Extracellular vesicles, found in all biofluids, include exosomes (30 nm to 150 nm) from endosomes/multivesicular bodies and microvesicles (150 nm to 1000 nm) from the plasma membrane. Multiple biotech companies are exploiting exosomes as a delivery modality [4]. Various methods for the isolation of exosomes from biological fluids have been developed. They include centrifugation, chromatography, filtration, polymer-based precipitation and immunological separation. Recent technical improvements in these methods have made the isolation process faster and easier. Contamination of isolated exosomes with non-exosomal particles, such as apoptotic bodies, small apoptotic vesicles, exomeres, and lipoproteins can cause wrong conclusions about biological activities of obtained exosomes and therefore should be avoided [5]. Isolation methods affect the purity and yield of exosomes. S Shu et al obtained 58-fold more exosomes using ultrafiltration and size exclusion chromatography than ultracentrifugation with up to 836-fold lower concentrations of co-purified soluble factors [6]. Exosomes from different specimens can possess different protein/lipid and luminal contents and different sedimentation characteristics. For example, exosomes from adipose tissue contain high lipid content, and necessitate adjustment in their isolation methods [7]. If exosomes are to be isolated from cultured media, one very important consideration is to use either serum-free media or exosome-free fetal bovine serum.

Differential centrifugation remains one of the most common techniques of exosome isolation [8]. The method consists of several steps, including 1) low-speed centrifugation to remove cells and apoptotic debris, 2) higher speed spin to eliminate larger vesicles and finally, 3) a high-speed centrifugation to precipitate exosomes (Figure 1). Importantly, the viscosity of the biofluids has a significant correlation with the purity of isolated exosomes [9]. Moreover, biological samples with high viscosity require longer ultracentrifugation step and higher speed of centrifugation. For example, A Hoshino et al went through 500 x g for 10 min, 3,000 x g for 20 min, 12,000 x g for 20 min and 100,000 x g for 70 min [10]. Capello M et al purified exosomes from cultured cells in serum-free media with sequential centrifugation steps of 800xg and 2000×g and finally pelleted with an ultracentrifugation at 100,000×g [11]. Exosomes from primary cortical neurons were obtained through sequential centrifugation of supernatants at 300 x g for 10 min, at 2000 × g for 10 min, 10,000 × g for 30 min, and 100,000 × g for 90 min at 4°C and the last pellet was re-suspended and centrifuged again at 100,000 × g for 90 min [12].

Figure 1. Differential centrifugation. Several rounds of centrifugation are applied to isolate exosomes. The centrifugation time is increased to pellet smaller particles in consecutive rounds. After each centrifugation, pellets containing cell debris are removed, and the supernatant is used for the next step. In contrast, after the 100 000 g centrifugation, the pellets with exosomes are kept for further analysis, and supernatants are discarded [1].

This approach combines ultracentrifugation with sucrose density gradient [13]. More specifically, density gradient centrifugation is used to separate exosomes from non-vesicular particles, such as proteins and protein/RNA aggregates. Thus, this method separates vesicles from the particles of different densities. The adequate centrifugation time is very important, otherwise contaminating particles may be still found in exosomal fractions if they possess similar densities. Capello M et al used a density gradient flotation approach to purify exosomes from blood preparations [11]. Recent studies suggest the application of the exosomal pellet from ultracentrifugation to the sucrose gradient before performing centrifugation [14].

Figure 2. Ciliated porous structures designed to isolate exosomes. The structures do not let the cells and other particles larger than 1 μm to enter the wired area. Some smaller particles and cell debris can enter the micropillar area, but are excluded by the nanocilia, which form pores with diameter 30-200 nm. The ciliated structures selectively capture exosomes and small extracellular vesicles [2].

A protocol of the modified version of density gradient-based method, introduced as Cushioned Density Gradient Ultracentrifugation has recently been described [15]. This method provides maximal recovery and high purity of the isolated exosomes and maintains their structure and functions.

Size-exclusion chromatography (SEC) is used to separate macromolecules on the base of size, not molecular weight. The technique applies a column packed with porous polymeric beads containing multiple pores and tunnels. The molecules pass through the beads depending on their diameter. It takes a longer time for molecules with small radii to migrate through pores of the column, while macromolecules elute earlier from the column. Size-exclusion chromatography allows precise separation of large and small molecules. Moreover, different eluting solutions can be applied to this method. Chromatography isolation has been shown to have more advantages compared to centrifugation methods, since the exosomes isolated by chromatography are not affected by shearing force, which can potentially change the structure of the vesicles [16]. Currently, the SEC is a widely accepted technique for isolation of exosomes present in both blood and urine [17, 18].

In addition, a combination of SEC method with ultrafiltration has been used for isolation and analysis of urine-derived exosomes [19]. Also, flow field-flow fractionation combined with a UV analyzer and light-scattering detector has been applied to analyze the size and pureness of the exosomes. The flow field-flow fractionation combines parabolic and cross-flow to isolate exosomes. The obtained exosomes have been detected by electron microscopy and mass spectrometry. In addition, a recent article by Lane et al has presented updated protocols for the purification of exosomes, including protocols for ultracentrifugation, SEC and density gradient centrifugation [20].

Ultrafiltration membranes can also be used for isolation of exosomes. Depending on the size of microvesicles, this method allows the separation of exosomes from proteins and other macromolecules. Exosomes may also be isolated by trapping them via a porous structure (Figure 2). Most common filtration membranes have pore sizes of 0.8 µm, 0.45 µm or 0.22 µm and can be used to collect exosomes larger than 800 nm, 400 nm or 200 nm. In particular, a micropillar porous silicon ciliated structure was designed to isolate 40-100 nm exosomes [2]. During the initial step, the larger vesicles are removed. In the following step, the exosomal population is concentrated on the filtration membrane. The isolation step is relatively short, but the method requires pre-incubation of the silicon structure with PBS buffer. In the following step, the exosomal population is concentrated on the filtration membrane. This method has not yet been tested using clinical samples. In addition to the standard filtration techniques, tangential flow filtration showed promising results for the effective isolation of exosomes and can be applied in both basic research and clinical analysis [21, 22]. This method is used for isolation of exosomes with well-determined size by removing free peptides and other small compounds. In addition, a combination of ultrafiltration with the SEC was shown to be very efficient for isolation of exosomes in in vitro studies [23] and from adipose tissue [7].

Figure 3. Sieving models for exosome filtration. Sieving is done by (a) making pressure on an on-chip filter or (b) creating an electric field, forcing the exosomes to move through the porous membrane, combining cross flow and electrophoresis sorting [3].

Polymer-based precipitation technique usually includes mixing the biological fluid with polymer-containing precipitation solution, incubation at 4 C and centrifugation at low speed. One of the most common polymers used for polymer-based precipitation is polyethylene glycol (PEG). The precipitation with this polymer has a number of advantages, including mild effects on isolated exosomes and usage of neutral pH. Several commercial kits applying PEG for isolation of exosomes were generated. The most commonly used kit is ExoQuick™ from System Biosciences, Mountain View, CA, USA [24, 25]. This kit is easy and fast to perform and there is no need for additional equipment. Recent studies demonstrated that the highest yield of exosomes was obtained using ultracentrifugation with ExoQuick™ method [26]. However, contamination of exosomal isolates with non-exosomal materials remains a problem for polymer-based isolation methods. In addition, the polymer substance present in the isolate may interfere with downstream analysis.

A recent study by Niu et al has compared the application of ultracentrifugation, ultrafiltration and polymer-based precipitation for exosomal isolation from human endometrial cells and found that polymer-based method showed the lowest protein contamination [27].

Several techniques of immunological separation of exosomes have been developed. The immuno-chip method is based on surface exosomal receptors, which are used to isolate exosomes depending on their origin. Obtained exosomes are analyzed directly or used for DNA or total RNA isolation [28]. Exosomal intracellular proteins can be used as specific markers for isolation of exosomes [29]. Antibody-coated magnetic beads were effectively used to isolate exosomes from antigen presenting cells [30]. Also, exosomes of tumor origin were isolated from tumor cells using antibodies against tumor-associated HER2 [31] and EpCAM [31]. Isolated bead-exosome complexes can be analyzed by flow cytometry, Western blotting and electron microscopy [32]. Moreover, Western blotting is applied to detect the exosome-specific proteins, including tetraspanins and the endosomal sorting complexes required for transport (ESCRT) proteins Alix and TSG101 [33]. However, the isolation using antibody-coated beads is not suitable for obtaining exosomes from large volumes [32].

In addition, ELISA-based ExoTEST™ was demonstrated to be effective for isolation of exosomes [34]. Using ExoTEST™ plates coated with exosomal antibodies, exosomes can be isolated from various biological fluids. The method is applied for detection, analysis and quantification of both common and cell type-specific exosomal proteins.

A recent study has applied immunoaffinitive superparamagnetic nanoparticles (ISPN) to bind the exosomes. The researchers generated ISPNs by connecting anti-CD63 antibodies and nanoparticles and used them to isolate exosomes from body fluids [35].

This technique isolates exosomes by sieving them from biological liquids via a membrane and performing filtration by pressure or electrophoresis [3] (Figure 3). The method requires a shorter separation period, but gives higher purity of isolated exosomes [36]. This method is considered to be non-selective with regard to the specific types of exosomes. The only disadvantage of the sieving separation is the low recovery of isolated exosomes.

Secreted exosomes have important functions in the pathogenesis of various diseases including tumors. Several methods have been developed to capture and isolate exosomes from biofluids. Centrifugation techniques remain very common, however, other methods, such as filtration, immunological separation and sieving, show promising results and can be effectively applied both in laboratory research and clinical medicine.

Various technologies applied for exosome characterization include biophysical, molecular and microfluidic methods. Biophysical methods characterize the exosomal size range. One of the biophysical approaches, optical particle tracking, measures the size distribution of exosomes from 10 nm to 2 µm and concentration of exosomes. The exosomal movement trajectories are detected in order to measure the velocity of the particles [37]. Other biophysical characterization methods include photon correlation spectroscopy [38], resistive pulse sensing [39], atomic force microscopy [40, 41], transmission electron method [42], cryo-electron microscopy [43], and field-flow fractionation [44]. Various models of NanoSight from Malvern Panalytical are commonly used [7, 40, 45]. Kapogiannis D et al measured and evaluated the size of blood exosomes through nanoparticle tracking analysis with Nanosight NS500 instrument from Malvern Instruments and transmission electron microscopy with a Zeiss Libra 120 [45]. Flaherty SE et al analyzed the particle concentration and size distribution of adipocyte-derived exosomes with NanoSight NS300 from Malvern Instruments Ltd and ViewSizer 3000 from MANTA Instruments, Horiba Scientific [7]. Capello M et al quantified exosome preparations using ZetaView Nanoparticle-tracking analysis instrumentation from Particle Metrix [11].

Molecular approaches are used for characterization of exosomal surface proteins. In particular, flow cytometry allows the measurement of the size and structure of exosomes [46]. Another molecular method of exosome characterization is Raman spectroscopy based on the illumination of analyzed samples by laser light. This technique creates a specific spectrum of the molecules, such as peptides and nucleic acids, and thus provides the chemical structure of the exosomes [47]. The microfluidic-based methodologies can also be used for characterization by binding of exosomes to specific antibodies on microfluidics channels flowed by the elution of bound vesicles [48].

Several methods have been developed for analysis of exosomal RNA content. Those methods include microarray analysis, next-generation sequencing (NGS) and digital droplet PCR (ddPCR) [49]. In particular, microarrays have been used to study miRNAs and toluene-mediated gene expression in the exosomes derived from leukemic cells [50]. Also, the expression of exosomal microRNAs in human stem cells has recently been evaluated by NGS [51]. In addition, the absolute quantification of microRNAs has been achieved by ddPCR [52]. With regard to proteins, the protein content of exosomes is analyzed by Western blotting, proteomic technology and fluorescence-based cell sorting [53-55].

Exosomes are secreted by cells in both normal and pathological conditions and contain various membrane and cytosolic proteins. Thus, exosomal proteins can be potentially used for clinical diagnostics (Figure 2). Top ten proteins found in the exosomes include heat shock protein 8 (HSPA8), CD63 antigen (CD63), beta actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase 1 alpha (ENO1), cytosolic heat shock protein 90 alpha (HSP90AA1), CD9, CD81, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ), muscle pyruvate kinase (PKM2) [29]. Exosomal proteins belong to various functional groups, such as tetraspanins (CD9, CD63 and CD81), heat shock proteins (HSC70 and HSC90), membrane transporters (GTPases) and lipid-bound proteins. For example, Wang J et al isolated exosomes from cell culture media with Amicon Ultra-15 Centrifugal Filter Device and Total Exosome Isolation Kit from Life Technologies [40]. Kapogiannis D et al evaluated plasma exosomes and neuronal-enriched plasma exosomes through positive markers Alix, CD9, cD81, TSG101, and a negative marker GM130 [45].

Tetraspanins are common exosome specific markers. They include CD9, CD63 and CD81 membrane proteins. Tetraspanins are involved in the production of exosomes. In antigen-presenting cells, the functions of MHC-II molecules are regulated by their integration into the cytoplasmic membrane regions enriched in the tetraspanin CD9 [69]. Tetraspanins may be used for diagnostics of various tumors and infectious diseases. In particular, CD63+ exosomes were shown to be significantly increased in patients with melanoma [34] and other cancers [62]. Thus, CD63 has been suggested to be a protein marker of cancer. Also, CD81, another member of the tetraspanin family, plays an important role in cell entry of hepatitis C virus. Exosomal CD81 was demonstrated to be significantly increased in the serum of patients with chronic hepatitis C [63], indicating that CD81 may be a marker for the diagnosis of hepatitis C viral infection.

Several exosomal proteins may be efficiently used in the diagnosis of brain tumors and other diseases of the central nervous system. Glioblastoma-specific epidermal growth factor receptor vIII (EGFRvIII) was suggested to be a specific marker of glioblastoma [64]. With regard to the central nervous system, EGFR, EGFRvIII and TGFβ were also detected in the exosomes isolated from the serum of patients with brain tumors [70].

Moreover, exosomes were suggested to be involved in the pathogenesis of neurodegenerative pathologies. Several aggregated proteins specific for Alzheimer’s, Parkinson’s and Creutzfeldt-Jakob diseases were detected in the cerebrospinal fluid and peripheral blood of individuals suffering from these diseases [71]. In particular, tau phosphorylated at Thr-181 was detected in the exosomes obtained from the cerebrospinal fluid samples of patients with Alzheimer’s disease [72]. Tau phosphorylated at Thr-181 is an established biomarker for Alzheimer’s disease. Also, alpha-synuclein, a peptide playing an important role in the pathogenesis of Parkinson’s disease, was found in the exosomes obtained in an in vitro model of Parkinson’s disease [73]. Besides, prion proteins were detected in the exosomes produced by prion-infected neurons [74].

Various exosomal markers have been identified in urinary exosomes and may be applied for the diagnosis of urinary tract diseases. Two urinary exosomal markers, fetuin-A and activating transcription marker 3, were found to be increased in patients with acute kidney trauma [58, 59]. Also, exosomal markers found in urine can also be markers of bladder and prostate tumors. Several urinary exosomal molecules detected in urinary exosomes were shown to be increased in patients with bladder cancer. They include retinoic acid-induced protein 3 (GPRC5A) and resistin [66]. In addition, two urinary exosomal proteins, PCA-3 and TMPRSS2:ERG, were demonstrated to be associated with prostate cancer [68]. Also, recent studies suggested that urinary exosomal proteins alpha 1-antitrypsin and histone H2B1K may be specific urinary markers of urothelial carcinoma [75].

In addition to the brain and urinary tract tumors, exosomal proteins can also be specific markers for pancreatic cancer. A recent study by Melo et al has shown that a cell surface proteoglycan, glypican-1 (GPC1), is enriched on the exosomes derived from pancreatic cancer cells [65]. Moreover, a decrease in GPC1+ exosomes was suggested to be a prognostic marker for patient survival. The findings were confirmed using mice with spontaneous pancreatic tumors. With regard to gastrointestinal malignancies, exosomal protein markers of colorectal cancer include EpCAM, cadherin-17, mucin 13 (MUC-13), keratin 18, claudins and ephrin B1 [76].

In addition to proteins, exosomes also contain RNAs [77]. Exosomal miRNAs may be applied for diagnostics of various tumors (Figure 3). Eight miRNAs were determined to be specific markers of ovarian cancer [78]. Also, miRNAs were reported to be increased in serum and tumor samples from patients with lung adenocarcinoma, which makes them useful for the diagnosis of lung adenocarcinoma [79]. In addition, elevated levels of exosomal miRNAs miR-141 and miR-375 in serum were found to correlate with the progression of prostate cancer [80]. Besides, exosomal miRNAs can be specific biomarkers for esophageal squamous cell cancer (ESCC). Recent studies showed increased serum levels of exosomal miRNA-21 [81] and miRNA-1246 [82] in patients with ESCC. Moreover, increased miRNA-1246 correlated with metastasis stage and thus poor survival. Interestingly, exosomal miRNAs may be potential diagnostic markers for renal fibrosis [83] and cardiovascular disease [84]. In addition, urinary exosomal miRNA miR-320c and miR-6068 were shown to be significantly upregulated in diabetic nephropathy patients [85]. With regard to the progressive degenerative disorders, high expression levels of several miRNAs, such as miR-9, miR-107,miRNA-128, miRNA134 and miRNA-137 miRNA124, were found in biological fluids from patients with Alzheimer’s disease [86].

In addition to serum and urine, other biological fluids may be considered as sources for exosomes. In particular, different types of RNAs were identified in salivary exosomes [92]. Furthermore, exosomes isolated from saliva were shown to contain diagnostic biomarkers of pancreatic cancer [93]. In addition, exosomes have been isolated from amniotic fluid, indicating their potential application in neonatal diagnostics [94]. Several miRNAs were detected in amniotic fluid and showed a correlation with pregnancy stage [95].

In addition the traditional isolation techniques, commercial precipitation solutions and column-based purification kits have become available and they are already commonly used. Such products are easy-to-use and do not require specialized equipment. However, their mode-of-action is proprietary, and has neither been disclosed nor validated. A few comparative studies, in which the performance of commercial exosome extraction products is analyzed together with traditional isolation methods or with other similar products have been published [53, 96-102].

Table 4 lists available commercial exosome isolation kits together with some of their characteristics based on published comparative studies. The kits’ performance is indicated relative to the most commonly used traditional purification method: gradient ultracentrifugation (GUC). Overall, the commercial kits show higher yields but less pure exosomes when compared with traditional purification methods. They are recommended as quick and reliable, especially, when working with small samples [53, 97], for total exoRNA analysis [99], small RNA sequencing [99], and transcriptomic analyses [96]. For example, Kapogiannis D et al isolated exosomes from blood samples through Exoquick exosome solution from System Biosciences and further enriched neuronally-derived exosomes by immunoprecipitation with L1CAM antibodies [45].

Antibodies against receptors—tetraspanins, heat shock proteins, or MHC antigens - bound to a fixed phase are commonly used in immunoaffinity capture methods to isolate exosomes selectively. Table 5 includes immunoaffinity methods used to isolate exosomes from different source materials based on specific markers.

The earlier version of this article was titled "Exosomes: isolation methods and specific markers". Dr. Georgeta Basturea contributed to the section "Commercial exosome-isolation kits" in September 2018.