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Tech Note: Raising the Bar on Single-Cell and Ultra-Low Input mRNA Sequencing

TECH NOTE

Improve isolated exosome purity and yield with Capturem technology

Introduction

Exosomes, a type of extracellular vesicle secreted by most cell types, were initially thought to be a means of ridding cells of unwanted macromolecules—proteins, lipids, DNA, RNA, and microRNAs, depending upon the cell type. Despite their small size (diameters range from 50 to 200 nm), recent research has found that these vesicles play an important role in intercellular communication for normal physiological processes including immune response, neuronal function, and stem cell maintenance (H. Rashed et al. 2017). More recently, many studies have pointed to the genomic and proteomic cargo of exosomes as potential contributors to the progression of cancer, liver disease, and neurogenerative diseases (H. Rashed et al. 2017). Given their implication in the pathology of diseases, exosomes represent a new source of biomarkers that can be explored for use in disease diagnosis, prognosis, and monitoring.

The genomic content of isolated exosomes is often assessed by next-generation sequencing or RT‑PCR, while the protein content is typically assessed via protein digestion followed by mass spectrometry. A key bottleneck in exosome research is the isolation of the vesicles. Historically, ultracentrifugation has been the primary method for exosome isolation. However, ultracentrifugation is time-consuming, is not scalable, requires specialized equipment, may damage vesicles during the high-speed spins, and suffers from low yield. Moreover, ultracentrifugation often pulls down protein aggregates, nucleic acids, and other cellular debris that can lead to background noise in the isolated exosome preps. Precipitation solutions have more recently been utilized in an attempt to simplify exosome isolation protocols, but these techniques are often inconsistent, with low yield and reduced purity. Thus, there is a strong need for a method to rapidly isolate exosomes that does not compromise purity or yield.

The Capturem Exosome Isolation Kit (Cell Culture) provides a complete solution optimized for the simple and rapid isolation of exosomes from cell culture media. Exosomes are isolated using specially designed Capturem spin columns on which a proprietary, exosome-binding compound that is not antibody-based has been immobilized. The kit contains all of the components and reagents needed to isolate exosomes in under 30 minutes: preclearing columns to remove large cellular debris, isolation columns to specifically bind exosomes, as well as optimized wash and elution buffers for the isolation protocol. As a whole, the system enables researchers studying exosomes to accelerate the pace of their research by obtaining high yields of non-contaminated exosomes in a simple and rapid manner.

Results

Capturem columns enable fast and easy isolation of appropriately sized exosomes

The exosome isolation protocol requires only a regular low-speed benchtop centrifuge and is completed in under 30 minutes. Prior to collecting the cell culture supernatant, HEK293T cells were cultured in conditioned media (CM) containing 10% exosome-free FBS for 72 hours. We used 10 ml of precleared supernatant for the isolation of exosomes using our Capturem kits (see Methods for full exosome isolation protocol details). The eluted exosomes were analyzed with an LM10 NanoSight to examine their physical properties (i.e., size) via nanoparticle tracking analysis (NTA). Our results show that Capturem-isolated exosomes have a size distribution with a mode of 95 nm and a D90 value of 211 nm, which signifies that the majority of exosomes are 95 nm and 90% of the isolated exosomes are below 211 nm (Figure 1). This size range agrees with the experimental values reported in the exosome field and published studies (H. Rashed et al. 2017).

Figure 1. NTA data of isolated exosomes from HEK293T cells using the Capturem Exosome Isolation Kit (Cell Culture). Panel A. Size and particle concentration distribution plot of exosomes from HEK293T cells (peak at 95 nm). D90 value is shown. Panel B. Three-dimensional graph illustrating size versus intensity (relative frequency of each size range in the eluted sample) versus concentration (particles/ml).

Capturem columns isolate more exosomes than ultracentrifugation or precipitation solutions

Next, we compared the yield and average size of exosomes isolated using three methods: Capturem isolation columns, precipitation solution (Competitor T), or ultracentrifugation. Despite using the same amount of input, our quantitative analysis characterizing the exosomes obtained from the different isolation methods showed that Capturem columns yielded significantly higher exosome concentrations in comparison with Competitor T’s precipitation-based method (Figure 2A). Further, the ultracentrifugation yield was significantly lower in comparison to all other methods. Most importantly, NTA analysis showed that Capturem‑column‑isolated exosomes were of similar size compared to those isolated by the other two methods (Figure 2B). Taken together, these data demonstrate that while all methods isolate similarly sized exosomes, Capturem columns gave the highest yield.

Figure 2. Quantitative determination of yield and mean size of exosomes isolated through different methods. NTA data of particle concentration (Panel A) and size (Panel B) for precleared CM (input) and isolated exosomes (yield) from HEK293T cells using the Capturem kit, precipitation reagent (Competitor T), and ultracentrifugation. The isolation methods are described in the Methods section. The exosomes were isolated from 10 ml of precleared CM supernatant for each method. The total exosome yield was divided by input volume to obtain yield (particle/ml of input).

Biochemical characterization of Capturem-isolated exosomes identifies key protein markers

Studies profiling the contents of exosomes have identified several consensus proteins known to be found in most exosomes: CD81 and Alix. Western blot analysis on Capturem-isolated exosomes identified the presence of these characteristic exosome-associated proteins (Figure 3). The densitometric analysis revealed enrichment of CD81 by approximately 8-fold and Alix by approximately 2-fold in eluted exosomes as compared to input CM supernatant containing unconcentrated exosomes. The enrichment of both CD81 and Alix in Capturem-isolated exosomes was similar to that observed by the ultracentrifugation method. This data indicates that exosomes isolated using Capturem columns are concentrated and contain key proteins found in exosomes.

Figure 3. Enrichment of exosomal markers CD81 and Alix in Capturem-isolated exosomes. Western blot analysis of CD81 and Alix as per the protocol described in the Methods section. The band densities were calculated using Image Lab software.

Exosomes isolated with Capturem columns are intact and functional

Exosomes can potentially be used as a tool to deliver specific therapeutics to target cells. However, isolated exosomes must be intact to later be internalized by target cells to exert their biological effect, and ultracentrifugation can often damage isolated exosomes. Therefore, we investigated the ability of isolated exosomes from either Capturem isolation columns or ultracentrifugation to be taken in by HEK293T cells and deliver a dye. The exosomes were labeled with PKH26 dye in a tube, added to cultured cells, and incubated for 1.5 hours (Figure 4). Our results demonstrate that Capturem-isolated exosomes were internalized by all cells in the field, similar to exosomes isolated using ultracentrifugation. However, since Capturem isolation provided a significantly higher yield of exosomes, as compared with ultracentrifugation, only 2% of the isolated exosome eluate was needed to conduct the cellular uptake application while 18% of the ultracentrifugation yield was required. The fluorescence intensity of cells challenged with labeled vesicles in precleared input CM supernatant was significantly lower than eluted exosomes, which is another indication of enrichment of exosomes in the Capturem sample and ultracentrifugation pellet. Thus, Capturem columns isolate a higher amount of intact and functional exosomes in comparison to ultracentrifugation.

Figure 4. Cellular uptake of exosomes in HEK293T cells. Isolated exosomes from the Capturem kit, ultracentrifugation, and input CM supernatant were labeled with PKH26 dye and uptake studies were carried out as described in the Methods. The percentage of total yield required for cellular uptake assay is displayed to provide a comparison of the yield differences between the two methods.

Capturem columns outperform ultracentrifugation when isolating exosomes from HT1080 cells

We also evaluated the performance of Capturem isolation columns against ultracentrifugation for the isolation of exosomes from a second cell type, HT1080 cells. The isolated exosomes from 8 ml of precleared CM supernatant of each method were subjected to NanoSight analysis. The general particle size observed was similar between the isolation methods, while yield, expressed as particles/ml of input, was almost 2.5‑fold higher for the Capturem columns (Figure 5). Capturem-isolated exosomes also showed an improved D90 value (205 nm) as compared with ultracentrifugation (257 nm). Thus, Capturem isolation columns again isolated more exosomes overall than ultracentrifugation.

Figure 5. NTA data for quantitation of isolated exosomes from HT1080 cells using the Capturem kit or ultracentrifugation. Size and particle concentration distribution plot of exosomes using Capturem column (peak at 115 nm) and ultracentrifugation (peak at 125 nm). D90 values and yields of the isolated exosomes CM is shown. In this experiment, we used 8 ml of precleared CM supernatant for the isolations.

Cell culture growth parameters affect the yield and potential aggregation of exosomes

Since cell culture practices vary greatly across different cell types and users, we investigated the effect of cell culture growth parameters, such as cell confluency and incubation time in exosome‑free FBS media, on the isolation of exosomes. We performed three different experiments using HEK293T cells, with each experiment involving a different cell culture parameter as shown in the table below. The exosomes were isolated from 8 ml of precleared CM supernatant for each experiment. In Experiment I, cells were 80% confluent when switched to exosome-free FBS for 72 hours. In contrast, Experiment II switched 80% confluent cells to exosome-free FBS for only 48 hours. In both experiments, the cells were overgrown when collecting supernatant for exosome isolation. Our results show that while exosome yield is high in both experiments, an additional peak is observed between 200 and 400 nm for both experimental sets and skewed D90 values of 327 nm and 331 nm, respectively. This extra peak most likely represents a phenomenon related to exosome biology that causes the formation of aggregates when exosomes are present at high concentrations, such as in these experiments where the cells were too dense.

To further test the hypothesis that highly confluent cells lead to an extra peak representing aggregated exosomes, we performed Experiment III, in which cells were only 40% confluent when switched to exosome-free FBS for 48 hours. Notably, cells were only 90% confluent when the supernatant was collected. Accordingly, the yield was approximately 3- to 6-fold lower when compared to Experiments I and II, due to the lower cell input. However, when cells were less confluent, we did not identify a second, larger peak. Instead, the isolated exosomes made a uniform peak of 50–200 nm and exhibited a D90 value of 176 nm. These data highlight the dynamic role cell culture conditions, especially cell density, can have on exosome production and aggregation in vitro. Further, these data demonstrate that the Capturem isolation protocol can identify altered exosome behavior that could occur when cells become overgrown, when exosomes aggregate, or due to inherent differences between cell types.

Figure 6. Effect of cell culture growth parameters on exosome yield and size. HEK293T cells were grown in 150-mm cell culture dishes until the indicated (see table) approximate cell confluency before incubation with conditioned media for the indicated duration. Following incubation, CM supernatant was harvested, centrifuged, precleared with Capturem preclearing columns, and then 8 ml of precleared CM supernatant was used for the exosome isolations using the Capturem kit as described in the Methods. NTA analysis was performed using an appropriate dilution to satisfy the 20–100 particles per frame criterion for reliable data acquisition. The total exosome yield was divided by input volume to obtain particles/ml of precleared CM supernatant input.

Effect of cell culture parameters on exosomes size and yield: HEK293T cells
Parameter Experiment I Experiment II Experiment III
Approximate confluency before incubation (%) 80 80 40
Incubation time in CM with exosome-free FBS (hr) 72 48 48
Approximate confluency at harvest (%) Overgrown Overgrown 90
Yield (particles/ml of input) 1.9 x 109 9.4 x 108 3.1 x 108
D90 size (nm) 327 331 176

Conclusions

As researchers strive to translate their findings from exosome studies into diagnostics and therapies, it will be critical to have reliable results. This requires consistently isolating functional exosomes of high purity in a straightforward manner. The Capturem Exosome Isolation Kit (Cell Culture) provides a unique and complete solution for the simple, rapid isolation of exosomes from cell culture. The protocol is performed using a benchtop centrifuge with an incubation-free workflow in under 30 minutes. The Capturem-isolated exosomes display the physical, biochemical, and biological properties, considered to be common exosome characteristics.

Methods

Cell culture and exosome isolation

HEK293T and HT1080 cells were grown in DMEM with high glucose (4500 mg/l) and supplemented with 10% FBS in 150-mm culture dishes for 24–48 hr until 40–60% confluent. Then, the cells were washed twice with DMEM-base media followed by incubation in DMEM media supplemented with 10% exosome-free FBS for 48–72 hr. The cell culture conditioned media (CM) was then collected and centrifuged at 3,000g for 10 min to remove any dead cells or larger cell fragments. Next, the CM supernatant was precleared using either a 0.8-µM Minisart syringe filter (Sartorius) or Exosome Isolation Pre-Clearing Columns (Capturem kit, purple inserts).

Exosome isolation

Unless indicated, for isolation of exosome from CM supernatants, syringe-precleared CM was used for the precipitation (Competitor T) and ultracentrifugation methods, while the Capturem preclearing columns were used for the Capturem exosome isolation protocol. We followed the supplier’s protocol for Competitor T’s kit.

For ultracentrifugation, precleared CM supernatant was centrifuged at 100,000g for 1.5 hr at 4°C using an Optima MAX-E Ultracentrifuge (Beckman Coulter) to collect exosomes. The exosomal pellet was washed in PBS and further ultracentrifuged at 100,000g for 1.5 hr at 4°C. The final exosomal pellet was resuspended in 1 ml PBS and stored at –80°C until use.

For the Capturem kit, precleared CM supernatant was loaded onto Capturem Maxiprep Exosome Isolation Columns (Cell Culture) and centrifuged at 1,000g for 2–4 min at room temperature. The column was washed once with the Exosome Isolation Wash Buffer and exosomes were eluted using the Exosome Isolation Elution Buffer supplied in the kit.

Nanoparticle tracking analysis (NTA)

NTA is a light scattering method widely used for the rapid assessment of exosome size and concentration. Since exosomes are polydisperse (50–200 nm) in nature and most studies use polystyrene beads size standards for NTA standardization, this method doesn’t provide satisfactory estimation as these beads have higher refractive index and are much brighter than the exosomes to be measured. To address this issue, we optimized the camera settings for accurate measurement of polydisperse exosomes. For reliable NTA measurement, we used an appropriate exosome sample dilution that provides approximately 20–100 particles in the field of view. The optimized video capture setting parameters included a screen gain set to 6 or 7 and camera level set to 12 or 13. The chosen tracking settings were a screen gain between 8–10 and a detection threshold of 2 or 3. We recorded three 30 sec videos on the NanoSight LM10, which were analyzed using tracking software. The NTA software plots particle size range vs. particle number in the sample.

Western blotting

Isolated exosomes, either with the Capturem kit or ultracentrifugation, were concentrated using 100‑kDa Vivaspin filter (Sartorius), washed once with PBS, and approximately 40 µl was recovered. The concentrated exosomes were mixed with 10 µl of 5X RIPA lysis buffer (Alfa Aesar) containing 1X protease inhibitor cocktail (Roche) and vortexed, which was then followed by sonication at 20°C for 5 min in a water sonicator (Ultrasonic Cleaner, JSP). After sonication, the tube was incubated on ice for 20 min with intermittent mixing. Next, the mixture was centrifuged at 10,000g for 10 min at 4°C. The isolated clear lysate was transferred to a fresh tube. The protein amount was estimated using the Micro BCA Protein Estimation Kit (Thermo Fisher) and 10 µg of protein was loaded per lane for Western blotting. The samples were resolved by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. The blots were then either probed with Exosome-Anti-CD81 antibody (Thermo Fisher) at 2 µg/ml or mouse monoclonal Alix antibody at a 1:1,000 dilution (SCBT). The primary antibody incubation was conducted at 4°C overnight. Following washing, the membranes were incubated with secondary anti-mouse IgG HRP at a 1:5,000 dilution for 1 hr at room temperature. The membranes were developed with chemiluminescence using SuperSignal West Pico substrate (Thermo Fisher) and bands were visualized on a BioRad ChemiDoc Touch Image System. The band density was calculated using Image Lab software (BioRad).

Exosome labeling and uptake into HEK293T cells

The exosomes were isolated using either the Capturem Exosome Isolation Kit (Cell Culture) or ultracentrifugation as described above. An aliquot of frozen eluted exosomes was resuspended in 1 ml of PBS and labeled using PKH26 Fluorescent Cell Linker Kits (Sigma-Aldrich) according to the manufacturer’s instructions, with slight modifications in the washing step. A 2X PKH26-dye solution (4 µl PKH26 dye into 1 ml of Diluent C) was prepared and mixed with 1 ml of exosomal solution for a final dye concentration of 2 x 10-6 M. The samples were immediately mixed gently for 5 min, and staining was stopped by adding 2 ml of 1% BSA to capture excess PKH26 dye. The labeled exosomes were transferred into a 100-kDa Vivaspin filter (Sartorius) and spun at 4,000g then washed with PBS twice, and approximately 50 µl of sample was recovered for analysis of exosome concentration using NTA prior to storage at –80°C. PBS was used as negative control for the labeling reaction. For input sample, 1 ml of precleared conditioned media was used for the labeling reaction. To perform the uptake studies, HEK293T cells were plated in 8‑well chamber slides (1 x 104 cells/well) using regular medium. After 24 hr, the slides were washed twice with PBS, and incubated with DMEM media containing 10% exosome-free FBS for 24 hr. Following this, fresh DMEM media with 10% exosome-free FBS (200 µl) containing each labeled exosome sample, corresponding to 2 x 109 exosomes, was added to each well and incubated for 1.5 hr in a cell culture incubator. After incubation, the slides were washed twice with PBS (500 µl) and fixed with 4% paraformaldehyde solution for 20 min at room temperature. The slides were washed twice with PBS (500 µl), dried, and mounted using a ProLong Gold Antifade Reagent with DAPI (Life Technologies). The cells were visualized using an Axioskop microscope (Carl Zeiss).

References

H. Rashed, M. et al. Exosomes: From Garbage Bins to Promising Therapeutic Targets. Int. J. Mol. Sci. 18, 538 (2017).

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