Evaluation of immune and chemical precipitation methods for plasma exosome isolation.

Exosomes are a type of extracellular vesicles (EVs) secreted by multiple mammalian cell types and involved in intercellular communication. Numerous studies have explored the diagnostic and therapeutic potential of exosomes. The key challenge is the lack of efficient and standard techniques for isolation and downstream analysis of nanovesicles. Conventional isolation methods, such as ultracentrifugation, precipitation, filtration, chromatography, and immune-affinity-based approaches, rely on specific physical properties or on surface biomarkers. However, any of the existing methods has its limitations. Various parameters, such as efficacy, specificity, labor input, cost and scalability, and standardization options, must be considered for the correct choice of appropriate approach. The isolation of exosomes from biological fluids is especially challenged by the complex nature and variability of these liquids. Here, we present a comparison of five protocols for exosome isolation from human plasma: two chemical affinity precipitation methods (lectin-based purification and SubX™ technology), immunoaffinity precipitation, and reference ultracentrifugation-based exosome isolation method in two modifications. An approach for the isolation of exosomes based on the phenomenon of binding and aggregation of these particles via clusters of outer membrane phosphate groups in the presence of SubX™ molecules has been put forward in the present study. The isolated EVs were characterized based upon size, quantity, and protein content.

Introduction

Extracellular vesicles (EVs) are nanoscale size bubble-like membranous structures secreted by most cell types and present in blood, urine, saliva, breast milk and cerebrospinal fluid [1]. EVs contain lipids, proteins, RNA, metabolites and are thought to be involved in intercellular communication [2]. There are several categories of EVs: apoptotic bodies (500–1000 nm), which are released from cells undergoing apoptosis, microvesicles (100–350 nm), which are released by evagination of the plasma membrane, and exosomes (40–150 nm) [3]. Exosomes are a subtype of EVs that are released by the fusion of multivesicular bodies with the plasma membrane. Exosomes differ from other types of EVs by a relatively small size and the expression of specific exosomal markers (CD9, CD63, CD81 and others) [3,4].

Extracellular vesicles in general and especially exosomes play a role in cell-to-cell signaling and serve as possible biomarkers for disease diagnosis, prognosis, and therapy [5,6] Their pathophysiological roles are being decoded in various diseases including cancer. In addition, growing data also suggests that exosomes are involved in facilitating oncogenesis by regulating angiogenesis, immunity, and metastasis [7-9]. This provides a growing demand for simple, efficient, and affordable techniques to isolate exosomes. At the moment, the isolation of exosomes with reliable quality and substantial concentration is still a major challenge.

To date, several approaches for exosome isolation have been developed. These include differential ultracentrifugation, size-based ultrafiltration, and microfluidics-based platforms [10,11]. Besides, there are specific exosome precipitation methods, such as immunoaffinity capture-based techniques or lectin-based purification, and non-specific precipitation by PEG, alginic acid and hydrophobic binding [12-14]. According to the International Society for Extracellular Vesicles (ISEV) guidelines, isolated EVs are considered to be exosomes if they are in size range 30–200 nm, have a typical spherical form, contain a bilayer membrane, and are enriched with exosomal markers [15]. Here we compared EVs extracted from human plasma by five different methods: differential ultracentrifugation, sequential ultracentrifugation in a sucrose cushion, sedimentation of EV lectin aggregates, immunoprecipitation of exosomes and SubX™ technology. This new approach to the isolation of vesicles is based on using the proprietary bi-functional compound (SubX™) that can bind clusters of phospholipids on the vesicular surface and thus oligomerize vesicles directly in biological liquids [16]. The subsequent centrifugation precipitates [oligoEVs-SubX™] complex. The pelleted EVs dissociate back to monomers in the reconstruction buffer in a ready-to-use form for downstream applications. The vesicles isolated by the five techniques were characterized based upon size, quantity, CD63, CD81 and Calnexin protein expression, and total protein quality using several complementary methods: nanoparticle tracking analysis, dynamic light scattering, atomic force microscopy, cryo-electron microscopy, and flow cytometry.

Materials and methods

The following reagents were used in the study: FITC-conjugated antibodies to CD63 and APC-conjugated anti-CD81 antibody (Beckman Coulter, USA); exosome isolation kits and kits for detection of the surface exosomal markers by flow cytometry (Lonza, Estonia), SubX™-Exo-DNA Plasma isolation kit (Capital Biosciences, USA), bovine pulmonary surfactant derived liposomes (Surfactant-HL, BIOSURF Ltd.), Bradford reagent and Clarity Western ECL Blotting Substrate (BioRad, USA); rabbit polyclonal antibodies to Calnexin (Abcam, ab22595). All other reagents used in the study were obtained from Sigma-Aldrich (USA).

Plasma sampling and isolation of extracellular vesicles

The plasma samples from healthy donors were obtained from Blood Transfusion Unit of N.N. Petrov National Medical Research Center of Oncology; in accordance with the legislation of the Russian Federation, written informed consent was obtained from each donor. Clinical data were depersonalized. The study protocol (AAAA-A18-118012390156-5) was approved by the Ethics Committee of N.N. Petrov National Medical Research Center of Oncology. Blood samples were collected using EDTA-coated vacutainers. Plasma samples were isolated from peripheral blood using centrifugation at 3,000 rpm at 4 °C for 20 minutes and stored at -80 °C. Samples were thawed on ice immediately before analysis.

Extracellular vesicles were isolated from equal volumes (1 mL) of initial plasma after the preliminary removal of cellular debris and large vesicles by centrifugation (2,000 g for 30 min, and then 16,000 g for 30 min). Within the framework of the study, EVs were isolated by the following methods:

Sequential ultracentrifugation (UC) was performed using the method described earlier [17,18]; it included the UC of plasma (diluted 1:5 with PBS) on a Beckman Coulter centrifuge (SW 55Ti rotor) at 110,000 g for 2 h. After centrifugation, the supernatant was removed, and the pellet was re-suspended in 1 mL of phosphate-buffered saline (PBS) for at least 1 h at 4°C; then the volume was adjusted to 5 mL and re-centrifuged at 110,000 g for 2 h. The resulting pellet was dissolved in 100 μL of PBS.

Sequential UC in a sucrose cushion (UC + Suc) was carried out as described earlier [17]. Briefly, EVs were concentrated by the UC of plasma (diluted 1:5 with PBS) at 110,000 g (SW 55Ti rotor) for 2 h. After centrifugation, the supernatant was removed and the pellet was re-suspended in 4 mL of PBS for at least 1 h at 4°C. The resulting suspension was applied onto 1.5 mL of the sucrose cushion (30% sucrose/Tris/D2O, pH 7.4) and re-centrifuged at 110,000 g for 2 h. After centrifugation, 1 mL of the cushion containing vesicles was gently taken by a syringe from the bottom of the tube. EVs were then concentrated and transferred to PBS by using Amicon-Ultra concentrators, 100 kDa (Millipore, USA). The final volume of the EV suspension was 100 μL.

Exosome isolation based on sedimentation of their lectin aggregates was carried out by the procedure described earlier [19,20]. Concanavalin A (Con-A) was used as a lectin binding sugar residue on the EV surface. This isolation method included the following steps: a) overnight incubation of the supernatant, in the presence of Con-A (1.0 μg/mL) at 4°C under constant stirring to form the EV aggregates; b) precipitation of EV aggregates by centrifugation at 15,000 g for 30 min; c) washing the EV precipitate with PBS and repeating the previous step; d) EV disaggregation in an excess of monosaccharides (40% glucose in PBS) for 4 h; e) ultrafiltration (Amicon-Ultra, 100 kDa, Millipore) and washing with PBS for purification of isolated EVs from Con-A and concentration of the preparation. The final volume of the isolated EV preparation was 100 μL.

Isolation of exosomal EVs by immunoprecipitation from plasma (1 mL) was carried out using the exosome isolation kit (Lonza, Estonia), which contains antibodies to the surface exosomal marker CD9, in accordance with the manufacturer’s recommendations. Briefly, 10 μl of pre-coupled beads were incubated overnight at 4°C in rotator with 1 mL of precleared plasma. After exosome binding, the beads were washed twice with 1 mL of PBS and centrifugated at 5,000 g for 10 min. Exosome elution from beads was performed by incubation with 10 μl of Exosome Elution Buffer for 5 min. Then exosomes were eluted in 100 μL of PBS by centrifugation at 5,000 g for 10 min.

Isolation of EVs by the SubX reagent included the following steps: a) centrifugation of plasma (1 mL diluted 1:20 with PBS) at 10,000 g for 1 h.; b) 30 min incubation of the supernatant, in the presence of 100 μL SubX reagent (1/10 of the plasma initial volume) at room temperature to form the EV aggregates; c) precipitation of EV aggregates by centrifugation at 10,000 g for 30 min; d) washing the EV precipitate with PBS and repeating the previous step; e) EV disaggregation in 300 mM NaCl for 1 h; f) re-centrifugation of the obtained suspension at 10,000 g for 30 min to remove possible contaminations of large aggregates; at this step, the pellet is thrown away, and the supernatant is used for the following step; g) ultrafiltration (Amicon-Ultra, 100 kDa, Millipore) and washing with PBS for the purification of isolated EVs from SubX reagent and the concentration of the preparation. The final volume of the isolated EV preparation was 100 μL.

Thus, five EV preparations (100 μL) isolated by different methods from identical samples of plasma (1 mL) were obtained. The EV samples were aliquoted, rapidly frozen in liquid nitrogen and stored at -80°C until analysis.

Methods for analysis of isolated extracellular vesicles

The EV size distribution was evaluated by the method of dynamic light scattering (DLS) using the particle size and zeta potential analyzer Photocor Compact-Z (Photocor Ltd., Russia) or Zetasizer Nano-ZS (Malvern Instruments, UK) according to the manufacturer’s recommendations. All measurements were carried out at +25°C. For each sample the particle size distribution curves were plotted by results of three measurements.

The EV size and concentration were determined by nanoparticle tracking analysis (NTA) using the NTA NanoSight® LM10 (Malvern Instruments, UK) analyzer, equipped with a blue laser (45 mW at 488 nm) and a C11440-5B camera (Hamamatsu Photonics K.K., Japan). Recording and data analysis were performed with the use of the NTA software 2.3. The following parameters were evaluated during the analysis of recording monitored for 60s: the average hydrodynamic diameter, the mode of distribution, the standard deviation, and the concentration of vesicles in the suspension.

The detection of EVs was carried out by the atomic force microscopy (AFM). Briefly, the EV solution in PBS was diluted 50 times in deionized water, and 10 μL were put on freshly cleaved mica. After 1min incubation at room temperature the mica surface was thrice washed by water to remove salt. The sample topography measurements were performed in semi-contact mode on atomic force microscope “NT-MDT-Smena B”. A probe NSG03 was used (NT-MDT, Russia). The images were analyzed using “Gwyddion” software [21].

Morphology of the isolated EVs was assessed using cryo-electron microscopy (cryo-EM) as described [22]. The initial volume of plasma for the study of EVs using the cryo-EM was 10 mL. The study was carried out on a Titan Krios 60–300 TEM/STEM (FEI, USA) transmission electron microscope equipped with a highly sensitive direct electron detector (DED) Falcon II (FEI) and a spherical aberration corrector (CEOS, Germany). Electron microscopy copper grids coated with a thin layer of amorphous carbon were treated in a Pelco easiGlow unit to produce a hydrophilic surface. After that, 3 μL of an analyzed sample was then applied onto the grids, and the sample was immediately frozen by a VitrobotMarkIV (FEI) unit in liquid ethane cooled to liquid nitrogen temperature (-196°C). The samples were thus fixed in a thin layer of amorphous ice, which allowed the investigation of EVs in their native state. In order to minimize radiation damage, the data set was performed using the EPU (FEI) software operated at a low dose mode.

Analysis of the exosomal markers (tetraspanins CD63 or CD81) on the surface of the isolated EVs was carried out using Exo-FACS ready-to-use kit for plasma exosome analysis (Lonza, Estonia) according to the manufacturer’s recommendations. Bead-coupled EVs were assayed using FITC-conjugated anti-CD63 antibody (Beckman Coulter, USA) and APC-conjugated anti-CD81 antibody (Beckman Coulter, USA). Analysis was performed with CytoFlex instrument (Beckman Coulter, USA).

The presence of the negative exosomal marker Calnexin in the samples of isolated vesicles was determined by western blotting. The 5 samples of isolated EVs from the same amount of plasma were incubated at 4°C for 30 minutes with 20 μL of lysis buffer (7M urea, 2M thiourea, 4% CHAPS, 5 mM PMSF, 1% DTT). The protein samples were diluted in Laemmli buffer (BioRad, USA), subjected to 10% SDS-PAGE containing 0.1% SDS, and transferred to the PVDF membrane (Thermo Scientific) using the Trans-Blot Turbo Transfer System (BioRad, USA). Rabbit polyclonal antibodies to Calnexin (Abcam, ab22595) at dilution of 1:200 were used as primary antibodies. Horseradish peroxidase-conjugated goat antibodies against mouse immunoglobulins from Sigma were used as secondary antibodies at dilutions of 1:10,000. Chemiluminescent detection of the protein bands was performed with Clarity Western ECL Blotting Substrate (Bio-Rad, USA) and Thermo Scientific CL-XPosure Films (Thermo Fisher Scientific, USA). U-87 MG сell lysate (in the same amount of total protein) was used as a positive immunodetection control.

The total protein content in EV samples was determined using a colorimetric method (Bradford) after lysis of vesicles in a buffer containing urea (7M urea, 2M thiourea, 4% CHAPS, 1% DTT). Analysis was performed with BioRad SmartSpec Plus instrument (BioRad, USA).

Results

SubX™ technology validation

An approach to isolating EVs based on the phenomenon of binding and aggregation of these particles via clusters of outer membrane phosphate groups in the presence of SubX™ reagent (Capital Biosciences, USA) has been put forward in the present study. It is assumed that, since vesicular membrane phospholipids contain multiple phosphate groups, this can lead to the formation of [SubX+EVs] aggregates. We first tested this feature on a model system. Thus, to confirm phosphate group clusters mediated mechanism of lipid vesicle aggregation via SubX™ anchoring, we performed a model experiment, which includes SubX™ addition to phospholipid-based and phosphatidylcholine-based vesicles with blocked phosphate groups. As phospholipid-based vesicles we employed commercially available liposomes manufactured from natural bovine lung extract (Surfactant-HL, BIOSURF Ltd.). As phospholipid particles with blocked phosphate groups we employed an in-house prepared phosphatidylcholine emulsion of exosomal size. Vesicles were analyzed by DLS to determine the particle size distribution in the total light scattering of the analyzed liquid sample. As seen from Fig 1, the liposomes derived from the emulsion of bovine lung alveolar liquid with phospholipide content of 80% have a bimodal particle size distribution with the maximum at 100 nm and 500 nm (Fig 1A, red line). SubX™ efficiently binds and oligomerizes phospholipid enriched vesicles into aggregates with the mean size of ~1000 nm (Fig 1A, blue line). In contrast, phosphatidylcholine-derived vesicles with blocked phoshate residues (Fig 1B, red line) do not interact with SubX™, and their size distribution pattern remains the same after the addition of SubX™ (Fig 1B, blue line). These data strongly confirm the suggestion that SubX™ captures phosholipid-containing vesicles through phosphate moietie clusters displayed on outer membrane.

Fig 1

Effect of SubX™ on phospholipid-based vesicles (A) and phosphatidylcholine-based vesicles (B).

Vesicles were analyzed by dynamic light scattering (DLS) to determine the particle size distribution in the presence (blue line)/absence (red line) of the SubX™ reagent.

Next, we tested the assumption that clusters of phospholipids on the surface of plasma vesicles can also be bound by the SubX™ reagent, leading to the aggregation of the EVs. To do this, we tried the following procedure: the blood plasma (1mL) diluted at 1:20 by PBS was centrifuged at 10,000 g for 1 h to remove large particles and cellular debris; then 100 μL of SubX™ reagent (1/10 of the plasma initial volume) was added to the supernatant and incubated 30 min at room temperature; after that, the sample was centrifuged at 10,000 g for 30 min, the supernatant was removed, and the pellet was re-suspended in PBS; then the sample was centrifuged at 10,000g for 30 min again, and the final pellet was re-suspended in a 300 mM solution of NaCl. At each stage of this procedure, an aliquot of the sample was analyzed using DLS. The results are shown in Fig 2.

Fig 2

Aggregation of plasma exosome-like vesicles by a SubX™ reagent.

Particle size distribution in: the initial plasma pre-cleaned from cell debris (A); the supernatant (B) and resuspended pellet (C) obtained after incubation of precleaned plasma with SubX™SubX and the first centrifugation at 10,000 g; the final pellet after the second round of centrifugation at 10,000 g and resuspension of the precipitate in a 300 mM solution of NaCl (D). Data obtained by dynamic light scattering (DLS). The X-axis is the hydrodynamic diameter of the particles in nm, the Y-axis is the contribution to the scattering in %.

In the supernatant obtained after the first centrifugation of diluted plasma, only a small number of particles with exosomal size were observed. Most likely, this is due to the masking of exosome-like vesicles by a large number of proteins present in the plasma and identified on the histogram as a peak with a particle size of about 10 nm (Fig 2A). Most of these proteins remain in the untargeted supernatant after the incubating of the sample with the SubX™ reagent and the first centrifugation at 10,000 g (Fig 2B). At the same time, particles with sizes of about 300 nm were observed in the re-suspended pellet obtained after the incubation with the SubX™ reagent (Fig 2C). In addition, the peak corresponding to particles with sizes of about 10 μm was significantly larger than that in all analyzed aliquots (Fig 2C). These data suggest that the addition of SubX™ to diluted plasma resulted in the appearance of the peaks corresponding to molecular aggregates substantially larger than exosomes. These aggregates could be sedimented by centrifugation at low speed (10,000 g). The addition of 300 mM NaCl to the final pellet resulted in the destruction of the bonds between the molecules of SubX™ and phospholipid clusters on the surface of EVs and the release of single vesicles from the aggregates. Disaggregated exosome-like vesicles have been detected by DLS as a peak with the size of about 90 nm (Fig 2D). These results clearly indicate the presence of the sites for the binding of SubX™ on the outer membrane of the vesicles, which confirms the possibility of EV separation from the plasma using SubX™ technology. A small peak corresponding to a particle size of about 10 nm indicates insignificant contamination of the EV sample with supposedly phosphorylated molecules (Fig 2D).

In order to confirm the vesicular nature of particles isolated with SubX™, these samples were analyzed by cryo-EM. As seen in Fig 3, isolated particles are indeed formed by a characteristic lipid bilayer, which has an average thickness of ∼10 nm, and have a round-shaped vesicular morphology. The visualized particles with a lipid bilayer generally varied in size from 50 to 220 nm (Fig 3).

Fig 3

Cryo-EM images of extracellular vesicles isolated from plasma by SubX™ reagent.

The white arrows point to the vesicles with a double membrane. Scale bars are 100 nm.

Comparison of SubX technology with conventional methods (physical properties)

The isolation of the circulating nano-vesicles (exosomes) from plasma is usually performed by one of the following methods: ultracentrifugation (UC), sequential UC in a “sucrose cushion”, agglutination by natural or synthetic polymers, capture by immune-beads with antibodies against known exosomal markers. Further in this study, we isolated EVs from equal samples of human plasma (1 mL) using SubX™ technology and the four methods mentioned above in parallel. Five samples of isolated vesicles were analyzed by DLS, nanoparticle tracking analysis (NTA) and atomic force microscopy (AFM). Presence of the exosomal surface markers CD63 and CD81 in membrane of isolated vesicles was evaluated by flow cytometry.

DLS was used to estimate the averaged hydrodynamic radius of isolated vesicles (Fig 4). Thus, a peak of 100 nm corresponding to the size of exosome-like vesicles was observed in all samples of EVs isolated by different methods. However, only the SubX™ protocol resulted in the isolation of predominantly 100 nm vesicles with minor contamination by plasma proteins (about 5–20 nm) (Fig 4A). Different particles sized from 50 nm to 200 nm contaminated by proteins (about 5–20 nm) and large (>10 μm) particles were isolated by UC (Fig 4B). UC with a “sucrose cushion” yielded a population of vesicles with a predominant fraction sized 100 nm (Fig 4C); however, small (<1 nm) and large (>10 μm) particles are still present. Thus, the use of “sucrose cushion” during UC increases the purity of the exosome-like fraction of isolated vesicles. The method of EV isolation involving agglutination by lectins, followed by sedimentation and dis-agglutination with monosaccharides, resulted in the isolation of particles of different sizes, including 100 nm. However, smaller particles and large aggregates, probably stemmed from non-complete disaggregation of EVs, were observed (Fig 4D). Isolation with immune-bead bearing antibodies against exosomal marker CD9 results in the isolation of an almost pure population of exosomes. A large peak in Fig 4E corresponding to the size of several micrometers reflected the presence of immune-beads in the sample.

Fig 4

Evaluation of the distribution of extracellular vesicles (EVs) in size by the method of dynamic light scattering (DLS).

Designation of the isolation methods: SubX™ technology (A); Ultracentrifugation–UC (B); ultracentrifugation in the cushion of 30% sucrose—UC + Suc (C), precipitation of lectin aggregates—Con-A (D); immunoprecipitation–IP (E). The X-axis is the hydrodynamic diameter of the particles in nm, the Y-axis is the contribution to the scattering in %.

At the next stage, NTA was applied to confirm measurement of EV size and to estimate their concentration (Fig 5). The mode size of vesicles isolated by all methods was in a range from 63 to 99 nm, which corresponded well with the size of exosomes. However, concentrations of vesicles isolated by different methods were quite different. Ultracentrifugation allowed to obtain the highest concentration of vesicles–(7.8 ± 0.7) x1011 particles/mL (Fig 5B). The use of “sucrose cushion” considerably reduced the yield of vesicles–(2.3 ±0.4) x1011 particles/mL (Fig 5C). An even lower concentration of vesicles was obtained by other methods: immune capturing (1.1±0.3) x1011 particles/mL (Fig 5E), SubX™ (0.7±0.2) x1011 particles/mL (Fig 5A) and agglutination by lectins (0.4±0.1) x1011 particles/mL (Fig 5D).

Fig 5

Nanoparticle tracking analysis (NTA): Size distribution (nm) and particle concentration (x1011 particles/mL) in preparations of extracellular vesicles.

Designation of the isolation methods: SubX™ technology (A); Ultracentrifugation–UC (B); ultracentrifugation in the cushion of 30% sucrose—UC + Suc (C), precipitation of lectin aggregates—Con-A (D); immunoprecipitation–IP (E).

Surface topology of plasma nanovesicles was estimated by AFM. In five samples of vesicles isolated by different methods, we have observed individual particles of a spherical shape that corresponds to vesicular topology (Fig 6A and 6C–6F). The population of vesicles isolated by SubX™ was analyzed in more details. Individual vesicles examined under AFM show characteristic cup-shaped morphology, appearing as flattened spheres with diameters ranging from 30 to 70 nm (Fig 6B). The cup-shaped morphology is most likely originated from the sample preparation process of conventional AFM microscopy, during which the vesicles are extremely dehydrated.

Fig 6

The surface topography of vesicles isolated by SubX™ technology (A), ultracentrifugation (C), ultracentrifugation in 30% sucrose (D), precipitation of lectin aggregates (E); immunoprecipitation (F). The characteristic “cup-shape” particle profile, presented on (B), “h”–vesicular height (nm) and “l”–diameter (nm). The scale bars are 300 nm. On the right of (A)-(F) is the pseudo color ruler indicating the particles’ height (nm).

Comparison of SubX™ technology with conventional methods (molecular content)

As suggested previously by the international society of EVs [15], we have analyzed a set of proteins, which should either be present in or excluded from the exosome population. In order to confirm the exosomal nature of isolated vesicles, they were non-specifically absorbed on the flow cytometry beads (4 μm) followed by incubation with antibodies to exosomal markers (CD63 or CD81). The presence of exosomal markers on the surface of vesicles was estimated in parallel in five samples of vesicles isolated by different methods using the same exosomal standard as positive control and beads non-incubated with exosomes as a negative control. The presence of CD63 and CD81-positive vesicles was confirmed by flow cytometry in all samples including the sample of EVs isolated by SubX™ (Fig 7A and 7B).

Fig 7

Analysis for positive and negative exosome markers in the samples of vesicles isolated by 5 methods.

Flow cytometry analysis of isolated vesicles for the surface expression of CD63 (A) and CD81 (B) tetraspanins classically used as exosome markers. Immunobeads blocked with BSA and stained with anti-CD63 or CD81 antibodies were used as negative control (–control). The exosomal standard included in the exosome cytometric assay kit (Lonza) was used as a positive control (+ control). Western blot analysis for exosome negative marker, calnexin, in isolated samples of vesicles and U-87 MG cell lysate sample (C). Designation of the isolation methods: SubX™ technology; Ultracentrifugation–UC; ultracentrifugation in the cushion of 30% sucrose—UC + Suc, precipitation of lectin aggregates—Con-A; immunoprecipitation–IP.

Proteins associated with compartments other than plasma membrane or endosomes should not be detectable within exosomes [17]. In fact, we were able to show that Calnexin, a protein associated with the endoplasmic reticulum, was found in whole U-87 MG cells, to a much lesser extent also in the sample of vesicles isolated with Con-A, but was excluded from all samples of vesicles isolated by other methods (Fig 7C).

Finally, total protein content of the vesicles isolated from the same amount of plasma by different approaches was tested (Table 1). Protein concentration seems to reflect the concentration of vesicles in the samples determined by NTA: the highest concentrations of proteins were detected in samples isolated by UC (0.45±0.08), with the highest concentration of vesicles being 7.8 x 1011, and UC with “sucrose cushion” (0.18±0.03), with the concentration of vesicles being 2.3 x 1011. Isolation of vesicles by lectin and SubX™ yielded to low concentrations of both vesicles and proteins.

Table 1

Total protein content in vesicles isolated by methods: Ultracentrifugation—UC; ultracentrifugation in the cushion of 30% sucrose—UC + Suc; precipitation of lectin aggregates—Con-A; immunoprecipitation—IP; SubX™ technology.

	Total protein (mg/ml) (Bradford assay)
UC	0.45±0.08
UC+Suc	0.18±0.03
Con-A	0.10±0.01
IP	0.15±0.05
SubX™	0.12±0.07

Discussion

As cell-derived extracellular vesicles, exosomes play a significant role in intercellular communication by serving as a carrier for the transfer of proteins, lipids, and RNA between cells [3]. Exosomal components have been found to be related to certain diseases and treatment responses, including cancer, neurodegenerative, and cardiovascular diseases. Therefore, they are considered to be crucial for the discovery of biomarkers for clinical diagnostics [23-25]. In addition, several studies have reported that EVs can be employed as a drug delivery system for targeted therapies with drugs and biomolecules [26,27]. The isolation of EVs or pure exosomes is critical for their subsequent analysis and applications in biomedical sciences. Various techniques have been adopted to facilitate the purification of exosomes [24,27]. Nevertheless, isolation of exosomes is still a state-of-art technique due to their vesicular nature–heterogeneity of particle size and shape pattern, variable membrane composition, and bio-liquid specificity. Here we compared the EVs isolated from equal volumes of human plasma by five different methods: differential ultracentrifugation, sequential ultracentrifugation in a sucrose cushion, sedimentation of EV lectin aggregates, immunoprecipitation of exosomes using commercial kit, and by SubX™ technology.

Ultracentrifugation-based EV isolation is considered to be the gold standard and is one of the most commonly used and reported techniques for exosome isolation [28,29]. Here we used two types of preparative ultracentrifugation–differential ultracentrifugation and sequential ultracentrifugation in a sucrose cushion. Based on a comparative analysis of the five differently isolated samples from plasma, we can conclude that the highest yield of exosomes was achieved by means of ultracentrifugation. However, exosomes obtained by this method are characterized by the highest level of contaminations with non-vesicular particles. Combination of the UC with the sucrose cushion reduces the level of non-vesicular contamination, but the number of isolated vesicles drastically decreases. Similar data were shown in a number of previous studies that analyzed the quality and quantity of vesicles isolated from various biological fluids by these methods [30-32].

Most exosomes contain proteins that are common among all exosomes regardless of the types of cells which secrete them [33,34]. These characteristic proteins therefore may serve as convenient biomarkers for the isolation and quantification of exosomes. However, several earlier studies demonstrated variability of vesicular populations secreted even by the same cells in terms of membrane protein content and biological properties [35-37]. Different approaches to separate vesicles on the basis of their physical properties (by differential centrifugation [38] or asymmetric flow field-flow fractionation [39]) also revealed distinct subsets of extracellular vesicles. Moreover, proteomic analysis [40] as well as deep RNA sequencing [41] have confirmed the existence of heterogeneous populations of extracellular vesicles of exosomal nature. Here we used plasma exosome purification kit (Lonza, Estonia) for immunoprecipitation of exosomes. Based on the results, we can state that the vesicle preparation obtained in this way is the most enriched with CD63 or CD81-positive vesicles; however, the number of particles released from immune-beads is extremely low compared to other approaches. It may be correct to say that immunoprecipitation allows to isolate specific populations of exosomes enriched with a certain surface marker, but not complete fraction of 100 nm exosomal vesicles. Thus, such extreme specificity of the isolation procedure is not always desirable.

Recently, a number of non-immunological precipitation procedures have been launched to isolate and study vesicles for various purposes [24,27]. Compared to ultracentrifugation, these procedures are less time consuming, less technique sensitive, and do not require special equipment. Most of these isolation methods are based on the composition of microvesicle membranes. For example, the outer surface of EVs and exosomes is rich in saccharide chains, such as mannose, polylactosamine, alpha-2,6 sialic acid, and N-linked glycans [42]. For our comparative analysis we used a preparative technique for isolation of EVs based on their ability to aggregate in the presence of lectins. The method for lectin-based isolation of exosomes from different biological fluids was tested earlier for exosome-based protein or miRNA biomarker researches [12,19,20]. In the comparative analysis of the methods for isolating vesicles that we conducted in this study, the isolation of EVs by precipitation of lectin aggregates proves significantly inferior to all other methods in terms of purity and quantity of the final sample. These data are consistent with the observations we made earlier when exosomes were isolated from the culture medium [32]. The advantages of the Con-A method are technical simplicity, the ability to handle large initial volumes of biological fluid, and a relatively low cost.

SubX™ is the alternative innovative technology based on affinity capture of membrane phosphate moieties of two neighbor vesicles via bifunctional SubX™ molecule. Since vesicular membrane phospholipids contain multiple phosphate groups, it results in an assembly of [SubX™+EVs] oligomers that precipitate from a complex bio-liquid by conventional bench-top centrifugation step. The pelleted EVs dissociate back to monomers in the reconstruction buffer in a ready-to-use form for downstream applications. The basic characteristics of SubX™ technology and four other methods are compared in Table 2.

Table 2

Summary of different exosomes isolation methods.

	Yield*	Purity**	Time, hours	Scalability	Standardization
UC	7.8 ± 0.7	55,3	7–8	Hardly	Hardly
UC+Suc	2.3 ±0.4	79,1	8–9	Very hardly	Very hardly
Con-A	0.4±0.1	42,6	18–20 (ON)	Intermediate	Hardly
IP	1.1±0.3	96,1	14–16 (ON)	Well	Well
SubX™	0.7±0.2	92,8	5–6	Well	Well

*The yield of exosome isolation reflecting efficacy of method is presented as results of NTA (particles concentration, x1011particles / mL);

** The purity of isolated exosomes is presented as results of DLS (% of ≈ 100 nm particles in the whole measured population of particles), the peak corresponding to immunobeads used for IP is excluded from calculation. ON is an overnight incubation.

The results of NTA or DLS demonstrate the presence of particles with a characteristic exosome size in the final sample isolated by SubX™. Comparison Sub-X technology with other methods indicates its relatively low efficacy (yield), yet high specificity (purity). In terms of other characteristics, SubX™ technology is not labor consuming, is well scalable and can be easily standardized. Cytometric detection of the CD63 and CD81 markers on the surface of the particles confirms the exosomal nature of the vesicles isolated from human plasma by SubX™. Cryo-electronic and atomic force microscopy analyses demonstrate that SubX™ technology allows obtaining homogeneous exosomal-size particles in the final preparation. Cryo-EM does not suffer from the effects of dehydration and fixation issues. Thus, Cryo-EM is considered the best method for visualizing extracellular vesicles and proteins without dehydration artifacts [22,43]. In this study, we capture images of exosomes isolated by SubX™, with membrane structures and lumens.

Conclusion

Summarizing all the data, we can assume that SubX™ allows obtaining relatively pure populations of exosomes, while the concentration of isolated vesicles is rather low. Undoubtedly, this method is suitable for the isolation of EVs from human blood plasma. The method is rather simple and does not require complex, expensive equipment. Its scope is limited to a relatively low yield of the target product; however, a more comprehensive analysis of SubX™-isolated EVs may help to further our insight into the complex composition of plasma exosomes.

(TIF)

Click here for additional data file.

4 Aug 2020

PONE-D-20-14332

Evaluation of immune and chemical precipitation methods for plasma exosome isolation

PLOS ONE

Dear Dr. Shtam,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Sept 22,2020. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Girijesh Kumar Patel, PhD

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2.In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript by Tatiana Shtam et al., the authors evaluated several different methods for isolation of plasma derived exosomes.

The experiments were expertly performed and were appropriately controlled. The data presentation is good. The experimental design is appropriate and the data are convincing. The manuscript is clearly written.

However, there are some major concerns:

- To estimate the purity of isolated exosome fractions it would be necessary to provide the analysis of non-EV-associated molecules, such as Apolipoproteins, to see how much contaminants are co-isolated in each method.

In lines 272-274 the authors write e.g. "Without contamination by plasma proteins", but just determined this on the basis of size.

The MISEV 2018 guidelines provide a table with categorized proteins that should be used to analyze the nature and purity of EVs (https://www.tandfonline.com/doi/full/10.1080/20013078.2018.1535750, table 3). This was not fully considered by the authors, since only CD63 was analyzed (e.g. line 431-433: Analysis of CD63 alone is not sufficient to confirm that these particles are exosomes).

- The authors did not use a uniform nomenclature for the vesicles they isolated. Sometimes the term “EVs” was used, then “exosomes” or “exosome-like vesicles”.

- The English could be improved, sometimes the authors use wrong words, e.g. in line 421 “adventures” instead of “advantages”.

Reviewer #2: In this manuscript, authors present a comparison of five protocols for exosome isolation from human

plasma: two chemical precipitation methods (lectin-based purification and SubX TM

technology), immunoaffinity precipitation, vs. basic ultracentrifugation-based exosome

isolation method in two modifications.

The over all manuscript is well written and informative. The authors should proofread manuscript. Discussion section can be improved further by removing redundancy.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

2 Oct 2020

We thank the Editor and the Reviewers for the interest in our work and constructive comments. We tried to satisfy these remarks, if it was possible. All major modifications are highlighted in red inside of the manuscript. Besides, Fig.2-7 were modified, additional panels were added to Fig.7., Table2 was included, and additional refs (4 and 14) were added in Reference Section.

The detailed replies to Reviewer’s comment are below.

Reviewer #1:

In this manuscript by Tatiana Shtam et al., the authors evaluated several different methods for isolation of plasma derived exosomes. The experiments were expertly performed and were appropriately controlled. The data presentation is good. The experimental design is appropriate and the data are convincing. The manuscript is clearly written.

However, there are some major concerns:

- To estimate the purity of isolated exosome fractions it would be necessary to provide the analysis of non-EV-associated molecules, such as Apolipoproteins, to see how much contaminants are co-isolated in each method.

>>Thank you very much for your fair comment. We have added Western blot analysis for the presence of Calnexin in isolated vesicle samples. Calnexin, a protein associated with the endoplasmic reticulum, is one of the negative markers of exosomes. The corresponding additions are made to Figure 7 and the revised manuscript to the Results and Materials & Methods sections.

In lines 272-274 the authors write e.g. "Without contamination by plasma proteins", but just determined this on the basis of size.

>>We absolutely agree that a too strong statement was made, not supported by additional methods. We have corrected the wording.

The MISEV 2018 guidelines provide a table with categorized proteins that should be used to analyze the nature and purity of EVs (https://www.tandfonline.com/doi/full/10.1080/20013078.2018.1535750, table 3). This was not fully considered by the authors, since only CD63 was analyzed (e.g. line 431-433: Analysis of CD63 alone is not sufficient to confirm that these particles are exosomes).

>>We agree with the comment of the reviewer about the insufficient determination of only one exosomal marker, and we followed their suggestions. We have added a flow cytometric analysis of the presence of tetraspanin CD81 on the surface of the five-way isolated vesicles. The corresponding additions are made to Figure 7 and the revised manuscript.

The authors did not use a uniform nomenclature for the vesicles they isolated. Sometimes the term “EVs” was used, then “exosomes” or “exosome-like vesicles”.

>>Thank you for your comment, we have tried to correct the nomenclature variety.

The English could be improved, sometimes the authors use wrong words, e.g. in line 421 “adventures” instead of “advantages”.

>>English corrected throughout the text

Reviewer #2:

In this manuscript, authors present a comparison of five protocols for exosome isolation from human plasma: two chemical precipitation methods (lectin-based purification and SubX TM technology), immunoaffinity precipitation, vs. basic ultracentrifugation-based exosome isolation method in two modifications.

The over all manuscript is well written and informative. The authors should proofread manuscript. Discussion section can be improved further by removing redundancy.

>>Thank you for your good evaluation of our study and helpful comments. The Discussion section is shortened in the revised manuscript. Language correction have been made across all the text of manuscript.

Reviewer #3:

The authors carry out a reasonably-designed comparison of 5 different methods of EV isolation from human plasma. Included is a stated ‘novel’ method based on a phosphate-driven aggregation based on the compound SubXTM.

>>We thank the reviewer for the attention and suggestions that we believe will improve our manuscript.

Below is our point-by-point description of the revisions according to the reviewer’s comments.

We are never told the identity of the compound, and so are immediately put off as this should be an open description of science. We are never informed as to the commercial source of this reagent nor is there a specific reference to the origins of the methodology described on page 6. We find this to be disingenuous and to be grounds itself for rejection.

>>In our early studies, we used the SubXTM Plasma cfDNA Isolation Kit to isolate extracellular DNA from plasma. The description of this reagent published on the company's website (https://www.capitalbiosciences.com/cfdna-isolation-reagents), namely the mention of the ability of SubXTM to bind phosphate residues, gave us the idea of using this reagent to isolate exosomes, the membrane of which is enriched in phospholipids. The presented study is devoted to the experimental verification of this idea.

The technique for isolating exosomes using SubXTM reagent was tested and modified in the course of experiments. This technique was based on the procedure developed by us for the isolation of vesicles from biological fluids using the precipitation of lectin aggregates of exosomes. (Lectin‐induced agglutination method of urinary exosomes isolation followed by mi‐RNA analysis: Application for prostate cancer diagnostic. Prostate. 2016;76: 68–79. doi:10.1002/pros.23101; Shtam TA et al Aggregation by lectins as an approach for exosome isolation from biological fluids: Validation for proteomic studies. Cell tissue biol. 2017;11: 172–179; Samsonov et al., Shtam TA et al., Isolation of Extracellular Microvesicles from Cell Culture Medium: Comparative Evaluation of Methods. Biochem Suppl Ser B Biomed Chem. 2018;12: 167–175. doi:10.1134/S1990750818020117, ref. 19, 20, 32).

SubXTM technology and line of isolation kits have been obviously developed over the past years. And now, an exosome isolation SubX reagent is also commercially available from Capital Biosciences (https://www.capitalbiosciences.com/exosome-isolation-kits). To our request to the company, we received the expected response that SubXTM is the trade secret of Capital Biosciences, as many commercially available kit components from different vendors. Information concerning SubXTM features are available on Capital Biosciences website (https://www.capitalbiosciences.com/liquid-biopsies ). But, as far as we know, there is no information in the scientific literature on the use of this kit for the isolation of extracellular vesicles. The studies presented in the manuscript demonstrate the effectiveness of the isolation of exosome-like vesicles by SubXTM reagent. This technology obviously has its advantages and disadvantages, some of which are considered in our study. We hope that the obtained data will be published and may become useful for research in the field of studying extracellular vesicles.

The comparative methods are all well known in the field. Many of the typical comparators are evaluated, though in the end it is not clear which metric sets this approach apart from the others. Indeed, there should have been a summary table inclusive of the metrics which were set out in the Abstract, “ . . efficacy, specificity, labor input, cost and scalability and standardization . .“.

It appears that the developed method may provide a high level of particle (assumed exosome) size homogeneity, but at the high expense of overall recoveries and much longer and complex processing protocols.

>>We agree with these comments and, as suggested by the reviewer, we have included a comparison Table2 in the Discussion section of the revised manuscript.

As the method described here assumes to take advantage condensing vesicles together based on surface phosphate groups, the potential for co-condensation of phosphorylated proteins is never discussed nor experimentally ruled out. This is a fatal omission. Taken a step farther, a primary ‘contaminant’ in exosome isolation protocols is the inclusion of phospholipids. This point is never addressed.

>>Yes, indeed, the issue of contamination of extracellular vesicles with other plasma components, including proteins, is one of the main issues in the development of new approaches to isolating vesicles. However, contamination of vesicles with non-vesicular proteins should be taken into account in proteomic analysis of vesicular samples. However, the presence of additional proteins obviously does not affect the analysis of the miRNA profile of the isolated vesicles. It all depends on the ultimate goal of isolating exosomes from biological material. In this study, we convincingly demonstrate that using the SubXTM reagent, it is possible to isolate vesicles having a characteristic size (Fig. 2.5) and shape (Fig. 6) of exosomes, a bilipid membrane (Fig. 3) and exosomal markers on the surface (Fig. 7) (with complete absence of a negative marker of exosomes (Fig. 7)). A detailed study of the contamination of final vesicle samples, unfortunately, is beyond the scope of this study and is virtually impossible to implement using the methods used. The data obtained only allow us to assume the presence of minor protein contamination, based on the results of DLS shown in Figures 2 and 4. The corresponding minor additions were made to the Results section. In addition, available reagent descriptions indicate that for anchoring target molecule/microvesicle SubXTM requires oligophosphate cluster of at least 20 residues. This indirectly indicates the ineffectiveness of the SubXTM reagent to form complexes with proteins and phospholipids containing fewer phosphate groups, which in turn minimizes the potential for contamination of vesicles with phosphorylated molecules. According to the company website data SubXTM does not bind non-membrane integrated phosphoproteins and phospholipids molecules and aqueous micelles (https://www.capitalbiosciences.com/liquid-biopsies).

We also find the referencing to be sorely lacking as the series of recent papers from the group of Marcus et al., wherein polymer fibers are used with very high recoveries and purities, low-cost materials, and <15 min processing times for human plasma is ignored.

>>Thank you for drawing our attention to the very interesting works of the Marcus group. In the revised manuscript, we quoted their data in the Introduction section (Ref.14: Bruce TF, Slonecki TJ, Wang L, Huang S, Powell RR, Marcus RK. Exosome isolation and purification via hydrophobic interaction chromatography using a polyester, capillary-channeled polymer fiber phase. Electrophoresis. 2019;40(4):571-581. doi:10.1002/elps.201800417). In our paper we did not discuss published, patented, and on-marked dominated group of “non-specific” methods and kits. Instead, we have compared three “affinity” techniques (immuno-, glicosacharide-, and oligophosphate-targeted) for exosome isolation along with two “golden standard” ultracentrifugation ones.

Our more specific comments regarding the manuscript and methods follow (noted with respect to line number).

50. Should be the plural (EVs)

>>corrected

58. The last sentence of the paragraph should include references.

>>Done

70. While methods based on “hydrophobicity” are listed, none of the references relates to that approach.

>>Thank you for your fair comment, we have added the appropriate reference.

79. We understand the intent, but is “disaggregate” a real term? “Dissociate” is probably more correct.

>>corrected

82-84. These methods should not be capitalized.

>>corrected

99. What approach was used to isolate the plasma? Referencing.

>>A brief description of the procedure is included in the Materials and Methods section.

115. Is deuterated water (D2O) really used? If so, why?

>>Yes, indeed we used deuterium water to prepare the sucrose cushion (30% sucrose/Tris/D2O, pH 7.4) exactly following the method detailed in ref. 17 of the revised manuscript.

116. The phrase “a fraction of pure vesicles” is totally vague. What size fraction? How “pure” are the vesicles? Pure is a very strong word.

>>corrected

111-119. If this method used a sucrose cushion UC method and an ultra-filtration concentrator, how can you compare the recoveries of the "UC cushion technique" to the other isolation methods that did not employ this additional concentration step? You don't know if the EV recoveries are efficiently isolated by the UC sucrose cushion itself, or by the ultra-filtration concentrator. If this technique is to be truly compared to the others, the recoveries after the sucrose cushion UC method should be used for quantitative purposes, and if still necessary, the ultra-filtration should be used as an additional isolation method, where these techniques are employed in tandem, then compared to others. 3 of the 5 presented methods mentioned used ultrafiltration techniques. How can you account for this with comparison to others?

>>A sucrose cushion eliminates more contaminants, such as proteins nonspecifically associated with exosomes, or large protein aggregates, which are sedimented by centrifugation (Szatanek R, Baran J, Siedlar M, Baj-Krzyworzeka M. Isolation of extracellular vesicles: Determining the correct approach (Review). Int J Mol Med. 2015;36(1):11-17. doi:10.3892/ijmm.2015.2194). Isolation of vesicles by ultracentrifugation into a sucrose cushion requires an additional sample concentration procedure. Repeated ultracentrifugation is required according to the procedure described in reference 17. To minimize isolation time, we modified this technique using ultrafiltration at this step.

127. Again, the previously mentioned concerns with ultrafiltration apply.

>>Undoubtedly, the ultrafiltration procedure makes both positive (additional purification of the sample) and negative (additional loss of a certain amount of vesicles) contributions. But here we used ultrafiltration (Amicon-Ultra, 100 kDa, Millipore) for purification of isolated EVs from Con-A and concentration of the preparation.

130-133. There are not details about the methodology here. In order to compare practical aspects such as method complexity and turnaround time, these need to be included.

>>A brief description of the technique is included in the Materials and Methods section.

105-144. Based on the individual descriptions, all of the methods require multiple hours to perform. This is a key point. A comprehensive tabulation is necessary.

>>A table comparing the different vesicle isolation techniques used in our study is now included in the Discussion section of the revised manuscript.

179. The term “quantitative” is used quite loosely here. The data reported is presented as a number of “events”, and not converted to real numbers of particles.

>>corrected

186. How did you ensure the quantitative removal free proteins before the lysation of the vesicles? This technique specifically quantifies total protein. How do you know that impure EV recoveries are not positively skewing the quantification of EVs? We consider this a major flaw in the characterization.

>>As rightly noted, we measured total protein in all samples, including non-vesicular contaminating proteins. The corresponding clarification was introduced into the text.

190. While the study presented in this section is quite convincing, what is not mentioned in subsequent sections is the potential that non-specific complexation of proteins will be an issue here because they also contain phosphate groups. How do the authors account for that?

>>The issue of contamination with phosphorylated proteins of vesicle samples isolated by the SubXTM reagent was discussed above. Based on the results and the methods used in this study, we can only make assumptions about the possibility of contamination of the final EV samples. Corresponding minor additions have been made to the Results section of the revised manuscript.

Figs 2 and 4. It should be pointed out that the DLS data are presented as relative populations on normalized scales, not absolute number densities.

>>Yes, the dynamic light scattering method allows one to determine the presence of particles of a certain size in a suspension, but does not allow an accurate estimate of their number. Figures 2 and 4 reflect the contribution of the scattering particles of a certain size population. Corresponding changes have been made to the both figures and their legends.

Fig. 2. Do you have DLS data of the sample after the incubation and before centrifugation? Because maybe a lot of exosomes didn't resuspend and still in the aggregates. DLS data couldn't give you the concentration information of exosomes.

>>DLS measurements of the sample after the incubation of diluted plasma with SubX and before centrifugation show the particle aggregation. We believe that it is more informative to represent the particle size distribution in the supernatant (2B) and resuspended pellet (2C) obtained after incubation with SubX and the first centrifugation at 10,000 g., as shown in Figure 2.

The DLS data presented in Figure 2D are completely identical to the data obtained after the last centrifugation. These results demonstrate the effectiveness of 300 mM salt in breaking down the formed aggregates of vesicles.

237. The results of Fig. 2A clearly shows how the quantification of EVs based on Bradford assays can be skewed by the presence of free protein and protein aggregates.

>>Figure 2А shows the particle size distribution in the blood plasma pre-cleaned from cell debris. Indeed, this sample contains a significant amount of plasma proteins that can potentially contaminate extracellular vesicles isolated from this pre-cleaned plasma during further isolation procedures. But the presence of free protein in the initial plasma sample does not at all correlate with its presence in the final samples of isolated vesicles. It is possible that the legend for panel A of figure 2 was not clear. The revised manuscript has been modified accordingly.

Fig. 3. Why are there figures A and B? What is the difference?

>>corrected

Fig. 5E. Mislabeled as “IB”.

>>corrected

Fig. 6. The order of sub-figure items is presented opposite of the other figures, causing confusion.

>>corrected

347. Potentially total protein could be used to quantify EVs, but you have to account for free protein contaminants.

>>One of the most used techniques to quantify the EVs is the quantification of the total proteins of the isolated samples of EVs [17]. But indeed, protein measurements of EV-containing samples are inadequate to quantify EVs as pellets from a high-speed spin or isolated by any other methods may contain protein complexes/aggregates, lipoprotein particles, and other contaminants. However, the quantification of the total proteins of the isolated samples of EVs is an additional characteristic of the final sample of extracellular vesicles, along with other methods. In our opinion, the use of this approach in a comparative assessment of the effectiveness of several isolation methods is quite appropriate in addition to other methods of characterizing isolated vesicles used in the study.

402. The recoveries of the IP method are the same as the proposed method, so this needs to be noted.

>>Exosome elution from IP beads was performed by incubation with Exosome Elution Buffer (Lonza), but [oligoEVs-SubXTM] complexes were dissociated back to monomers in the reconstruction buffer (300 mM NaCl).

421. Should “adventures” be “advantages”?

>>corrected

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

9 Nov 2020

Evaluation of immune and chemical precipitation methods for plasma exosome isolation

PONE-D-20-14332R1

Dear Dr. Shtam,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Girijesh Kumar Patel, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: In this manuscript, authors compared different methods for the isolation of exosomes from the plasma. This manuscript is well written with appropriate experiments. However, there are some concerns like-

1. Authors should provide explanation/evidence for selecting CD63 to isolate exosomes, since exosomes express so many other markers on their surface.

2. Figure legend is poorly written, need an improvement.

3. Abstract should be finding focused, so should be revised as it contains so much background information.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

12 Nov 2020

PONE-D-20-14332R1

Evaluation of immune and chemical precipitation methods for plasma exosome isolation

Dear Dr. Shtam:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Girijesh Kumar Patel

Academic Editor

PLOS ONE