Extracellular vesicles (EVs) contribute to cellular biology and intercellular communications. There is a need for practical assays to visualize and quantify EVs uptake by the cells. The current protocol proposes the EV uptake assay by utilizing three-dimensional fluorescence imaging via confocal microscopy, following EV isolation by a nano-filtration-based microfluidic device.
There is a need for practical assays to visualize and quantify the cells’ extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular communication in various research fields; cancer biology, neuroscience, and drug delivery. Many EV uptake assays have been reported in the literature; however, there is a lack of practical, detailed experimental methodology. EV uptake can be assessed by fluorescently labeling EVs to detect their location within cells. Distinguishing between internalized EVs in cells and the superficial EVs on cells is difficult, yet critical, to accurately determine the EV uptake. Therefore, an assay that efficiently quantifies EV uptake through three-dimensional (3D) fluorescence confocal microscopy is proposed in this work. Fluorescently labeled EVs were prepared using a nano-filtration-based microfluidic device, visualized by 3D confocal microscopy, and then analyzed through advanced image-processing software. The protocol provides a robust methodology for analyzing EVs on a cellular level and a practical approach for efficient analysis.
Extracellular vesicles (EVs) are nano-sized, lipid membrane-bound particles that are categorized by their sizes: ectosomes (100-500 nm) and exosomes (50-150 nm)1. EVs contain various biomolecules, such as proteins, nucleic acids, and lipids. These biomolecules originate from the cells before being encapsulated as cargo and released into the extracellular space via EVs1,2,3.
Due to the variety of their cargo, EVs are believed to play an active role in intercellular communication. The release and uptake of EVs by cells allow the transfer of biomolecules between the cells4,5. The introduction of EV cargo to a cell may alter the recipient cell's functions and homeostatic state4,5,6. EVs are internalized through multiple pathways; however, the exact mechanisms have not been accurately demonstrated.
The majority of the EV uptake assays, such as genetic tagging, fluorescently label individual EVs7. The resulting signal can be measured by microplate photometer, flow cytometry, or microscopy, with each technology having substantial limitations. Microplate photometers, flow cytometry, or standard two-dimensional (2D) microscopy cannot distinguish between internalized and superficially attached EVs8,9. Additionally, the necessary sample preparation for each of these techniques may introduce additional issues to EV uptake evaluation. For example, lifting adhered cells with trypsin before EV uptake analysis may cleave some superficially attached EVs on the cell's surface10,11. Trypsin may also interact with the cell surface, affecting cell and EV phenotype. Additionally, trypsin may not detach superficial EVs entirely, skewing isolated populations.
To accurately label EVs with fluorescent dyes, additional wash steps are required to remove the residual dye7. Accepted isolation techniques can also contribute to false-positive signals due to coagulation that occurs during EV isolation. For example, serial ultracentrifugation (UC) is widely used to isolate EVs and remove the immobilized dye. However, UC may co-precipitate EVs, and the residual dye may lead to a false-positive signal12,13. Other nano-filtration methods, such as column-based filtration, are also widely used for non-immobilized dye removal. The complex nature of EVs and dye interacting within the column matrix may lead to incomplete removal of residual dye due to the molecular cut-off of the column being altered by the complex input14,15,16.
The current protocol proposes a nano-filtration-based microfluidic device to isolate and wash fluorescently labeled isolated EVs. The nano-filtration-based microfluidic device can provide efficient filtration via fluid-assisted separation technology (FAST)17,18. FAST reduces the pressure drop across the filter, thus reducing potential aggregation between EVs and dyes. By efficiently removing residual dye, it is possible to enhance the quality of fluorescently labeled EVs and the assay's specificity.
Confocal microscopy can distinguish between internalized and superficially attached EVs on the cell surface and comprehensively investigate the cellular mechanisms of EV uptake in a spatiotemporal resolution19,20,21,22,23,24,25. For example, Sung et al. described the visualization of the exosome lifecycle using their developed live-cell reporter. The location of the internalized EVs was detected and analyzed using a confocal microscope in three-dimension (3D) and post-image processing tools20. Although the size of small EVs (40-200 nm) is below the resolution limit of the optical microscope, the fluorescently labeled EVs can be detected by confocal microscopy since the photodetector can detect the enhanced fluorescence emission. Therefore, the subcellular localization of the fluorescently labeled EVs within a cell can be precisely determined by acquiring multiple z-stacked images of the EVs and the surrounding cellular organelles.
Additionally, 3D reconstruction and post-data processing can provide further insight into the positioning of the internalized, superficial, and free-floating EVs. By utilizing these processes in conjunction with the time-lapse live-cell imaging offered by confocal microscopy, the level of EV uptake can be precisely evaluated, and the real-time tracking of EV uptake is also possible. Further, EV trafficking analysis can be performed using confocal microscopy by assessing the co-localization of EVs with organelles, a first step to determine how internalized EVs are involved in the intracellular function. This protocol describes the methodology for performing an EV uptake assay using the nano-filtration-based microfluidic device17,26, confocal microscopy, and post-image analysis.
1. EV isolation and on-chip immuno-fluorescent EV labeling
2. Incubation of the cells with fluorescently labeled EVs for the EV-uptake assay
3. Confocal microscopy
4. Image processing
Using a nano-filtration-based microfluidic device, EVs were isolated from PC3 CCM and labeled with a fluorophore-conjugated EV-specific (CD63) antibody (Figure 1). The labeled EVs were successfully visualized by the 3D confocal microscopy (Figure 2). The labeled EVs were incubated with cells for several hours in exosome-depleted media. Following incubation, cells were washed with exosome depleted media. The remaining EVs were internalized or adhered to cells during incubation. The cell area was labeled. Internalized EVs were visualized as puncta19,20,24,27 and an individual EV (Figure 3 and Figure 4A). Post-processing of these images allows the visualization and quantification of EV internalization into the cells (Figure 4). The steps in conjunction allow accurate EV uptake assay performed efficiently. Figure 5 shows the representative results of the EV uptake assay. The assay indicates that the level of EV uptake is dependent on the length of the incubation period. The procedure allows for the systematic exclusion of non-internalized EVs (Figure 4C) to precisely mea+sure the number of internalized EVs. The size distribution of internalized EV dots was calculated (Figure 6). Furthermore, the number of internalized EVs can be normalized to the recipient cells' volume to determine the actual rate of EV uptake for the specific cell. Normalization accounts for the heterogeneous cell size and represents the number of internalized EVs regarding the cellular surface area. Cellular surface area is defined as the cell area in contact with EV-spiked, exosome-depleted culture media during incubation.
Figure 1: Schematic illustration of the EV isolation and on-chip labeling using a nano-filtration-based microfluidic device. (A) EVs isolation from CCM. (B) On-chip Immunofluorescent labeling of EVs. (C) Removal of unbound antibodies. Please click here to view a larger version of this figure.
Figure 2: Imaging of fluorescently labeled EVs. The fluorescently labeled (anti-CD63-Alexa Fluor 488) EVs were detected using the confocal microscope (40x objective). (A) Positive sample (anti-CD63-Alexa Fluor 488 labeled EVs). (B) Negative control 1 for the EV labeling (EVs with 2nd antibody (Alexa Fluor 488) only, without 1st antibody). (C) Negative control 2 (EVs with the mouse (MS) IgG antibody and 2nd antibody (Alexa Fluor 488)). Please click here to view a larger version of this figure.
Figure 3: Imaging of the internalized EVs into cells in a 2D image. (A) The fluorescently labeled (anti-CD63-Alexa Fluor 488, green) EVs and the cells (CMTMR, red) were detected by using the confocal microscope (20x objective) after the incubation. (B) A separate image of the fluorescently labeled EVs only. (C) A separate image of the fluorescently labeled cells only. The excitation/emission laser wavelengths for CMTMR and Alexa Fluor 488 are 560.6/595 (±50) nm and 487.8/525 (±50) nm. Laser power settings are 3.0 % for CMTMR and 10.0 % for Alexa Fluor 488. Please click here to view a larger version of this figure.
Figure 4: Quantification of the internalized EVs by the post-imaging process. (A) Raw confocal image obtained from the EV-uptake assay. (B) Virtual rendering of the EVs as a dot (green) and the cells as a surface (red) by using the image-processing software. (C, i-iv) Discrimination of the internalized EVs (yellow dots) and non-internalized EVs (green dots, white arrow) using the software provided algorithm. Please click here to view a larger version of this figure.
Figure 5: The amount of EV uptake as a function of incubation time. (A) The number of internalized EVs per cell. (B) The number of internalized EVs per cell volume. The number of internalized EVs was increased depending on the incubation time. Please click here to view a larger version of this figure.
Figure 6: The size distribution of internalized EV dots. The size of EV dots was measured and plotted to a distribution. Please click here to view a larger version of this figure.
Supplementary Figure 1: NTA measurement of anti-CD63-Alex Fluor 488 labeled EVs. Please click here to download this File.
Supplementary Figure 2: The amount of co-localized EVs with lysosomes as a function of incubation time. Please click here to download this File.
Supplementary Figure 3: The amount of EV uptake as a function of incubation time. (A) The number of internalized EVs per cell. (B) The number of internalized EVs per cell volume. The number of internalized EVs was increased depending on the incubation time. The EV sample was labeled by RNA staining dye (see Table of Materials). Please click here to download this File.
An EV uptake assay based on 3D fluorescence imaging via confocal microscopy provides an efficient methodology and sensitive analysis. This fluorescent EV labeling facilitates the visualization of EVs and successfully performs a precise EV uptake assay. Previous methods for labeling EVs and removing the residual dye have been reported by removing precipitation using ultracentrifugation (UC); however, UC may co-precipitate EVs, and the immobilized dye may lead to a false-positive signal12,13. Nano-filtration-based microfluidic devices eliminate this co-precipitation of EVs and dye, thus enhancing the quality of the fluorescently labeled EVs and the assay's specificity. EV uptake was measured by volumetric analysis to distinguish and quantify the internalized EVs separate from superficial EVs on the cell surface. The volumetric analysis of EV uptake allows for the normalization of EV uptake by cell size. The live-cell EV uptake assay was achieved by utilizing the on-stage confocal microscope incubator. The protocol applies to studies requiring live cell cultures and EVs. Real-time intracellular EV tracking can be performed across a 3D window. Additionally, EV trafficking analysis of spatial-temporal resolution along with the co-localization of EVs with subcellular organelles can be achieved through the protocol (Supplementary Figure 2). The protocol described here is a powerful tool for the cellular analysis of EVs.
Despite all advantages of the protocol, there are potential limitations. The nano-filtration microfluidic device utilized in this study may co-isolate other contaminants during EV isolation. Low-density lipoproteins are similar in size to EVs and may be caught in the membrane during isolation28. Although EV-specific markers can specify EVs, the co-isolated contaminants may affect downstream analysis. In this manuscript, EVs were labeled with a CD63 conjugated Alexa Fluor 488 dye. This labeling increases the specificity for EV detection; however, it also binds the EV surface molecule and increases competitive binding. Additionally, the level of EV uptake may be dependent on the cell line and CCM. The labeling methods detailed in this protocol can be adapted to other staining dyes such as lipophilic dyes, cytosolic dyes, and RNA dyes (Supplementary Figure 3). The protocol uses a light scanning confocal microscope (LSCM) to detect the tiny signal of EVs. LSCM relies on intense lasers and, as a result, living cells may be damaged or altered by long-term laser excitation. The fast acquisition speed can decrease photodamage by utilizing a spinning disc confocal microscope (SDCM). SDCM can also be used in EV tracking that requires short time-lapse imaging to visualize short-distance movements.
Despite these potential limitations, the proposed protocol provides an efficient method for EV-uptake assessment and has the potential for further live-cell EV-tracking analysis.
The authors have nothing to disclose.
This work was supported by NCI grant nos. U54CA143803, CA163124, CA093900, and CA143055 to K. J. P. This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI19C1122). Work by J. Kim and Y.-K. Cho was supported by Institute for Basic Science (IBS-R020-D1), funded by the Korean Government. The authors thank the current and past members of the Brady Urological Institute, especially members of the Pienta-Amend laboratory, for the critical reading of the manuscript.
Alexa Fluor 488 anti-human CD63 Antibody | Biolegend | 353038 | Fluorescent dye conjugated EV-specific antibody |
CellTracker Orange CMTMR Dye | Thermo Fisher Scientific | C2927 | Live cell (cytoplasm) fluoresent labeling reagent |
CFI Apo Lambda S 40XC WI | Nikon | MRD77400 | Objective for confocal imaging, NA=1.25 |
CFI Plan Apo VC 20X | Nikon | MRD70200 | Objective for confocal imaging, NA=0.75 |
Exodisc | Labspinner Inc. | EX-D1001 | A nano-filtration based microfluidic device for EV isolation |
ExoDiscovery | Labspinner Inc. | EX-R1001 | Operation device for Exodisc |
Exosome-depleted FBS | Thermo Fisher Scientific | A2720801 | Nutrient of cell culture media for PC3 cell line derived EV collection |
Fetal bovine serum (FBS) | VWR | 1500-500 | Nutrient for cell cultivation |
Goat Anti-Mouse IgG H&L preadsorbed | abcam | ab7063 | Mouse IgG antibody for negatvie control of EV labeling |
Ibidi USA U DISH μ-Dish 35 mm | Ibidi | 81156 | Culture dish for confocal imaging |
Imaris 9.7.1 | Oxford Instruments | 9.7.1 | Post-image processing software |
Incubator System+ CO2/O2/N2 gas mixer | Live Cell Instrument | TU-O-20 | Incubator system for live cell imaging |
Nikon A1 HD25 / A1R HD25 camera | Nikon | NA | Camera for confocal imaging |
Nikon Eclipse Ti microscope | Nikon | NA | Inverted microscope for confocal imaging |
NIS-Elements AR 4.50.00 | Nikon | 4.50.00 | Image processing software for Nikon microscope |
NTA, NanoSight NS500 | Malvern Panalytical | NS500 | Measurement device for EV concentration |
OriginPro 2020 | OriginLab | 9.7.0.185 | Graphing software |
Penicillin-Streptomycin | Thermo Fisher Scientific | 15140122 | Antibiotics for cell cultivation |
RPMI 1640 | Thermo Fisher Scientific | 21875034 | Cell culture media for PC3 cell line cultivation |
SYTO RNASelect Green Fluorescent cell Stain – 5 mM Solution in DMSO | Thermo Fisher Scientific | S32703 | RNA staining fluorescent dye for the EV labeling |