Method Article

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

DOI:

10.3791/62836

⸱

February 14th, 2022

 ,  ,  ,  ,  ,  , 
1The Brady Urological Institute, Johns Hopkins University School of Medicine, 2Department of Biomedical Engineering, School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), 3Department of Urology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, 4Center for Soft and Living Matter, Institute for Basic Science (IBS)

* These authors contributed equally

In This Article

Summary

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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.

Abstract

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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.

Introduction

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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.

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Protocol

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1. EV isolation and on-chip immuno-fluorescent EV labeling

  1. Collection of cell culture media (CCM) and pre-processing of CCM for EV isolation
    1. Seed PC3 cells at 30% confluency in a 75 cm2 cell culture flask. Allow control cells to grow to 90% confluency (~48 h) in standard media and cell-line-specific supplements.
      NOTE: To prevent EV-containing components from affecting cellular uptake (i.e., fetal bovine serum), use exosome-depleted media and supplements.
    2. Harvest the CCM.
    3. Centrifuge the CCM at 1000 x g for 10 min at room temperature (RT) to pellet any unattached cells and large debris harvested with the media. Transfer the supernatant to a new conical tube.
    4. In the new tube, centrifuge the supernatant at 10,000 x g for 20 min at 4 °C to pellet smaller debris and apoptotic bodies remaining in the media. Some larger EVs will pellet. Transfer the supernatant to a new tube.
    5. Filter the supernatant through a 0.45 µm hydrophilic Polyvinylidene fluoride (PVDF) membrane syringe filter.
      NOTE: If not immediately processing CCM for EV isolation, store the pre-processed CCM at -80 °C until isolation is performed. If frozen, limit freeze-thaw cycles to one.
  2. EV isolation from CCM using a nano-filtration based microfluidic device
    1. If frozen, completely thaw CCM and vortex for 30 s before step 1.2.2.
    2. Inject 1 mL of pre-processed CCM (step 1.1) into the sample chamber of the nano-filtration-based microfluidic device (see Table of Materials)17,26.
      NOTE: Follow the standard operating procedure for the nano-filtration-based microfluidic device17,26.
    3. Spin at 3000 rpm for 10 min in the bench-top spinning machine (see Table of Materials) to operate the microfluidic device.
      NOTE: If CCM remains on the sample chamber following the initial run, perform additional spins until all CCM has emptied from the sample chamber.
    4. Remove the fluid from the waste chamber by pipetting and repeat steps 1.2.1, 1.2.2, and 1.2.3 twice.
      NOTE: In total, 3 mL of CCM will be processed for EV isolation.
    5. Inject 1 mL phosphate-buffered saline (PBS) into the sample chamber to wash the isolated EVs. Spin in the bench-top spinning machine for operating the microfluidic device as mentioned in step 1.2.3. Locate the pure EVs on the membrane of the device.
      NOTE: The quality of EVs isolated from the nano-filtration based microfluidic device, specifically, was confirmed and compared to the conventional UC method by transmission electron microscopy (TEM), scanning electron microscope (SEM), nanoparticle tracking analysis (NTA), structured illumination microscopy, enzyme-linked immunosorbent assay and real-time PCR in the previous research17,26.
  3. Immunofluorescent labeling of EV using nano-filtration based microfluidic device (Figure 1)
    1. Select an EV-specific antibody according to the purpose of the assay (see Table of Materials).
      NOTE: Certain antibodies may interfere with ligand binding sites specific to EV-uptake pathways (i.e., endocytosis).
    2. Inject 1 µg/mL of the EV-specific antibody into the elution hole of the device containing 100 µL of isolated EVs.
    3. Incubate for 1 h in the dark at RT on a plate shaker to ensure the even distribution of the antibody across the sample.
    4. Attach an adhesive tape to the elution hole. Inject 1 mL PBS into the sample chamber to wash out any residual antibodies.
    5. Spin the device at 3000 rpm until the sample chamber is empty. Remove any fluid from the waste chamber by pipetting. Inject 1 mL PBS into the sample chamber.
      NOTE: Fluorescently labeled EVs will be located in the membrane chamber.
    6. Pipette the fluorescently labeled EVs (Figure 2) from the membrane chamber to an amber tube. Block from light until use.

2. Incubation of the cells with fluorescently labeled EVs for the EV-uptake assay

  1. Target cell seeding and culture on the cell-culture compatible dishes.
    1. Seed 1 x 104 PC3 cells into the microslide 8-well plate (9.4 x 10.7 mm for each well) with 0.2 mL of media or 4 x 104 PC3 cells into a 35 mm dish with 1 mL of media. Plate the cells into a cell-culture compatible dish consisting of a thin coverslip (thickness: 0.18 mm).
      NOTE: The thin coverslip minimizes the adverse scattering of light.
    2. Allow cells to adhere overnight in optimal cell-culture conditions (37 °C, 5% CO2 concentration, 90% humidity).
    3. Wash adhered cells twice with exosome-depleted media (described in step 1.1.1).
  2. Cell incubation with fluorescently labeled EVs.
    1. Measure the concentration of fluorescently labeled EVs (step 1.3.5) by nanoparticle tracking analysis (NTA, Supplementary Figure 1). Determine the optimal concentration of fluorescently labeled EVs to be added to the cultured cells (step 2.1.2.).
    2. Dilute the fluorescently labeled EVs with exosome-depleted media to match the desired concentration measured in step 2.2.1. (i.e., 7.80 x 109 EVs (in NTA value) in 200 µL of exosome-depleted media.)
    3. Add the diluted EVs (Step 2.2.2) to the adhered target cells prepared at 2.1.2. Incubate for experimental time (i.e., 4, 8, or 12 h).
    4. Wash cells thrice with exosome-free media to remove any non-internalized EVs.
      NOTE: Optional: Cells can be fixated following wash.
    5. Label the cytoplasm of the adhered cells with 1 µg/mL of CMTMR ((5-(and-6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine) (see Table of Materials) and incubate in optimal cell-culture conditions (37 °C, 5% CO2 concentration, 90% humidity).
      NOTE: Cell area dyes should fluoresce separately from labeled EVs to aid in determining the spatial location (internalized or superficial) of the spiked EVs during the EV uptake assay.
    6. Wash labeled cells twice with exosome-depleted media to remove the residual dye. Add fresh exosome-depleted media to the cells in preparation for live-cell confocal imaging.

3. Confocal microscopy

  1. To perform live-cell imaging, utilize an on-stage incubator to maintain optimal cell-culture conditions (37 °C, 5% CO2 concentration, 90% humidity).
  2. Place the prepared cells in the on-stage incubator.
  3. Set the imaging parameters based on control samples.
    NOTE: Suggested control samples include: Fluorescently labeled EVs only, fluorescently labeled cells, unlabeled EVs, and unlabeled cells.
  4. Determine the depth of the target cells and the range of stacking size in the z-direction to acquire 3D confocal images.
    NOTE: The thickness of a Z-stack is 1 µm. The confocal 3D image acquisition lasted 2 min 34 s (each Z-plane image acquisition took approximately 8 s; a total of twenty Z-stack images).
  5. Set image acquisition to multiple z-stacked images of both cell-specific dye (i.e., red) and EV-specific dye (i.e., green) simultaneously (Figure 3 and Figure 4A).

4. Image processing

  1. Utilize automatic image-processing software to analyze the raw z-stacked confocal images and determine the EV uptake by cells (see Table of Materials).
  2. Set thresholding parameters to the fluorescent signal of the cells and EV-specific dyes. Build the virtual surfaces of cells (Figure 4A,B).
    1. To build the virtual surfaces of cells, click the button Add new Surfaces.
    2. Select Shortest Distance Calculation as "Algorithm Settings" to use the provided algorithm by the software, then click Next: Source Channel.
    3. Select Channel 2 - CMTMR as "Source Channel" in this experiment.
    4. Select Smooth and put the appropriate value into "Surfaces Detail" for surface smoothing.
      NOTE: 0.57 µm in this experiment since 1 pixel represents 0.57 µm in raw imaging data.
    5. Select Absolute Intensity as "Thresholding."
    6. To automatically threshold the fluorescent image by the provided algorithm, click Threshold (Absolute Intensity): The value is automatically set.
    7. Select Enable as "Split touching Objects (Region Growing)" and put the value of estimated cell size into "Seed Points Diameter," 10.0 µm in this experiment. Then click Next: Filter Seed Points.
    8. To configure the virtual cell surfaces, click + Add button, then select Quality as "Filter Type." Threshold the appropriate value (210 in this experiment) for the low limit by a visual inspection and the maximum value (1485) for the upper limit, then click the Finish button.
      NOTE: A visual inspection means that a researcher can discriminate the cellular area from a raw fluorescent image.
    9. Next, to build the virtual dots of EVs, click the button Add new Spots.
    10. Select Different Spot Sizes (Region Growing) and Shortest Distance Calculation as "Algorithm Settings," then click Next: Source Channel.
    11. Select Channel 1 - Alexa Fluor 488 as "Source Channel" in this experiment.
    12. Put the appropriate value into "Estimated XY Diameter" for the spot detection, 1 µm in this experiment. Then, click Next: Filter Spots.
    13. To configure the virtual EV dots, click + Add button, select Quality as "Filter Type," and set "Lower Threshold" by a visual inspection, 100 in this experiment. Then, click the Next: Spot Region Type button.
    14. Select Absolute Intensity as "Spot Regions Type," then click Next: Spot Regions.
    15. To threshold, the region of EV dots, put the appropriate value into "Region Threshold" by a visual inspection, 100 as "Region Threshold" in this experiment.
      NOTE: A visual inspection means that a researcher can discriminate the EVs area from a raw fluorescent image.
    16. Select Region Volume as "Diameter from," then click Finish.
  3. Use the software's provided algorithms to split the grouped spots inside the built surface at step 4.2 (Figure 4C, i-iv).
    1. Click the built Spots, then go into Filters.
    2. Click + Add button, then select Shortest Distance to Surfaces Surfaces = Surface 1 as Filter Type, then click Duplicate Selection to new Spots button. The lowest threshold (-7.0 in this experiment) for the low limit and the appropriate value (-0.5) for the upper limit.
      NOTE: Set the upper limit with the estimated radius of Spots. In this experiment, the estimated diameter of Spots,i.e., EV dots, was set to 1 µm in step 4.2.11.; thus, the upper limit can be 0.5.
  4. Automatic count of EVs inside the cells
    NOTE: The software will automatically count the number of EVs inside the cells, indicating the number internalized by the target cells.
    1. Click the built Spots 1 selection [Shortest Distance to Surfaces Surfaces = Surfaces 1 between -7.00 and -0.500].
    2. Go to the Statistics, and export the value from "Total Number of Spots"
      NOTE: The software's provided algorithms will automatically calculate the number and volume of cells.
  5. Determine the yield of EV uptake per incubation period based on the above-calculated values (Figure 5).
    1. To obtain the number of cells, click the built Surfaces 1, then go to the Statistics, and export the value of "Total Number of Surfaces" from Overall.
    2. Go to the Detailed into Statistics to export the Volume from Detailed.

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Results

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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 dur...

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Discussion

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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

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Disclosures

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Y.-K. Cho is an inventor of the patents on the nano-filtration-based microfluidic device, Exodisc, which are licensed to Labspinner (Ulsan, Korea). All other authors have nothing to disclose.

Acknowledgements

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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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Alexa Fluor 488 anti-human CD63 AntibodyBiolegend353038Fluorescent dye conjugated EV-specific antibody
CellTracker Orange CMTMR DyeThermo Fisher ScientificC2927Live cell (cytoplasm) fluoresent labeling reagent
CFI Apo Lambda S 40XC WINikonMRD77400Objective for confocal imaging, NA=1.25
CFI Plan Apo VC 20XNikonMRD70200Objective for confocal imaging, NA=0.75
ExodiscLabspinner Inc.EX-D1001A nano-filtration based microfluidic device for EV isolation
ExoDiscoveryLabspinner Inc.EX-R1001Operation device for Exodisc
Exosome-depleted FBSThermo Fisher ScientificA2720801Nutrient of cell culture media for PC3 cell line derived EV collection
Fetal bovine serum (FBS)VWR1500-500Nutrient for cell cultivation
Goat Anti-Mouse IgG H&L preadsorbedabcam ab7063Mouse IgG antibody for negatvie control of EV labeling
Ibidi USA U DISH μ-Dish 35 mmIbidi81156Culture dish for confocal imaging
Imaris 9.7.1Oxford Instruments9.7.1Post-image processing software
Incubator System+ CO2/O2/N2 gas mixerLive Cell InstrumentTU-O-20Incubator system for live cell imaging
Nikon A1 HD25 / A1R HD25 cameraNikonNACamera for confocal imaging
Nikon Eclipse Ti microscopeNikonNAInverted microscope for confocal imaging
NIS-Elements AR 4.50.00Nikon4.50.00Image processing software for Nikon microscope
NTA, NanoSight NS500Malvern PanalyticalNS500Measurement device for EV concentration
OriginPro 2020OriginLab9.7.0.185Graphing software
Penicillin-StreptomycinThermo Fisher Scientific15140122Antibiotics for cell cultivation
RPMI 1640Thermo Fisher Scientific21875034Cell culture media for PC3 cell line cultivation
SYTO RNASelect Green Fluorescent cell Stain - 5 mM Solution in DMSOThermo Fisher ScientificS32703RNA staining fluorescent dye for the EV labeling

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EV Uptake AssayConfocal MicroscopyFluorescent EV LabelingMicrofluidic Device3D Imaging AnalysisEV InternalizationImage Processing SoftwareCell CommunicationEV Tracking

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