as differential ultracentrifugation, size-exclusion chromatography, ultrafiltration, polyethylene glycol-based precipitation, immunoaffinity capture, or by using microfluidics [
1,
19]. Each method of isolation has inherent advantages and disadvantages, and provides a differential yield of exosomes that may be contaminated with EV subtypes and/or humoral protein aggregates. Here, we review the commonly used methods of exosome isolation, with a focus on size-exclusion chromatography (
Figure 1,
Table 1 and
Table 2).
3. Ideal Method for Exosome Isolation: SEC
Given the differences between the isolation techniques as mentioned above, each with respective advantages and disadvantages, an ideal method has yet to be established or agreed upon universally by researchers in the EV field. Nonetheless, a worldwide survey [
56] from one hundred and ninety-six members of ISEV was collected from an online questionnaire administered via email in October 2015 [
56]. The survey found that dUC was used in 85% of all cases to collect EVs from conditioned cell culture media. In contrast, UF, PEG-based precipitation, and SEC were respectively 18%, 14%, and 15% of all cases. A follow-up survey conducted in 2019 from 600 respondents by ISEV found that while dUC and density-gradient ultracentrifugation were still the most commonly used methods, the use of SEC has increased markedly, more than double its percent use from 2015 [
94]. None of these techniques are exclusively optimal for exosome isolation, thus there is an understanding that an inclusive approach using multiple techniques may create the best results. Following the MISEV guidelines of 2018 [
18], specificity and recovery are the main variables of each isolation technique, as summarized in
Table 1. This summary table captures the evidence accumulated to date, which supports the use of SEC as an ideal exosome isolation technique compared to others.
SEC has been described as the best method for separating exosomes from most proteins, simultaneously recovering morphologically and functionally intact exosomes from plasma [
95]. Exosomal preparations from SEC methodology have low levels of contaminants and co-precipitates, leading to a relatively homogenous final exosome isolation [
96]. This fact has popularized the use of SEC amongst its competitors for blood-based exosome-associated biomarker discovery research [
83,
89]. SEC has been used successfully to isolate, purify and enrich exosomes from a variety of biological fluids including plasma [
20,
21,
22,
23,
24,
25], serum [
26,
27,
28], bovine and human milk [
29,
30], urine [
31,
32,
33,
34], saliva [
35,
36], tears [
35], cerebrospinal fluid [
37,
38], synovial fluid [
39], nasal lavage [
41], seminal fluid, [
40] and stromal vascular fraction (SVF) from adipose tissue [
42,
43] (
Table 2). Recent findings reported at the ISEV Virtual Conference 2020 suggest SEC is superior to other techniques in isolation of pure exosomes from human body fluids (unpublished findings). Another noteworthy advantage of SEC is that it does not require an excessive volume of sample to isolate exosomes compared to some techniques, independent of the type of sample (
Table 2). It has also been used to document distinct proteomic and extracellular miRNA signatures in small exosomes isolated from conditioned media from amniotic fluid stem cells (AFSCs) [
45,
46], human-induced pluripotent stem cell (iPSC)-derived neurons [
47], and human umbilical cord mesenchymal stromal cells (MSCs) [
48], among others. An overview of the sample types that SEC has been able to isolate, purify, or enrich exosomes from is presented in
Table 2. Of note is the fact that all studies included in this table have adhered to MISEV 2014/2018 guidelines to ensure high rigor and reproducibility of their work.
The recovery and purity of SEC-based exosomal isolation is especially recognizable when compared to other isolation methods, as shown in comparative studies with PEG-based isolation and dUC and UF [
93,
97,
98]. dUC has been reported to potentially damage exosomes and can alter the exosome proteome, lipome, and/or genome [
50]; UF can lead to the deformation and break-up of larger vesicles due to the pressure and contact with filter membranes [
19]; PEG can co-precipitate non-EV components and alter exosomal protein signatures [
93]. SEC is able to reduce or completely circumnavigate these limitations, hence, it has earned its place as a minimally invasive, rapid, and high-purity isolation technique. This is specifically an asset in blood plasma exosomal fraction isolation, as low-density lipoproteins mimic the exosomes that would otherwise be derived from the sample through dUC, which would interfere with future analysis [
99]. Additionally, SEC columns are cost-effective, as one column can be washed and then reused a number of times, and can be purchased or made within the laboratory [
29,
89,
100]. Since a single exosome isolation technique cannot reach optimal yield and purity, coupling SEC with other isolation methods including dUC, UF, or PEG-based retrieval can precipitate intact, highly purified exosomes in a reproducible manner. However, the limitations of these approaches as detailed above will then accompany the chimeric protocol [
32,
37,
44,
49,
90,
101,
102,
103].
More recently, newer techniques have been used in combination with SEC to improve exosome purity [
104,
105]. A technique known as dual-mode chromatography (DMC) was used successfully to reduce the contamination of lipoprotein particles (LPPs) in plasma exosome preparations [
105]. This combination technique integrates two separation steps: the removal of high-density lipoproteins (HDLs) by SEC and the use of cation exchange to separate positively charged LPPs from negatively charged exosomes. Another technique combines SEC with fluorescence detection and is known as Flu-SEC or F-SEC. Here, SEC is combined with detection of fluorescently-labeled exosomes using high-performance liquid chromatography and a fluorescence detector to optimize exosome isolation [
104]. Other hybrid approaches that use both SEC with PEG or dUC exist as well. It is important to keep in mind that outside of the EV research, SEC is considered to be a standard technique for purification and fractionation of peptides due to its highly reproducible and stable features [
106].
Despite its prowess in recovery and specificity during exosome isolation, SEC still has its shortcomings as noted earlier in this review, and in details elsewhere [
107]. In addition to not being able to distinguish same-sized exosomes from microvesicles, SEC-based exosome isolation techniques must take care to avoid denaturation of the biological targets while also controlling for unwanted electrostatic and hydrophobic interactions between the mobile phase containing the vesicles and the stationary, porous phase. The number of samples that can be processed simultaneously is another limitation associated with use of SEC. Hence, being coupled with another technique [
108,
109], such as ultracentrifugation, ultrafiltration, or PEG-based precipitation, may be the optimal method of isolation. SEC-coupled techniques generate a high yield of exosomes [
19] that can be used for protein and RNA diagnostics as well as potentially used as a drug or drug delivery system [
110,
111].
4. Application of SEC for Isolation of Exosomes from Adipose Tissue
SEC-coupled techniques are specifically useful within the context of adipose tissue-derived exosomes [
42,
43,
112]. Flaherty et al. [
43] reported that standard isolation techniques are inefficient for extraction of exosomes from adipocytes given their high lipid content (which could affect vesicle density). This is a crucial caveat to using density-based dUC for purification of adipose-derived exosomes. A combination technique of SEC and UF was effectively utilized to isolate adipocyte-exosomes from SVF and adipose tissue-conditioned media [
42,
43]. Another group combined SEC with dUC to isolate adipocyte-exosomes (identified by presence of adipocyte markers) from platelet-free human plasma samples [
112]. Adipose-derived exosomes enriched via SEC contain both canonical markers of exosomes, as well as adipocyte-specific proteins such as adipose triglyceride lipase (ATGL), caveolin 1 (CAV1), fatty acid-binding protein 4 (FABP4), adiponectin, perilipin, and peroxisome proliferator-activated receptor gamma (PPARγ) [
43,
112]. Interestingly, ATGL in adipose-derived exosomes is resistant to proteinase digestion, indicating the lipid-droplet cargo is enclosed within the vesicles [
43]. However, FABP4, perilipin, and PPARγ are found as soluble proteins in circulating plasma, and FABP4 and PPARγ can be released from non-adipocytes as well [
112]. As noted earlier, SEC columns [
83,
113] facilitate effective and reproducible exosome isolation, eliminating over 95% of non-vesicular protein from biological fluids. This is in contrast to dUC, which co-pellets soluble plasma proteins with exosomes, leading to over-estimation of adipocyte-derived exosome signatures. Thus, SEC is a superior approach to adipose-exosome isolation.
Recent papers have reported that adipose-derived exosomes constitute a relatively small proportion of the circulating plasma EVs; the majority originate from platelets, leukocytes, erythrocytes, and vascular endothelial cells [
43,
112]. It is important to note that a small adipose-exosome fraction does not correlate with the effect or clinical relevance of these vesicles. Seven miRNAs contained within adipose tissue-derived EVs, involved in the regulation of adipogenesis, oxidative stress, inflammation, fibrosis, and fat metabolism, were differentially expressed in lipedema patients compared to healthy controls, implicating the applicability of adipose-exosomes as biomarkers of disease [
42]. Hence, using a purification technique that can use small starting volumes while maximizing yield of functionally intact exosomes free from non-vesicular protein contamination, renders the SEC-coupled approach to be the optimal method for adipose tissue-derived exosome isolation.
5. Conclusions
There is a lack of protocol standardization in exosome isolation methods. This void has led to the development of advanced pioneering techniques in order to optimize exosome isolation from a variety of biological fluids [
114]. Currently, the optimal EV isolation method is chosen based on the amount and type of starting material (e.g., plasma, milk, cell culture media, urine, etc.), availability of specialized equipment, intended therapeutic use, route of administration, as well as desired end product (e.g., total EVs, or CD81
+ exosomes). Considering the variable sources of exosomes, an optimized isolation method would minimize uncertainties and inconsistencies in exosomal research. Researchers need to find a balance between purity, efficiency, and downstream applications of the isolated vesicles. Through a systematic evaluation of dUC, UF, PEG-based precipitation, immunoaffinity capture, microfluidics, and SEC methods, a combined optimized protocol is advisable. Based on the efficiency, reliability, rigor, reproducibility, and ease of use, a SEC-coupled approach to exosome isolation for a high yield of homogenous, intact exosomes seems ideal. Consequentially, the data generated from such samples would expedite establishing novel diagnostic biomarkers and therapeutic drug applications of exosomes [
115]. Irrespective of chosen methodology, we recommend that researchers validate the exosome isolation technique before beginning experiments, especially if using novel biofluids or samples. Thus, while SEC is an outstanding candidate method to isolate exosomes, direct comparative studies are required to support this conclusion.