Effects of different separation methods on the physical and functional properties of extracellular vesicles

Hyungtaek Jeon; Su-Kyung Kang; Myung-Shin Lee

doi:10.1371/journal.pone.0235793

. 2020 Jul 7;15(7):e0235793. doi: 10.1371/journal.pone.0235793

Effects of different separation methods on the physical and functional properties of extracellular vesicles

Hyungtaek Jeon ¹, Su-Kyung Kang ¹, Myung-Shin Lee ^1,^*

Editor: Ramani Ramchandran²

PMCID: PMC7340315 PMID: 32634162

Abstract

Extracellular vesicles (EVs) are small vesicles secreted from cells. They have crucial biological functions in intercellular communications and may even be biomarkers for cancer. The various methods used to isolate EVs from body fluid and cell culture supernatant have been compared in prior studies, which determined that the component yield and physical properties of isolated EVs depend largely on the isolation method used. Several novel and combined methods have been recently developed, which have not yet been compared to the established methods. Therefore, the purpose of this study is to compare the physical and functional differences in EVs isolated using a differential centrifugation method, the precipitation-based Invitrogen kit, the ExoLutE kit, and the Exodisc, of which the latter two were recently developed. We investigated the properties of EVs isolated from non-infected and Kaposi’s sarcoma-associated herpesvirus-infected human umbilical vein endothelial cells using each method and determined the yields of DNA, RNA, and proteins using quantitative polymerase chain reaction and bicinchoninic acid assays. Additionally, we determined whether the biological activity of EVs correlated with the quantity or physical properties of the EVs isolated using different methods. We found that Exodisc was the most suitable method for obtaining large quantities of EVs, which might be useful for biomarker investigations, and that the EVs separated using Exodisc exhibited the highest complement activation activity. However, we also found that the functional properties of EVs were best maintained when differential centrifugation was used. Effective isolation is necessary to study EVs as tools for diagnosing cancer and our findings may have relevant implications in the field of oncology by providing researchers with data to assist their selection of a suitable isolation method.

Introduction

Extracellular vesicles (EVs) are nano-sized membrane vesicles (20–500 nm) of endocytic origin that are secreted by most cells under normal physiological conditions as well as by cells undergoing pathological processes. Based primarily on size and biological origin, EVs can be divided into three main types: (i) apoptotic bodies, which are greater than 800 nm in diameter and are secreted by cells during apoptosis; (ii) microvesicles, which are large membrane-bound vesicles (50–1,000 nm in diameter) that bud from the plasma membrane; and (iii) exosomes, which are 30–150 nm in diameter and of endocytic origin. In this study, EVs are defined as microvesicles and exosomes.

EVs contain a variety of molecules such as proteins, nucleic acids, and lipids [1–4], which are affected by environmental factors and health conditions [5–7]. EVs exist in body fluids such as cerebrospinal fluid, blood, breast milk, saliva, and urine. Tumor cells secrete more EVs than healthy cells and EVs from tumor cells may have certain tumor-specific markers [8, 9]. Therefore, EVs have been highlighted as potential cancer biomarkers [10, 11]. Furthermore, EV-containing bioactive molecules have an important role in intercellular communication [12, 13]. Many studies have focused on studying the biological activities of EVs and modulating them for therapeutic intervention [14]. For instance, in previous studies, we found that compared with EVs from non-infected cells, EVs from Kaposi’s sarcoma-associated herpesvirus (KSHV)-infected human endothelial cells have greater capacity to activate the complement system and that the complement activation activity of these EVs is a biological functional property [15]. Efficient methods of separating and analyzing EVs would provide the potential to better understand their functions, identify their roles in disease, and monitor therapeutic responses.

Despite the importance of EVs as intercellular communicators and potential biomarkers, there are distinct technical challenges in separating and purifying them from biological samples [16, 17]. Proteomics analyses or on-chip assays such as microarray and next-generation sequencing are frequently chosen by many researchers for EV analysis [18–20]. To perform these analyses, a large quantity of well-separated EVs are essential. Differential centrifugation, the classical and most-common method, is generally accepted as the standard [21, 22]. Since differential centrifugation can be applied to most biological fluids and has relatively good reproducibility, this method is most frequently used to isolate EVs from cell culture supernatants or biological fluids [21]. Unfortunately, differential ultracentrifugation cannot process large volumes of samples and its multi-step procedure may compromise the efficiency of processing samples [21]. Furthermore, the requirement of expensive equipment and the length of the processing time is a critical disadvantage for clinical diagnosis applications. In this study, we used differential centrifugation as a control to compare other methods.

Various EV isolation methods have been developed to obtain large, quantities of high-quality EVs. To overcome the drawbacks of ultracentrifugation, precipitation with hydrophilic polymers such as polyethylene glycol (PEG) is also frequently used [23]. Many commercial reagents, such as ExoQuick (System Biosciences) and Total Exosome Isolation reagents (Invitrogen), use PEG for EV isolation. Such precipitation methods do not require expensive equipment and are faster as well as simpler than differential centrifugation. However, the purity of the precipitate can be relatively low, as it may contain non-EV proteins such as albumin, apolipoprotein E, immunoglobulins, and immune complexes [24].

Filtration is the simplest method for separating EVs [25]. Ultrafiltration with membranes that filter out proteins with molecular weights exceeding 100 kDa is frequently used for EV separation [4, 24]. Microfiltration using filters with pore diameters of 0.8, 0.45, 0.1, or 0.02 μm can be used to separate EVs. Larger particles are removed using filters with pore diameters of 0.8 or 0.45 μm, and EVs are then separated using a 0.1 or 0.02 μm filter. Microfilters below 100 nm easily clog during centrifugation, which may cause EV deformation due to the high pressure. Exodisc is a recently developed method designed to separate EVs with low-speed centrifugation to prevent deformation [26].

It may be difficult for a single standard method to serve the purposes of all EV studies; it is likely that different methods are suitable for different EV studies. To investigate the differences in the EV fractions produced by the different methods, EVs were isolated from normal and KSHV-infected human endothelial cells using four different separation methods: 1) the standard differential ultracentrifugation method; 2) a precipitation-based Total Exosomes Isolation kit by Invitrogen; 3) ExoLutE, a recently developed multistep combined Exosome Isolation Kit involving size-exclusion chromatography by the Rosetta Exosome Company; and 4) a recently developed 20 nm size-selective nanofilter-based isolation method by Exodisc.

Materials and methods

Cell culture

Human umbilical cord vein endothelial cells (HUVECs) were purchased from Lonza (Allendale, NJ, USA) and cultured with the endothelial cell growth medium-2 (EGM-2) bullet kit (Lonza, Allendale, NJ, USA) in a humidified atmosphere of 5% CO₂ at 37°C. HUVECs up to passage 6 were used in this study.

Virus isolation and infection

iSLK BAC16 cells harboring recombinant KSHV BAC16 were used to produce virions [27]. Infectious KSHV BAC16 virions were induced from iSLK BAC16 cells by treatment with doxycycline and sodium butyrate for 3 d. The culture supernatant was collected, filtered through a 0.22 μm filter, and centrifuged at 100 000 × g for 1 h. The pellet was resuspended in phosphate-buffered saline (PBS) and stored at -70°C as infectious virus particles. HUVECs were infected with KSHV according to the methods used in a previous study [28]. For negative control, mock-infected cells were prepared with the same processes in place of phosphate-buffered saline (PBS) instead of the virus.

Separation of EVs

The culture supernatant was collected from non-infected and KSHV-infected HUVECs as described previously [15]. Briefly, equal volumes of the culture supernatant were used as the sources of EVs for each of the four different isolation methods. For differential centrifugation, the supernatant was centrifuged at 300 × g for 10 min to remove cellular debris and at 2000 × g for 10 min to remove apoptotic bodies. Subsequently, the supernatant was centrifuged at 10 000 × g for 30 min and then at 100 000 × g for 60 min. The pellet was dissolved with PBS to collect the EVs.

For the other commercial kits and EV separation equipment, we followed the procedures suggested by each manufacturer’s instructions. The schematic for the separation process is summarized in Fig 1. For the Invitrogen kit, the supernatant was centrifuged at 2000 × g for 10 min to remove cellular debris and apoptotic bodies. After adding the precipitation solution to the culture supernatant, the mixture was incubated overnight and then centrifuged at 10 000 × g for 60 min. The pellet was dissolved in PBS to collect EVs. For the ExoLutE kit, cellular debris was removed with a 0.45 μm syringe filter and crude EVs were precipitated in the solutions supplied with the kit. The dissolved pellet was processed in a spin-based size exclusion column to separate the EVs. For the Exodisc method, PBS and the culture supernatant were filtered through a 0.45 μm syringe filter. For priming, PBS was added to the filter chamber and centrifuged in a Labspinner centrifuge for 5 min to activate the filter. Then, the clear supernatant was transferred to filter chambers and centrifuged for 5~15 min to separate the EVs for enrichment. Finally, the collected EVs were washed by adding PBS to the filter chambers and centrifuging the solution in the Labspinner. The obtained EVs were used for further analysis.

Fig 1 — The culture supernatant was divided into four parts of equal volumes, each for a different method. (A) Differential centrifugation: the culture supernatant was put through four centrifugation steps to separate EVs. (B) Total Exosome Isolation reagent from Invitrogen: cell debris was removed by centrifugation and EVs were separated by precipitation. (C) ExoLutE Exosome isolation kit from Rosetta Exosome: Cell debris was removed by filtration and EVs were separated by multiple processes, including spin-based size exclusion chromatography. (D) Exodisc from LabSpinner: After filtration to remove cell debris, the supernatant was applied to Exodisc with a 20 nm size-selective nanofilter.

Nano-particle Tracking Analysis (NTA)

The number and size distribution of microparticles in the EV preparations were analyzed by the nanoparticle tracking analyzer ZetaView (Particle Metrix GmbH, Meerbusch, Germany). Preparations of EVs were diluted in PBS and passed through 0.8 μm filters before analysis. The analysis parameters were as follows: maximum area: 1000, minimum area: 10, minimum brightness: 25, sensitivity: 75, shutter: 100, and temperature: 25°C.

RNA isolation, cDNA synthesis, and quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) analysis

To investigate the quality of the mRNAs, we used an equal amount of mRNA (20 ng) from each preparation for the cDNA synthesis. To analyze the quality of the mRNA, the housekeeping genes GAPDH and β-actin were used as representatives. Subsequently, PCR amplification of different transcripts (GAPDH and β-actin) was performed using specific primer sets (Table 1). Total RNA was isolated using the easy-BLUE total RNA Extraction kit (iNtRON Biotechnology, Daejeon, South Korea) according to the manufacturer’s instructions and quantified using Nanodrop-1000 (Thermo Scientific, Waltham, MA, USA). Using a cDNA synthesis kit (Takara, Shiga, Japan), cDNA was synthesized from mRNA. Specific reverse transcription (RT) primers were used for the synthesis of U6 and miR-20a, while random hexamers were used for the synthesis of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin. The synthesized cDNA was used as a template for qRT-PCR using the CFX96 touch real-time PCR detection system (Bio-Rad, Hercules, CA, USA) and TB Green Premix Ex Taq qPCR kit (Takara, Shiga, Japan). The cycling conditions were as follows: 95°C for 30 s, 40 cycles of 95°C for 5 s, and 60°C for 10 s. The specificity of the amplified products was confirmed by analyzing the melting curves. All samples were tested in triplicate and normalized with GAPDH. The primers were synthesized by Genotech (Daejeon, South Korea); their sequences are described in Table 1.

Table 1. List of primers used for PCR.

Gene	Sense primer	Antisense primer
GAPDH	`GGT ATC GTG GAA GGA CTC`	`GTA GAG GCA GGG ATG`
β-actin	`AGA GCT ACG AGC TGC CTG AC`	`AGC ACT GTG TTG GCG TAC AG`
NADH sub1	`TTC TAA TCG CAA TGG CAT TCC T`	`AAG GGT TGT AGT AGC CCG TAG`
NADH sub5	`TTC ATC CCT GTA GCA TTG TTC G`	`GTT GGA ATA GGT TGT TAG CGG TA`
U6	`CTC GCT TCG GCA CAT ATA CT`	`ACG CTT CAC GAA TTT GCG TGT C`
miR20a	`TAA AGT GCT TAT AGT GCA GGT AG`	-
Universal	-	`GTC GTA TCC AGT GCA GGG TCC GAG GT-`

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Isolation of EV DNA

DNA was extracted from isolated EVs using the DNeasy genomic DNA isolation kit (Qiagen, Duesseldorf, Germany). The DNA pellet was resuspended in 100 μL of nuclease-free water. Due to the difficulties in measuring small amounts of DNA with Nanodrop, equal volumes of DNA extracted from the EVs separated by each isolation method were used as a template for qRT-PCR of GAPDH and NADH subunits 1 and 5 with the TB Green Premix Ex Taq qPCR kit (Takara, Shiga, Japan). The cycling conditions were as follows: 95°C for 30 s, 40 cycles of 95°C for 5 s, and 60°C for 10 s. The primer sequences are described in Table 1.

Protein-based EV quantification and western blot analysis

EVs were lysed in 1x RIPA buffer with the cOmplete^TM Protease Inhibitor Cocktail (Roche, Basel, Switzerland). The lysate was centrifuged and the supernatants were collected. Protein-based quantification of isolated EVs was performed using the Thermo Scientific protein microBCA assay kit (Rockford, IL, USA). Subsequently, equal volumes of the EVs isolated using different methods were denatured using a 5X sample buffer without dithiothreitol at 95°C for 10 min and then resolved on 10–12% SDS-acrylamide gel by electrophoresis.

To investigate the composition of known EV markers in EVs separated using each method, normalized quantities (2 μg) of proteins were analyzed by western blotting (S1 Fig). Resolved proteins were transferred onto nitrocellulose membrane from Amersham (GE Healthcare, Cheltenham, GB) and then blocked by incubation in 5% skimmed milk with 0.1% Tween-20 buffer to minimize the non-specific binding of antibodies. Blocked blots were treated with primary antibodies, subsequently washed three times with 1X Tris-buffered saline with 0.1% Tween-20 (TBST) buffer, and then incubated with HRP-conjugated secondary antibody. Unbound antibodies were removed by washing with 1X TBST buffer and were signal-recorded using the West Femto Maximum Sensitivity Substrate Kit under the Bio-Rad ChemiDoc Imager (Hercules, CA, USA).

The antibodies for mouse monoclonal anti-beta-actin (A5316, Sigma-Aldrich, St. Louis, MO, USA), mouse monoclonal CD81 (sc-23962, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-CD63 (sc-5275, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti-HSP70 (ab45133, Abcam, Cambridge, MA, USA), and rabbit polyclonal anti-TSG101 (bs-1365R, Bioss Antibodies Inc., Woburn, MA, USA) were used.

EV complement activation and C5b-9 cell-ELISA

In our previous study, we found that EVs from KSHV-infected human endothelial cells were able to activate the complement system when they were transferred into non-infected cells [15, 29]. After EVs separated using each method were transferred to human endothelial cells for 24 h, normal human serum was added to supply all complement factors needed to activate the complement system. We evaluated the activation of the complement system by analyzing the deposition of C5b-9 with cell-ELISA and compared the complement system activation potentials among the EVs isolated using the different methods.The cell-ELISA was performed as previously described [30], with modifications. Briefly, 10 000 cells/well were seeded in 96-well culture plates and incubated overnight at 37°C in 5% CO₂. Cells were then cultured in media containing 10% pooled human serum (Innovative Research, Novi, MI, USA) for 1 h to activate the complement system. Plates were washed with PBS and then fixed with 4% paraformaldehyde (PFA) for 15 min. The cells were incubated in blocking buffer (5% skim milk in Tris-buffered saline (TBS)) for 1 h at 37°C. A rabbit polyclonal C5b-9 antibody (Abcam, Cambridge, MA, USA) diluted in blocking buffer (1:4000) was added to the plate and incubated with the cells for 2 h. The plate was washed three times with TBST for 15 min, and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (GE Healthcare, Cheltenham, GB) was added. After incubation at room temperature for 1 h, 3,3′,5,5′-tetramethylbenzidine (TMB; KPL, Gaithersburg, MD, USA) was used as a substrate. The absorbance at 450 nm was measured using a microplate reader (Molecular Devices, Silicon Valley, CA, USA).

Statistical analysis

Each experiment was performed at least three times independently, and representative results are shown. Results are shown as the mean ± the standard deviation (SD). A two-tailed Student’s t-test and one-way ANOVA were used to assess the significance of the difference between groups. Microsoft Excel (version 16.37) was used for all statistical analyses. Statistical significance at p values of < 0.05 and < 0.01 is indicated by * and **, respectively.

Results

EVs separated using different methods demonstrated differences in particle number and size distribution

The number and size distribution of EV particles separated from non-infected and KSHV-infected HUVECs using the different methods are shown in Fig 2. The particle numbers of the isolated EVs differed according to the method used (Fig 2A). Differential centrifugation resulted in the lowest number of particles; the numbers of particles produced by the other methods were about two- to five-fold higher. The EV preparations from each separation method exhibited a range of particle sizes (from 20–500 nm) (Fig 2B). The median size distribution ranged from 120 nm to 140 nm in diameter. Interestingly, the EVs separated using the ExoLutE kit had a larger median size and a broader size distribution than those separated using the other methods.

Fig 2 — (A) The number of EV particles separated using each method. DC: EVs separated using differential centrifugation. Invitrogen: EVs separated using the Invitrogen Total Exosome Isolation reagent. eLutE: EVs separated using the ExoLutE exosome isolation kit. eDisc: EVs separated using Exodisc from LabSpinner. Data are shown as the mean ± SD, n = 3, *p < 0.05, **p < 0.01, ns: not significant. (B) Size distribution of EVs separated using each method. The red line indicates the median value of all EV sizes. Mock-HUVEC: mock-infected HUVECs. KSHV-HUVEC: KSHV-infected HUVECs.

Analysis of RNA from EVs separated by different methods

The quantities of RNA from the EVs obtained by the different isolation methods were analyzed (Fig 3A). Interestingly, total RNA quantity was not correlated with EV particle number. While the number of EV particles obtained using the Exodisc method was the highest, the quantities of total RNA from EVs separated using differential centrifugation and the Invitrogen kit were larger than that from EVs separated using the Exodisc method. No significant differences were detected between the CT values in the RNAs from different EV preparations, implying that they had qualitatively equal mRNA levels (Fig 3B).

To investigate the quality of non-coding small nuclear RNA (snRNA) and miRNA, total RNA was reverse-transcribed using specific primers for U6 and miR-20a, and the quantity of each type of RNA was analyzed using qRT-PCR (Fig 3C). Intriguingly, the quantities of snRNA and miRNA differed depending on the method. Even though qRT-PCR was conducted with the same quantity of total RNA, the Exodisc and Invitrogen methods produced larger quantities of U6 snRNA and miR-20a than the other methods.

Analysis of DNA from EVs separated by different methods

The quantities of genomic DNA and mitochondrial DNA analyzed by qRT-PCR using specific primers for GAPDH and NADH subunits 1 and 5, respectively, are shown in Fig 4. qRT-PCR results for GAPDH indicated that the EVs separated using Exodisc had the highest quantity of genomic DNA, followed by those of the Invitrogen kit, differential centrifugation, and the ExoLutE kit (Fig 4A). For mitochondrial DNA, the qRT-PCR results for NADH subunits 1 and 5 exhibited a similar pattern to that of the results for GAPDH (Fig 4B). All qRT-PCR reactions presented clear melting curve peaks in the qRT-PCR analyses, indicating that the EVs separated using all four methods contained a notable amount of DNA.

Fig 4 — EVs were separated from the supernatants of non-infected or KSHV-infected HUVECs using the four different EV separation methods. Equal volumes of DNA extracted from the EVs separated by each method were analyzed with qRT-PCR. qRT-PCR was used to analyze genomic and mitochondrial DNA for GAPDH (A) and NADH subunits 1 and 5 (B), respectively. The number above the bar graph indicates the average CT value from the qRT-PCR reaction. CT values from differential centrifugation were used as a control. DC: EVs separated using differential centrifugation. IN: EVs separated using the Invitrogen Total Exosome Isolation reagent. eLutE: EVs separated using the ExoLutE exosome isolation kit. eDisc: EVs separated using Exodisc from LabSpinner. Mock-HUVEC: mock-infected HUVECs. KSHV-HUVEC: KSHV-infected HUVECs. Data are shown as the mean ± SD, n = 3, **p < 0.01.

Analysis of proteins from EVs separated by different methods

To compare the quantities of proteins in the EVs separated by different methods, the proteins in each EV separation were analyzed by bicinchoninic acid assay (BCA) (Fig 5A). Compared to the protein quantity in EVs separated by differential centrifugation, more protein was found in EVs isolated with the Invitrogen kit and less in those isolated with ExoLutE. In the EV fraction separated by Exodisc, a significantly higher amount of protein was found compared to the amount found in EVs separated by the other methods. Although there was a larger amount of protein found in the EVs from KSHV-infected HUVECs than in EVs from non-infected HUVECs, the pattern of protein quantity extracted from EVs using each method was similar between the EVs from the non-infected and KSHV-infected cells.

Fig 5 — (A) Quantification of EV proteins isolated using the four different separation methods. The total quantity of proteins was calculated using BCA and is presented based on the EVs separated from 1 mL of culture supernatant. DC: EVs separated using differential centrifugation. IN: EVs separated using the Invitrogen Total Exosome Isolation reagent. eLutE: EVs separated using the ExoLutE exosome isolation kit. eDisc: EVs separated using Exodisc from LabSpinner. Data are shown as the mean ± SD, n = 3, *p < 0.05, **p < 0.01. (B) Western blot analysis of EV markers. Equal volumes of EVs separated by each method were loaded onto the gel, and EV markers were analyzed by western blotting. Mock-HUVEC: mock-infected HUVECs. KSHV-HUVEC: KSHV-infected HUVECs.

The proteins from EVs separated using the Exodisc method were determined to have the highest levels of all EV markers, which was consistent with the results of the BCA (Fig 5B). All EV markers were observed in the EVs separated using differential centrifugation, but only HSP70 was detected in the EVs separated using the Invitrogen kit. In the EVs separated with the ExoLutE kit, none of the EV markers were detected. A previous study showed that EVs from each separation method contain a different composition of EVs marker protein. To investigate the composition of known EVs markers in EVs separated from each method, proteins were analyzed in normalized quantity (2 μg each) by western blot analysis (S1 Fig). EVs from Exodisc showed the highest expression of all markers, and the expression of each marker was different according to the method, indicating that the composition of EVs extracted by each separation method may be different.

Complement activation of EVs separated by different methods

In the cells treated with EVs from non-infected cells, complement activation was not observed as expected. The EVs separated from KSHV-infected cells using the Exodisc method showed the highest amount of deposition. Interestingly, the EVs separated using differential centrifugation had a slightly lower amount of deposition compared to those separated using the Exodisc method. The EVs separated using the Invitrogen kit and the Exolute kit also activated the complement system, although the amounts of deposited C5b-9 were lower than those of the EVs separated using differential centrifugation and the Exodisc method (Fig 6A and 6B).

Fig 6 — (A) Schematic summary of the experimental process. Equal volumes of EVs separated by the four different methods from KSHV-infected HUVECs were applied to non-infected HUVECs with heated or normal human serum. The deposition of C5b-9 on the cells was analyzed by cell-ELISA. (B) The results of the cell-ELISA for C5b-9 in the non-infected HUVECs treated with separated EVs by various methods. DC: EVs separated using differential centrifugation. IN: EVs separated using the Invitrogen Total Exosome Isolation reagent. eLutE: EVs separated using the ExoLutE exosome isolation kit. eDisc: EVs separated using Exodisc of LabSpinner. Data are shown as the mean ± SD, n = 3, **p < 0.01.

Discussion

We found that the four different separation methods yielded EVs with different physical and functional properties. The characteristics of the separated EV fractions appeared to vary; the number and size distribution of the EV particles isolated using different methods exhibited some differences.

For instance, total RNA from the samples separated using the different methods was not consistent with the number of EV particles in each sample and miR20a from the same amount of total RNA was significantly higher in EVs separated using the Exodisc method. Since the quantity of DNA in the EVs was too small to measure with a spectrophotometer, qRT-PCR was used for analysis. The qRT-PCR results shown in Fig 4 demonstrated significant quantitative differences in genomic and mitochondrial DNA. EVs isolated with Exodisc had the highest quantity of DNA. However, relative to the EV particle number, the purity of the EVs isolated using the Invitrogen kit was higher.

In a previous study comparing the Invitrogen kit with differential centrifugation, the Invitrogen kit produced a higher yield of total protein and RNA than that produced by differential centrifugation due to the presence of non-EV proteins; the Invitrogen preparation also showed a broader size distribution [31]. Our results were consistent with this prior finding and also showed that Exodisc was more suitable than the other methods for obtaining larger quantities of EV proteins.

Notably, in our study, the highest yield of EV particles was obtained using the Exodisc method, possibly due to its ability to concentrate all nanoparticles with a diameter over 20 nm. Therefore, Exodisc might be the best method to obtain large quantities of EVs, as the particle number obtained using this method was significantly higher than those obtained using the other methods. This method would be very useful for identifying biomarkers in EVs because a large amount of EVs were separated from a comparatively small biological sample. The quantity of protein and DNA in the isolated EVs correlated positively with the particle number obtained by each method. These findings supported the results of a previous study showing that EVs separated by different methods contain different compositions of EV marker proteins [32].

In contrast to the other methods, the ExoLutE kit used in this study uses a combination of multiple methods, including precipitation and size-exclusion chromatography. Size-exclusion chromatography can produce an EV fraction with a higher purity by generating samples free of non-EV proteins and lipoproteins [32]. However, this method requires pretreatment and concentration of EV samples by ultracentrifugation or ultrafiltration. Furthermore, a relatively low yield is a disadvantage of this multi-step separation process; although the purity achieved was high, the yield of the ExoLutE kit was the lowest among the four methods.

In terms of biological function, the EVs separated using Exodisc showed the highest level of complement activation. In a previous study using differential centrifugation, KSHV-infected HUVECs exhibited EV production and complement system activation potential that increased relative to those of the non-infected HUVECs [15]. Surprisingly, in our study, EVs isolated using differential centrifugation had similar complement activity as those isolated with Exodisc, even though the number of EV particles isolated with differential centrifugation was much lower than that of EV particles isolated with Exodisc. This result indicated that the purity of the EV sample isolated using differential centrifugation would be higher than that of the same isolated using Exodisc.

A limitation of this study was that although the samples tested with each method were taken from the same source and should have had similar qualities, each sample had its own unique characteristics. EVs are a heterogenous population containing exosomes, microvesicles, and apoptotic bodies; even among EVs of the same size, individual EVs may have differing contents of DNA, RNA, and protein. Therefore, it may be impossible to isolate a homogenous population of EVs. Each EV separation method has a different theoretical background, and our results showed that EVs isolated using different methods had different physical and functional properties. However, it can be difficult to determine to what degree the differences in isolated EV samples are due to the effects of EV population heterogeneity and to what degree they can be attributed to the choice of isolation method. Additionally, while our study showed that the different methods produced separated EV fractions with significantly different physical and functional properties, further studies that include miRNA profiling, protein profiling, and more functional assays of EVs isolated with each method are necessary.

In conclusion, we provided novel data that showed how the recently developed ExoLutE and Exodisc methods compared to established methods. We demonstrated that the newly developed Exodisc method yielded the largest quantity of EVs; this indicated that Exodisc might be especially useful when searching for biomarkers. Selecting an appropriate separation method for a given purpose may be critical not only for producing useful results but also for reproducing experiments; our findings may help researchers determine which methods best suit their needs.

Supporting information

S1 Fig. Western blot analysis of markers from EVs normalized for quantification.

Two micrograms of protein lysate from each sample of EVs separated using the four different methods were loaded to the gel. The EV markers Tsg101, CD63, CD81, and HSP70 were analyzed by Western blotting.

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S2 Fig. Western blot uncut image of Fig 5B.

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S1 Dataset

(XLSX)

Click here for additional data file.^{(21.5KB, xlsx)}

Acknowledgments

We thank the members of Lee’s laboratory for technical assistance and helpful discussions.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This research was supported by the Mid-Career Researcher Program through the National Research Foundation of Korea (KNRF) funded by the Ministry of Science and ICT (NRF-2019R1A2C2083947, NRF-2017R1A2B4002405) to MSL. NO

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PLoS One. doi: 10.1371/journal.pone.0235793.r001

Decision Letter 0

Ramani Ramchandran

15 May 2020

PONE-D-20-09529

Effects of different separation methods on the physical and functional properties of extracellular vesicles

PLOS ONE

Dr. Lee,

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.

I require that all controls associated with western blots, EV isolation, and other techniques used in the paper must be provided in the revised submission. Additional revisions to the write up is recommended, especially the rationale for the study, and its objective. Statistical analysis needs to be performed throughout the manuscript, replicates (biological vs. technical), all details of the methods used must be included. I would encourage the manuscript be proofread by professional services before resubmission. Finally, data that helps determine whether the preferred EV isolation method is suitable for downstream analysis will greatly strengthen the manuscript findings. However, this data request is optional.

We would appreciate receiving your revised manuscript by August 15, 2020. If you need more time to make these revisions, please do not hesitate to contact us. When you are 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.

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Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Sincerely,

Ramani Ramchandran

Academic Editor

PLOS ONE

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[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: Partly

Reviewer #2: Partly

**********

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

Reviewer #1: No

Reviewer #2: I Don't Know

**********

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: No

**********

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: No

**********

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: Rationale for "Comments to the Author":

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

Please include or explain the lack of HUVECs treated with a vehicle control for the experiment associated with figure 6

Please alter lines 355-357 of page 16, as you are not determining which isolation method is "most suitable," but rather just documenting the physical and functional effects of each

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

What software and model is used? Also, t-tests are inappropriate for the number of experimental groups. Look towards one/two-way ANOVAs

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

The data statement is included

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

The manuscript is easy to follow

Additional Comments:

Please include citations (line 57-58 on page 3; lines 68-71 and 77 of page 4, and lines 381-383 of page 17) for these statements/ claims.

Please explain the types of EVs and which you will be isolating in your introduction (exosomes, micorvesicles, and/or apoptotic bodies)

Please move your list of antiboides located under "cell culture and antibodies" to the section titled "protein-based centrifugation and western blot analysis"

Please state the passage number used of HUVECs

Please move the latter portion of lines 232-234 to the discussion section (speculation belongs in the discussion)

Please briefly summarize the findings found in reference 29 (lines 357-358 on page 16)

Please also include in the discussion that it is difficult to make decisions without: miRNA profiling, protein profiling, and more functional assays

Reviewer #2: Summary

The goal of this research was to find the method that will best facilitate identifying EV biomarkers and thus identifying the method that is the most suitable for “follow-up studies involving EVs.” The authors isolated EVs by new and traditional approaches including differential ultracentrifugation, PEG precipitation with two new EV isolation technologies Exodisc and Exolute. For each isolation method then compared select DNA, RNA, and protein cargos as well as their ability to induce compliment activation. They found that each method distinct outcomes, isolating different amounts of particles with distinctive mean sizes, with different levels of select protein, RNA, and DNA cargos. They went on to show that the EV isolated from herpes-infected cells though the four methods induce different levels of compliment activation in naïve cells.

Major Concerns

As an EV biologist I can appreciate the findings in this manuscript. The Exodisc, and Exolute purification methods were news to me. It’s nice that they were able to directly compare these novel methods with two widely used purification methods. However, I was curious why they did not also compare immunoprecipitation as this is increasingly becoming the purification method of choice. The key finding of interest for me was that the Exodisc recovers way more EVs than other methods. Their results clearly show that the particles purified by the Exodisc contain traditional protein cargos of EVs and that these contain classical EV protein markers.

While the authors conducted rigorous experiments that each showed extremely little variation between biological replicates. As it stands, they are only quantifying a few select RNA, DNA transcripts. It would be nice to have seen a broader characterization of the protein and RNA. In the background they describe their motivation is to identify a purification method that empowers RNAseq and LC-MS-MS analysis of EVs. It would have been nice ideal to have included some -omic data. Later in the discussion they establish their aim to determine “which method is most suitable for a specific research, specifically, we investigated which method would be most suitable for identifying biomarkers in EVs.” Later they change and say “It may be difficult for a single standard method to serve the purposes of all EV studies. We hypothesized that different methods are suitable for different EV study purposes.” And concluded with “In this study, physical and functional properties were analyzed and compared in EVs obtained using the different separation methods to demonstrate which methods would be suitable for follow-up studies involving EVs.” This is pretty vague, but even given this broad goal it’s not clear how these experiments are anything but the first step towards that. While the experiments are well done I feel that while these results are useful and necessary for the research group to develop their experimental methodology I don’t think that the novelty and impact of them is sufficient for publishing in PlosOne.

Minor Concerns

The methods for EV purification reference other publications. It would be nice to have them at least described briefly. This seems especially important since the focus of the paper is on addressing the results of these methods.

The figures are difficult to follow without figure legends.

For each experiment it would be nice to indicate the number of biological replicates.

Nanoparticle tracking analysis of EVs can be notoriously prone to artifacts and strongly dependent on setting conditions. Therefore, it would have been nice to provide a complimentary characterization of EV abundance, such as also show electron micrographs of EVs.

Line 163 “random RT primers were used for synthesis of 164 glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin.” What do you mean by random? Primers of random sequence would not specifically amplify GAPDH or B-actin.

Also, the authors should make a case for why they chose to look at these transcripts.

They never state the number of particles they isolated with the different methods and the lo-res figures make it impossible to make out the exponential on the Y-axis.

There is little discussion on the significant differences they found in DNA and RNA cargo abundance. It would be nice to hear how finding differences in these nucleotide cargos furthers the overall goal of the research.

It would be nice to see Western blots also conducted on samples normalized for protein. As it is the Western blot results in figure 5B could be simply be due to the differential abundance of the starting samples.

The authors do not make clear if their biological activity of inducing complementation is definitively associated specifically with EVs or there may also be other secreted factors that induce it. This knowledge is critical to make any kind of determination of EV bioactivity. Also it is curious that the ultracentrifuged EVs, with many less particles and lower EV protein makers nevertheless had similar activity to the much more abundant Exodisc sample. This would seem to show that the ultracentrifuged EVs was much purer in terms of activity. Maybe this could be due to a ceiling effect and which they could address with a dilution series.

Grammar issues throughout.

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Jul 7;15(7):e0235793. doi: 10.1371/journal.pone.0235793.r002

Author response to Decision Letter 0

1 Jun 2020

CC: rramchan@mcw.edu

PONE-D-20-09529

Effects of different separation methods on the physical and functional properties of extracellular vesicles

PLOS ONE

Dr. Lee,

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. 

• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. 

• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. 

We look forward to receiving your revised manuscript.

Sincerely,

Ramani Ramchandran

Academic Editor

PLOS ONE

Authors’ response:

We thank the Academic Editor for the consideration and the favorable comments. We have revised our manuscript according to the comments of the Editor and the reviewers.

Controls associated with western blots, EV isolation, and other techniques

In this study, differential centrifugation was used as a control method; the results of the other methods were compared with the results of the differential centrifugation. In the KSHV infection experiments, we used mock-infected cells as a control and prepared them using the exact same process used to prepare the KSHV-infected cells. We apologize for the confusion caused by not clearly describing the mock-infected HUVECs; we have changed “HUVECs” in all figures to “mock-HUVECs” and corrected the manuscript text accordingly.

The objective and rationale for the study

We have revised the abstract and discussion as follows:

Abstract line 23-26 on page 2:

“Therefore, the purpose of this study is to compare the physical and functional differences in EVs isolated using a differential centrifugation method, the precipitation-based Invitrogen kit, the ExoLutE kit, and the Exodisc, of which the latter two were recently developed.”

Discussion line 462-468 on page 21:

“In conclusion, we provided novel data that showed how the recently developed ExoLutE and Exodisc methods compared to established methods. We demonstrated that the newly developed Exodisc method yielded the largest quantity of EVs; this indicated that Exodisc might be especially useful when searching for biomarkers. Selecting an appropriate separation method for a given purpose may be critical not only for producing useful results but also for reproducing experiments; our findings may help researchers determine which methods best suit their needs.”

Statistical analysis throughout the manuscript, replicates (biological vs. technical):

Statistical analysis information has been uploaded to the S1 Data set of Supplementary Information file.

Methods details:

Details of the methods have been extensively revised.

Line 138-150 on page 7:

“For the Invitrogen kit, the supernatant was centrifuged at 2000 × g for 10 min to remove cellular debris and apoptotic bodies. After adding precipitation solution to the culture supernatant, the mixture was incubated overnight and then centrifuged at 10 000 × g for 60 min. The pellet was dissolved in PBS to collect EVs. For the ExoLutE kit, cellular debris was removed with a 0.45 μm syringe filter, and crude EVs were precipitated with solutions supplied by the ExoLutE kit. The dissolved pellet was put into a spin-based size exclusion column to separate the EVs. For the Exodisc method, PBS and the culture supernatant were prepared by filtering with a 0.45 μm syringe filter. For priming, PBS was added to the filter chamber and centrifuged in a Labspinner centrifuge for 5 min to activate the filter. Then, for enrichment, the clear supernatant was transferred to filter chambers and centrifuged for ~5–15 min to separate the EVs. Finally, for washing, the collected EVs were washed by adding PBS to the filter chambers and centrifuging the solution in Labspinner. The obtained EVs were used for further analysis.”

Proofreading by a professional service before submission:

The manuscript has been edited by a professional English editing company. Please see the editing certificate.

-Omics data

We thank the Editor for the excellent suggestions and agree that -omics data would be ideal.

Unlike studies using cancer cells, it is challenging to obtain a large amount of EVs from KSHV-infected primary cells. Since it was difficult to obtain enough EV proteins for proteomics analysis using known EV separation methods, several newly developed methods were sought to overcome this limitation. In fact, we did conduct a proteomics study with EVs isolated from KSHV-infected cells using the Exodisc method, but it was difficult to obtain enough isolated EVs using the other methods for LC-MS-MS analysis. It would be ideal to present the -omics data of EVs extracted by different methods, but we are very sorry to report that we are unable to apply them this time due to our technical and financial limitations.

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

Authors’ response:

We have carefully edited the manuscript to meet PLOS ONE’s style requirements according to the templates.

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

Authors’ response:

We have uploaded the minimal data set as a Supporting Information file.

Authors’ response:

We have uploaded the uncut blot images as S2 Fig.

[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?

Reviewer #1: Partly

Reviewer #2: Partly

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

Reviewer #1: No

Reviewer #2: I Don't Know

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

Reviewer #1: Yes

Reviewer #2: No

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

Reviewer #1: Yes

Reviewer #2: No

5. Review Comments to the Author

Reviewer #1: Rationale for "Comments to the Author":

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

Please include or explain the lack of HUVECs treated with a vehicle control for the experiment associated with figure 6

Authors’ response:

We thank the Reviewer for the valuable suggestion. We apologize for the confusion regarding the control. As a control, we used EVs from mock-infected HUVECs (HUVECs treated with a vehicle control; indicated as just “EVs from mock-HUVEC” in Figure 6).

To activate the complement system, we used normal human serum. As a control, we used serum inactivated by heating at 56 °C for 30min. More detailed experimental procedures can be found in our previous papers (Jeon, H., Lee, J.S., Yoo, S. & Lee, M.S. Quantification of complement system activation by measuring C5b-9 cell surface deposition using a cell-ELISA technique. J. Immunol. Methods 415, 57–62 (2014), and Jeon, H. et al. Extracellular vesicles from KSHV-infected endothelial cells activate the complement system. Oncotarget (2017). doi:10.18632/oncotarget.21668).

To further assist with the reader’s understanding, a schematic diagram of the experimental process has been added to Figure 6 (Fig 6A).

Please alter lines 355-357 of page 16, as you are not determining which isolation method is "most suitable," but rather just documenting the physical and functional effects of each

Authors’ response:

We completely agree with the reviewer’s excellent observation and have changed the description of the study’s contribution (now at line 462-468):

“…we provided novel data that showed how the recently developed ExoLutE and Exodisc methods compared to established methods. We demonstrated that the newly developed Exodisc method yielded the largest quantity of EVs than other methods; this indicated that Exodisc might be especially useful when searching for biomarkers.”

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

What software and model is used? Also, t-tests are inappropriate for the number of experimental groups. Look towards one/two-way ANOVAs

Authors’ response:

We used Microsoft Excel for all statistical analyses. We agree that t-tests are not appropriate for the number of experimental groups. However, in this study, we tried to show the differences between EVs separated using newly developed methods and those separated using the standard method. To accomplish this purpose, we used EVs separated using differential centrifugation as a control. In this setting, the t-test is looking more useful than ANOVA to compare each method with differential centrifugation. Nevertheless, we have added the statistical results of the multiple groups using ANOVA to the data set in the Supporting Information file.

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

The data statement is included

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

The manuscript is easy to follow

Additional Comments:

Please include citations (line 57-58 on page 3; lines 68-71 and 77 of page 4, and lines 381-383 of page 17) for these statements/ claims.

Authors’ response:

We apologize for these mistakes. We have added the citations as the reviewer suggested.

Line 57-58 on page 3 � line 71-72 on page 4, references 21, 22

Lines 68-71 and 77 of page 4 � line 82-83 on page 4, reference 23

Lines 381-383 of page 17 � line 429-431 on page 20, reference 32

Please explain the types of EVs and which you will be isolating in your introduction (exosomes, micorvesicles, and/or apoptotic bodies)

Authors’ response:

We thank the reviewer for the insightful suggestion. We have added the following description of the types of EVs and those which we will be isolating to the Introduction section at line 44-49 on page 3.

“EVs can be divided into three main types, primarily based on their size and biological origin: (i) apoptotic bodies, which are greater than 800 nm diameter and are secreted by cells during apoptosis, (ii) microvesicles, which are large, membrane-bound vesicles (50–1,000 nm in diameter) that bud from the plasma membrane, and (iii) exosomes, which are 30–150 nm in diameter and of endocytic origin. In this study, EVs are defined as microvesicles and exosomes.”

Please move your list of antiboides located under "cell culture and antibodies" to the section titled "protein-based centrifugation and western blot analysis"

Authors’ response:

We have moved our list of antibodies to the “protein-based centrifugation and western blot analysis” section.

Please state the passage number used of HUVECs

Authors’ response:

We have added the passage number of HUVECs at line 115 on page 6.

“HUVECs up to passage 6 were used in this study.”

Please move the latter portion of lines 232-234 to the discussion section (speculation belongs in the discussion)

Authors’ response:

We have moved these lines to the discussion section (line 418-420 on page 19).

Please briefly summarize the findings found in reference 29 (lines 357-358 on page 16)

Authors’ response:

We have added brief summary of the findings of the indicated reference (now on page 19, lines 411-414):

“In a previous study comparing the Invitrogen kit with differential centrifugation, the Invitrogen kit produced a higher yield of total protein and RNA than that produced by differential centrifugation due to the presence of non-EV proteins; the Invitrogen preparation also showed a broader size distribution [31].”

Please also include in the discussion that it is difficult to make decisions without: miRNA profiling, protein profiling, and more functional assays

Authors’ response:

We have added this suggestion to the discussion section (line 457-460 on page 21).

“…while our study showed that the different methods produced separated EV fractions with significantly different physical and functional properties, further studies that include miRNA profiling, protein profiling, and more functional assays of EVs isolated with each method are necessary.”

Reviewer #2: Summary

Major Concerns

Authors’ response:

We thank the Reviewer for their positive comments. We hope this study will help researchers working with EVs and are encouraged that the Reviewer found our Exodisc findings to be of interest.

I completely agree that immunoprecipitation is also useful for separating EVs. However, as the Reviewer indicated, our key finding was that Exodisc was the best way to obtain a larger quantity of EVs from a limited sample volume. For our purposes, immunoprecipitation was excluded because the result was expectied to produce a low yield. Furthermore, a previous study had already compared immunoprecipitation with other classical methods (Patel GK, Khan MA, Zubair H, Srivastava SK, Khushman M, Singh S, et al. Comparative analysis of exosome isolation methods using culture supernatant for optimum yield, purity and downstream applications. Sci Rep. 2019;9(1):5335).

Authors’ response:

We thank the Reviewer for the excellent observations and agree that -omic data would be ideal. Unfortunately, we faced technical and financial limitations. In terms of technical limitations, we did in fact conduct a proteomics study with EVs from KSHV-infected cells using the Exodisc method, but it was difficult to isolate a sufficient amount of EVs using the other methods. In terms of financial limitations, proteomics analysis is financially burdensome, and we had insufficient funding. We apologize that we could present only the results from our general analysis, but we believe that our results will still be helpful to other researchers in this field.

Later in the discussion they establish their aim to determine “which method is most suitable for a specific research, specifically, we investigated which method would be most suitable for identifying biomarkers in EVs.” Later they change and say “It may be difficult for a single standard method to serve the purposes of all EV studies. We hypothesized that different methods are suitable for different EV study purposes.” And concluded with “In this study, physical and functional properties were analyzed and compared in EVs obtained using the different separation methods to demonstrate which methods would be suitable for follow-up studies involving EVs.” This is pretty vague, but even given this broad goal it’s not clear how these experiments are anything but the first step towards that. While the experiments are well done I feel that while these results are useful and necessary for the research group to develop their experimental methodology I don’t think that the novelty and impact of them is sufficient for publishing in PlosOne.

Authors’ response:

We thank the Reviewer for the helpful comments. According to the reviewer’s observation, we have revised the discussion section as follows:

Minor Concerns

Authors’ response:

We thank the Reviewer for the kind comments.

We have added a further description of the detailed process for each method (line 127-150 on page 6~7).

The figures are difficult to follow without figure legends.

Authors’ response:

To address this issue, we have added a schematic summary to Figure 6 (Figure 6A).

For each experiment it would be nice to indicate the number of biological replicates.

Authors’ response:

We have added the number of biological and technical replicates in the data set included in the Supporting Information file.

Authors’ response:

We agree that nanoparticle tracking analysis is dependent on the setting conditions. Therefore, we ran a nanoparticle tracking analysis with a constant setting condition, as described in the Materials and methods (maximum area: 1000, minimum area: 10, minimum brightness: 25, sensitivity: 75, shutter: 100, and temperature: 25 °C). With a constant setting condition, nanoparticle tracking analysis is a reliable tool for measuring the EV particle number.

EM is a good tool for visualizing the EVs, but the field is too small to measure the particle number. In our previous study, EVs from HUVECs and KSHV-HUVECs were shown using EM (Jeon H, Yoo S-M, Choi HS, Mun JY, Kang H-G, Lee J, et al. Extracellular vesicles from KSHV-infected endothelial cells activate the complement system. Oncotarget; Vol 8, No 59. 2017). Therefore, we excluded EM results from this manuscript.

Authors’ response:

Thank you for bringing our attention to this mistake. “Random RT primers” has been replaced with “random hexamers”.

Also, the authors should make a case for why they chose to look at these transcripts.

Authors’ response:

Thank you for the suggestion. We have added a sentence at line 173-174 on page 8:

“To analyze the quality of mRNA, the housekeeping genes GAPDH and β-actin were used as representatives.”

They never state the number of particles they isolated with the different methods and the lo-res figures make it impossible to make out the exponential on the Y-axis.

Authors’ response:

Please download the high-resolution image on the Plos One web page; it appears low-resolution only in PDF files.

Authors’ response:

We thank the reviewer for the insightful suggestion. RNA was discussed in lines 402-405. We have added the following (lines 405-409):

“The quantity of DNA in the EVs was too small to measure with a spectrophotometer, so qPCR was used for analysis. The qRT-PCR results shown in Fig. 4 demonstrated significant quantitative differences in genomic and mitochondrial DNA. EVs isolated with Exodisc had the highest quantity of DNA. However, relative to the EV particle number, the purity of the EVs isolated using the Invitrogen kit was higher.”

Authors’ response:

We thank the Reviewer for the insightful suggestion. We have uploaded western blots conducted on normalized protein samples as S1 Fig. and added a description at line 212-213 on page 10 and line 358~362 on page 17.

Authors’ response:

In our previous study (Jeon H, Yoo S-M, Choi HS, Mun JY, Kang H-G, Lee J, et al. Extracellular vesicles from KSHV-infected endothelial cells activate the complement system. Oncotarget; Vol 8, No 59. 2017), we showed that complement activation is specifically induced by EVs from KSHV-infected HUVECs.

We agree that it was surprising that the ultracentrifuged EVs did not have better complement activation than the EVs in the Exodisc sample. Therefore, we described this issue in the discussion section at line 440-445.

We were unable to determine which factor in the EVs stimulates the complement system. We suggested that differential centrifugation may be more effective for maintaining EV properties because of the higher purity.

Grammar issues throughout.

Authors’ response:

The manuscript has been edited by a professional English editing company. Please see the editing certificate.

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PLoS One. doi: 10.1371/journal.pone.0235793.r003

Decision Letter 1

Ramani Ramchandran

23 Jun 2020

Effects of different separation methods on the physical and functional properties of extracellular vesicles

PONE-D-20-09529R1

Dr. Lee,

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Reviewer #1: All points raised regarding the manuscript were sufficiently addressed. The manuscript is now technically sound, and statistical analysis have been included in a supplemental file. Additionally, minor corrections raised regarding lack of citations and definition of certain terms have been corrected.

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PLoS One. doi: 10.1371/journal.pone.0235793.r004

Acceptance letter

Ramani Ramchandran

25 Jun 2020

PONE-D-20-09529R1

Effects of different separation methods on the physical and functional properties of extracellular vesicles

Dear Dr. Lee:

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.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Western blot analysis of markers from EVs normalized for quantification.

(TIF)

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S2 Fig. Western blot uncut image of Fig 5B.

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S1 Dataset

(XLSX)

Click here for additional data file.^{(21.5KB, xlsx)}

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Effects of different separation methods on the physical and functional properties of extracellular vesicles

Hyungtaek Jeon

Su-Kyung Kang

Myung-Shin Lee

Roles

Abstract

Introduction

Materials and methods

Cell culture

Virus isolation and infection

Separation of EVs

Fig 1. The schematic summary of the EV separation methods.

Nano-particle Tracking Analysis (NTA)

RNA isolation, cDNA synthesis, and quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) analysis

Table 1. List of primers used for PCR.

Isolation of EV DNA

Protein-based EV quantification and western blot analysis

EV complement activation and C5b-9 cell-ELISA

Statistical analysis

Results

EVs separated using different methods demonstrated differences in particle number and size distribution

Fig 2. NTA of EVs separated by different methods.

Analysis of RNA from EVs separated by different methods

Fig 3. Analysis of RNA from EVs separated using each method.

Analysis of DNA from EVs separated by different methods

Fig 4. Analysis of DNA from EVs separated using each method.

Analysis of proteins from EVs separated by different methods

Fig 5. Analysis of proteins from EVs separated using each method.

Complement activation of EVs separated by different methods

Fig 6. Activation of the complement system by EVs isolated from KSHV-infected cells using different methods.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Ramani Ramchandran

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Author response to Decision Letter 0

Decision Letter 1

Ramani Ramchandran

Roles

Acceptance letter

Ramani Ramchandran

Roles

Associated Data

Supplementary Materials

Data Availability Statement

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