The local landrace of Korean Panax ginseng, Jakyung, was used in this work. To isolate exosomes from ginseng, 1 g of root, stem, and leaf samples were collected from one-year-old and three-year-old ginseng plants, which were grown in an open field and purchased from a local farm at Jangseong, Korea (35°18′45.7″N, 126°45’37.8″E). For the isolation of Arabidopsis exosomes, Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used. One gram of leaf samples was collected from four-week-old Arabidopsis plants grown in a growth chamber with a light regime of 16 h/8 h (light/dark) at 23°C.

Exosome isolation by ultracentrifugation was carried out as previously described with slight modification (Wang et al., 2014). Briefly, the ultracentrifugation method used in this study included two experimental steps: crude extraction by centrifugation and exosome isolation by ultracentrifugation. To obtain crude extracts from ginseng and Arabidopsis, 1 g of plant samples was homogenized using an HG-15D homogenizer (DAIHAN, Korea) and 10 mL of 1x phosphate-buffered saline (PBS, pH 7.4) (Biosesang, Korea). The PBS was filtered through a 0.2 μm membrane (Sartorius Stedim Biotech, Germany) before use in the exosome isolation method. Homogenized samples were centrifuged three times at 4°C to remove cell debris, fibers, and large particles (1^st centrifugation: 1,000 g for 10 mins, 2^nd centrifugation: 3,000 g for 20 mins, 3^rd centrifugation: 10,000 g for 60 mins). After the final centrifugation, supernatants were collected, and the volume was brought to 10 mL with PBS buffer. To isolate exosomes from the crude extracts, the extracts were ultra-centrifuged three times, which included a sucrose density gradient ultracentrifugation process. The first ultracentrifugation was performed at 150,000 g for 90 mins at 4°C using an SW 41 Ti rotor and an Optima XE-100 ultracentrifuge (Beckman Coulter, USA). The pellets formed by the first ultracentrifugation were resuspended in 1 mL of PBS buffer. Then, the resuspended samples were transferred to a sucrose gradient solution (8, 15, 30, 45, and 60% [w/v] in PBS) and centrifuged at 150,000 g for 90 mins at 4°C. The band between the 30% and 45% sucrose solutions was collected, which corresponds to the density of exosomes (1.12–1.20 g/cm³). The volume of the collected layers was brought to 10 mL with PBS buffer, and then ultra-centrifuged at 150,000 g for 90 mins at 4°C. The exosome pellets were resuspended in 100 μL of PBS buffer.

Ginseng and Arabidopsis exosome isolation using the ExoQuick system (ExoQuick-TC™) was performed according to the manufacturer’s instructions with minor modifications (System Biosciences Inc., USA.). The ExoQuick method used in this study included two experimental steps: crude extraction by centrifugation and exosome precipitation by ExoQuick solution treatment. The crude extraction step in the ExoQuick method was identical to that in the ultracentrifugation method. To precipitate ginseng and Arabidopsis exosomes from crude extracts, 2 mL of the ExoQuick solution was added to 10 mL of crude extracts (crude extracts:ExoQuick solution = 5:1) and mixed well by inverting. The mixtures were incubated at 4°C overnight without rotation or mixing. The incubated mixtures were centrifuged at 1,500 g, 4°C for 30 mins. Exosome pellets were resuspended in 100 μL of PBS buffer.

The ultracentrifugation-ExoQuick combination method includes three experimental steps: a crude extraction by three rounds of centrifugation, exosome isolation by three rounds of ultracentrifugation, and exosome purification by ExoQuick solution treatment (Supplementary Figure 1). The crude extraction by centrifugation and exosome isolation by ultracentrifugation steps were identical to those in the ultracentrifugation method described above. In the combination method, the exosome pellets formed by the third ultracentrifugation were resuspended in 10 mL of PBS buffer and treated with 2 mL of ExoQuick solution. The subsequent precipitation step was performed identically to that in the single ExoQuick method.

To investigate the total number and size distribution of extracellular vesicles including exosomes, Nanoparticle Tracking Analysis (NTA) was performed using Nanosight NS300™ (Malvern Instruments, UK). To do this, isolated exosome samples were diluted 1:10 with PBS buffer, and 1 mL of the diluted samples was injected into the sample chamber. NTA results were analyzed using the NTA 3.2 software (Malvern Instruments, UK). To investigate the colloidal stability of the ginseng and Arabidopsis exosomes isolated by the three different methods (ultracentrifugation, ExoQuick, and combination methods), zeta potential values were measured using the ELSZ-2000ZS zeta potential and particle size analyzer (Otsuka Electronics, Japan). Exosome samples were diluted 1:2 with PBS buffer, and 200 μL of samples were measured at 25°C. The results were analyzed using the ELSZ-2000 version 7.0 software (Otsuka Electronics, Japan).

To visualize ginseng and Arabidopsis exosomes, transmission electron microscopy (TEM) was used. Isolated exosome samples were fixed in 4% paraformaldehyde for one hour, and 5 μL of fixed samples were placed on the carbon support film of a nickel grid and incubated to allow the sample to be adsorbed to the grid. Grids were washed with 60 μL of 2% uranyl acetate. TEM observation was performed using a JEM-2100F Field Emission Transmission Electron Microscope (JEOL Ltd., Japan) at 200 kV and images were captured using an UltraScan 4000 CCD camera (Gatan Inc. USA.). For cryo-transmission electron microscopy observation (Cryo-TEM), 5 μL of the isolated exosome samples were loaded onto copper Quantifoil grids with a 1.2 μm diameter hole and an inter-hole distance of 1.3 μm (Electron Microscopy Sciences, USA). Prior to freezing, the grids were glow discharged for 45 sec on each side. Grids were frozen using a Vitrobot, and the frozen samples were observed using the JEM 3200FS (JEOL Ltd., Japan) transmission electron microscope at 120 kV.

Data are averages of at least two biological replicates with three technical replicates for each. The statistical analysis was performed using Microsoft Excel 2016, and the statistical difference between the samples and their control was determined using a two-tailed Student’s t-test with a P <0.01.

Ultracentrifugation is the most widely used method to isolate exosomes (Théry et al., 2006; Li et al., 2017; Coughlan et al., 2020). To determine the purity of ginseng exosomes isolated by the ultracentrifugation method, we extracted exosomes from one-year-old ginseng roots using this method, which includes two experimental steps: crude extraction by three rounds of centrifugation, and exosome isolation by three rounds of ultracentrifugation. To obtain crude extracts, 1 g of ginseng roots collected from one-year-old ginseng plants was homogenized in 10 mL PBS buffer (Figure 1) and centrifuged three times at 4°C. To isolate ginseng exosomes from the crude extracts, a total of three rounds of ultracentrifugation was performed, and the second round of ultracentrifugation, which used sucrose density gradient solutions, formed two yellow layers (Figure 1A). The upper layer, which corresponds to the density of exosomes (1.12–1.20 g/cm³), was collected, and exosome pellets were obtained by the third round of ultracentrifugation. To examine whether the pellets contain exosomes, the pellets were observed by transmission electron microscopy (TEM) and Cryo-transmission electron microscopy (Cryo-TEM) (Supplementary Figure 2, Figure 1B). Although exosome-like nanoparticles were observed by TEM, this approach was not sufficient to clearly visualize whether the nanoparticles were exosomes (Supplementary Figure 2). To determine whether exosomes were present, we observed the samples using Cryo-TEM. The pellets obtained by the ultracentrifugation method contain ginseng exosomes, which are surrounded by a lipid bilayer and range in size between 50–150 nm, indicating that ginseng exosomes can be extracted by the ultracentrifugation method (Figure 1B).

To understand how the isolated EVs including exosomes are distributed in size, the samples were investigated by Nanoparticle Tracking Analysis (NTA) using Nanosight NS300™ (Figure 1C). The NTA approach revealed that the resuspensions contained 1.65×10¹² EVs and that EV size ranged between 29–499 nm (Figure 1C). Among the EVs, the number of exosomes (50–150 nm) was around 5.62×10¹¹. This suggested that ginseng exosomes make up 34.1% of the total EVs extracted by the ultracentrifugation method (Figure 1D).

The ExoQuick method uses a polymer-based precipitation solution, ExoQuick-TC, to isolate exosomes. ExoQuick-TC establishes a polymer network that allows exosomes to be isolated, and previous studies reported that the ExoQuick method improves exosome isolation purity in an animal cell system (Alvarez et al., 2012; Peterson et al., 2015; Jung et al., 2020). To address whether exosome isolation purity in ginseng can be improved by the ExoQuick system, we attempted to extract exosomes from one-year-old ginseng roots using this system. The ExoQuick method we designed in this approach included two experimental steps; crude extraction by three rounds of centrifugation, and exosome precipitation by ExoQuick solution treatment. The crude extraction step in the ExoQuick method was identical to that in the ultracentrifugation method. Crude extracts obtained from 1 g of one-year-old ginseng roots were treated with ExoQuick solution to precipitate exosomes (crude extract:ExoQuick solution = 5:1), and the pellets were resuspended in 100 μL PBS buffer (pH 7.4) (Figure 2A). When the resuspended samples were visually examined using Cryo-TEM, exosomes surrounded by a lipid bilayer were observed (Figure 2B). These findings suggested that EVs, including exosomes, can be isolated by the ExoQuick method.

To investigate the size distribution of isolated EVs, NTA was performed. The NTA result showed that the resuspended solutions contained 1.08×10¹² EVs, and their size ranged between 42–389 nm (Figure 2C). Among the total EVs isolated by the ExoQuick method, the number of exosomes (50–150 nm) was approximately 6.4×10¹¹. This finding indicated that ginseng exosomes make up 59.7% of the total EVs extracted by the ExoQuick system, suggesting that the ExoQuick method extracts exosomes from ginseng roots with 59.7% purity (Figure 2D). The exosome isolation purity of the ExoQuick method was approximately 75% higher than that obtained using the ultracentrifugation method (34.1%). These data indicate that exosome isolation purity is affected by isolation methods and that the ExoQuick system improves exosome isolation purity in ginseng. Also, this finding suggested that combining ultracentrifugation and the ExoQuick system may further improve the isolation purity of ginseng exosomes.

To test the hypothesis that exosome isolation purity in ginseng can be improved by applying a combination of ultracentrifugation and the ExoQuick method, we designed a combination method that includes three experimental steps: crude extraction by centrifugation, exosome isolation by ultracentrifugation, and exosome precipitation by ExoQuick-TC treatment. We then attempted to extract ginseng exosomes using this combination method (Figure 3A). Sucrose gradient ultracentrifugation of the crude extracts formed two yellow layers and the upper layer was collected and precipitated using ExoQuick-TC™ solution to obtain exosome pellets. When the pellets were observed using Cryo-TEM, 50–150 nm vesicles with membrane structure were detected (Figure 3B), indicating that the pellets included ginseng exosomes. To address how the combination method affects exosome isolation purity, we investigated the total number of EVs and their size distribution by an NTA assay. The total number of EVs in the samples was 1.87×10¹¹, and EV sizes ranged between 43–244 nm (Figures 3C, D). Among the total EVs, the number of exosomes was 1.56×10¹¹, indicating that approximately 83% of the total isolated EVs were exosomes. This result suggested that combining ultracentrifugation and the ExoQuick system significantly improves exosome isolation purity in ginseng. Exosome isolation purity using the combination method was approximately 2.4-fold and 1.5-fold higher than that of the ultracentrifugation and ExoQuick methods alone, respectively (Figures 3E–G; Supplementary Table 1).

To address whether the ultracentrifugation-ExoQuick combination method can extract high-purity exosomes from different tissues or different developmental stages of ginseng, we applied this method to leaves, stems, and roots of three-year-old ginseng plants, and then performed an NTA assay (Figures 4A, B). The total number of EVs isolated from 1 g of leaves, stems, and roots were 1.49×10¹², 1.27×10¹², and 2.79×10¹², respectively. Although EVs smaller than 50 nm or bigger than 150 nm were partially detected, most EVs in the extracts were between 50–150 nm. In the leaf, stem, and root extracts, the number of exosomes was 1.20×10¹², 1.00×1012 and 2.44×10¹², respectively, indicating that exosome isolation purity from these tissues was 80.1%, 78.9%, and 87.4%, respectively (Figures 4C, D). Furthermore, the exosome isolation purity of the three-old-year ginseng plants was almost identical to that of the one-year-old ginseng plants (Supplementary Figure 3). These findings suggested that the ultracentrifugation-ExoQuick combination method is a suitable technique to extract exosomes with high purity in plants including ginseng, and this finding was supported by an exosome isolation test using the model plant Arabidopsis (Figure 5).

To obtain Arabidopsis crude extracts, 1 g of leaves from four-week-old plants was homogenized using 10 mL PBS buffer (pH 7.4) and centrifuged three times (Figure 5A). Sucrose gradient ultracentrifugation of the crude extracts formed two green layers, and the upper layer was treated with ExoQuick-TC solution to precipitate exosomes (Figure 5B). As expected, Cryo-TEM visualization revealed that the Arabidopsis pellets contained membrane-bound vesicles 50–150 nm in size, suggesting that Arabidopsis exosomes can also be extracted by the combination method (Figure 5C). In addition, by comparing the NTA results of EV samples extracted by ultracentrifugation alone, ExoQuick alone, and the ultracentrifugation-ExoQuick combination method, it was found that the combined method improves exosome isolation purity even in Arabidopsis (Figures 5D–G). In the Arabidopsis EV samples extracted by the ultracentrifugation and ExoQuick methods alone, exosome purity was 75.9% and 78.7% respectively. In contrast, the combination method increased exosome purity to approximately 100% (Figure 5G). These findings indicated that the combination method increases exosome isolation purity by around 23% compared with that achieved by ultracentrifugation and ExoQuick methods alone in Arabidopsis. The data also suggest that the combination method can be widely used for the isolation of high-purity exosomes from plants including ginseng.

Colloidal stability is a key factor determining the functional maintenance and storage of isolated exosomes. The colloidal stability of isolated exosomes is greatly affected by isolation methods and sample types (Jeyaram and Jay, 2018; Midekessa et al., 2020). Zeta potential analysis, which measures the surface potential of colloidal particles, is widely used to estimate the colloidal stability of isolated exosomes. Previous studies suggested that the zeta potentials of isolated exosomes range between −6 and −30 mV, and values less than −20 mV indicate high colloidal stability (Lee, 1998; Soares Martins et al., 2018; Kim et al., 2021). The zeta potentials of Arabidopsis exosomes extracted by the three different methods were measured and compared. As expected, the zeta potentials were significantly different among the isolation methods, and we found that the ExoQuick system decreased zeta potentials (Figures 6A, B; Supplementary Table 2). The zeta potential of exosomes isolated by the ultracentrifugation method was −17.1 mV. Exosomes isolated by the ExoQuick method alone and by the combined ultracentrifugation-ExoQuick method had zeta potential values of −21.3 and −25.9 mV, respectively. This suggested that the ExoQuick system increased the colloidal stability of isolated exosomes, and that combining ultracentrifugation with the ExoQuick system is most effective to improve the colloidal stability of isolated Arabidopsis exosomes. This finding was further supported by the zeta potentials of ginseng exosomes. The zeta potential of ginseng exosomes extracted by the ultracentrifugation method was −20.61 mV. The zeta potentials of exosomes isolated by the ExoQuick and the combination methods were −28.88 and −29.54 mV, respectively (Figures 6C, D ). Collectively, these findings suggest that combining ultracentrifugation with the ExoQuick system is an effective method to isolate high-purity and high-stability exosomes from plants including ginseng.