Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles
Summary
This protocol provides a rapid and size-specific isolation method for small extracellular vesicles by optimizing the size of the air spray nozzle, sheath fluid pressure, sample flow pressure, voltage, gain, and triggering threshold parameters.
Abstract
Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the physiological and pathological state of a cell. However, there is no standard method for sEV isolation, which prevents downstream biomarker identification and drug intervention studies. In this article, we provide a detailed protocol for the isolation and purification of 50-200 nm sEV by a flow cell sorter. For this, a 50 µm nozzle and 80 psi sheath fluid pressure were selected to obtain a good sorting rate and stable side stream. Standard sized polystyrene microspheres were used to locate populations of 100, 200, and 300 nm particles. With additional optimization of the voltage, gain, and forward scatter (FSC) triggering threshold, the sEV signal could be separated from the background noise. These optimizations provide a panel of critical sort settings that enables one to obtain a representative population of sEV using FSC vs. side scatter (SSC) only. The flow cytometry-based isolation method not only allows for high-throughput analysis but also allows for synchronous classification or proteome analysis of sEV based on the biomarker expression, opening numerous downstream research applications.
Introduction
A cell releases extracellular vesicles (EVs) of varying sizes that result in signaling molecules and membrane inclusions, which are important for intercellular communication1. EVs of different sizes also play different biological roles, with 50-200 nm sEV being able to precisely distribute RNA, DNA, and proteins to the correct extracellular location. The sEV also helps determine their secretion mechanisms, involving not only the regulation of normal physiological processes such as immune surveillance, stem cell maintenance, blood coagulation, and tissue repair but also the pathology underlying several diseases such as tumor progression and metastasis2,3. Effective isolation and analysis of sEV are critical for identifying biomarkers and designing future drug interventions.
With continuous research on the clinical application of sEV, the isolation methods of sEV have put forward higher requirements. Due to the heterogeneity of sEV in size, source, and contents, as well as their similarity with other EVs in physicochemical and biochemical properties, there is no standard method for sEV isolation4,5. Currently, ultracentrifugation, size exclusion chromatography (SEC), polymer precipitation, and immunoaffinity capture are the most common sEV isolation methods6. Ultracentrifugation is still the gold standard for sEV isolation in research, despite being time-consuming, resulting in low purity with a wide size distribution of 40-500 nm and significant mechanical damage to the sEV after long-term centrifugation7,8,9. Polymer precipitation, which usually uses polyethylene glycol (PEG), suffers from unacceptable purity for subsequent functional analysis with concomitant precipitation of extracellular protein aggregates and polymer contamination10,11. Immunoaffinity capture-based methods require high-cost antibodies with varying specificity, as well as have problems with low processing volume and yields12,13,14. Particle size is one of the main indicators to evaluate the purity of isolated sEV. Although sEV purity remains an unattainable goal, SEC removes a considerable quantity of medium components, and the sEV particles extracted by the SEC method are mainly in the range of 50-200 nm15. The existing techniques have a few disadvantages, including but not limited to being time-consuming, low purity, low yield, poor reproducibility, low throughput of samples, and potential damage of sEV, which makes it incompatible with clinical utilization16. Thus, a rapid, inexpensive, and size-specified sEV isolation method applicable to diverse biofluids is an essential need in several research and clinical situations.
In flow cytometry, single particles are analyzed in a high-throughput, multiparametric manner, and subsets are sorted out17. Due to the heterogeneity of EVs, a single-particle flow cytometric measurement would be ideal, which has been used to investigate EVs following the paradigms of cell analysis, with light scatter and fluorescence labels being used to identify physiology-related features and protein components18,19,20. Nevertheless, conventional flow cytometry is challenged by the small size of sEV and low abundance of surface biomarkers. The sensitivity of flow cytometry could be improved by optimizing detection parameters to distinguish background noise and sEV regardless of using forward scatter (FSC), side scatter (SSC), or fluorescence threshold triggering parameter19.
With the present protocol, high-resolution flow cytometric sort settings were optimized using fluorescent beads as standard. By selecting the proper nozzle size, sheath fluid pressure, threshold triggering parameter, and voltages controlling scattered light intensities, we were able to isolate a specific subset of sEV from a complex mixture.
Protocol
1. Cell culture
- Prepare a culture medium of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Culture the human pancreatic cancer cell, PANC-1, in a 75 cm2 culture flask at a density of 1 x 106 cells/mL. Incubate the culture at 37 °C with 5% CO2.
- Subculture the cells when cell density reaches 75%-80% under microscopic observation. Remove the medium, and rinse 2x with 3-5 mL of phosphate buffer saline (PBS; 0.01 M, pH 7.2-7.4).
- Discard the solution, add 1-2 mL of trypsin-EDTA solution, and let the cells detach until they become rounded and suspended in the medium under the microscope. Add fresh medium, aspirate, and disperse into 75 cm2 culture flasks with 15 mL culture medium. Use a sub-cultivation ratio of 1:2 to 1:4 (recommended).
NOTE: All operations are performed in a biosafety cabinet to avoid cell contamination.
2. Culture medium collection
- Incubate the cells for 48 h at 37 °C with 5% CO2. Collect cell supernatant (90 mL) in two 50 mL centrifuge tubes and centrifuge at 300 x g for 10 min at 4 °C.
- Transfer the supernatant to new sterile tubes and centrifuge successively at 2,000 x g for 20 min, 10 000 x g for 30 min, and 12,000 x g for 70 min at 4 °C.
- Remove the supernatant and rinse the pellet with 10 mL of PBS by pipetting gently. Centrifuge at 12,000 x g for 70 min at 4 °C again and resuspend the pellet in 10 mL of PBS buffer to obtain a mixture of EVs.
- Perform nanoparticle tracking analysis (NTA) to quantify and size the EV mixture. Perform the NTA analysis according to the instrument operation reference manual and reference21.
3. Cell sorting
- Perform the standard startup procedure of the flow cell sorter. Turn ON the machine by pressing the Startup button. Click the Laser Power button on the laser control panel. Press the Change Tanks button in the smart sampler submenu to pressurize the fluidics.
NOTE: Ensure that the waste tank is empty, and the sheath tank is full before starting up. - Use an ultrasonically cleaned 50 µm jet-in-air nozzle and install it onto the nozzle assembly. Adjust the pressure to 80 psi for sheath fluid and 80.3 psi for sample flow on the pressure console.
- Press the Start Sheath Flow button to start the sheath stream flowing. Click the Bubble Button in the smart sampler submenu to automatically debubble for 10 min and then turn it off.
NOTE: For cell sorting, different sizes of air spray nozzles require different sheath fluid pressures to maintain liquid flow stability. In this method, a nozzle of 50 µm was used, and only a sheath fluid pressure greater than or equal to 80 psi could generate stable side streams. The pressure difference between the sample flow and the sheath fluid determines the loading speed of sorting. Under the setting condition of the method (the pressure difference between the sample flow and the sheath fluid is 0.3-0.6 psi), the liquid flow was relatively stable, and the loading speed enabled the sorting efficiency to increase to 80% from 50% for pressure difference less than 0.3-0.6 psi. - Align the stream and determine the laser spot. Turn ON the illumination chamber light to view the stream over the pinholes. Adjust the up-and-down, left-and-right, and front-and-back micrometers to center the stream on the pinhole and focus the stream.
- Select the Laser and Stream Intercept tab. Press the Green Arrow button continuously as prompted to complete the laser intercept and nozzle alignment calibration process.
- Access the fine laser alignment screen. Select 640-722/44 (H) channel as the X-axis parameter and 405-448/59 (H) channel as the Y-axis parameter. Press the Trigger Parameter Selector and choose the SSC Parameter of the 488 nm laser. Open all laser shutters.
NOTE: The X-axis and Y-axis parameters chosen depend on the laser configuration of the system. Other parameters are allowed if the system does not have a 640 nm or 405 nm laser. For best results, select lasers with the greatest spatial separation on the pinhole strip (not including the 355 nm laser). - Dilute the rainbow fluorescent particles by adding one drop into 1 mL of double distilled water for a final concentration of 1 x 106 beads per mL. Open the door of the sample chamber and load a tube of the prepared rainbow fluorescent particles.
- Press the Start Sample button. Adjust up-and-down micrometers to optimize fluorescence intensity, making each signal as intense as possible. Press the Start Quality Control (QC) button on the touchscreen control panel to perform QC automatically.
- Perform drop delay. Access the sort setup screen and select the drop delay button. Press the IntelliSort Initialization button. The instrument automatically adjusts the frequency and amplitude of droplet oscillation to obtain the drop delay of 25 ± 5 and be able to visualize the break-off point of the drop clearly. Press the Maintain button.
- Select tube as the sort output type. Place a 15 mL tube holder in the sort chamber. Turn the plate voltage ON and set the plate voltage to approximately 4000 V.
- Turn on the test pattern by selecting the Stream Setup button. Adjust the charge phase slider and deflection slider to ensure that the image of the stream is lined up with the collection tube. Select the Check Mark button.
- Log in to the Summit workstation on the computer. Create a new protocol from the File main menu. Create a dot plot by selecting the Histogram tab in the Summit software control panel.
- Right-click the axes of the new dot plot in the workspace and choose FSC as the X-axis parameter and SSC as the Y-axis parameter. Present the FSC and SSC in logarithmic form.
- Select the Acquisition tab and locate the acquisition parameters panel. Enable all signals in the submenu of enabling parameters. Choose FSC as the trigger parameter and set the threshold of 0.01. Adjust the voltage and gain of FSC and SSC at 250 and 0.6 to ensure events within the FSC vs. SSC plot and populations are separated.
NOTE: Threshold setting is equivalent to setting the particle size that can be detected by flow cytometry. The larger the threshold is, the larger the detected particles will be, and the small particles will be cut off by the threshold and cannot be detected. In this method, the threshold is set to 0.01, so all particles larger than electronic noise can be detected.
4. Isolation of sEV
- Dilute the fluorescent polystyrene microspheres to suspensions of 100 nm, 200 nm, and 300 nm. Add 1 µL of microspheres to 1 mL of double distilled water.
- Load the microsphere suspension successively. Select Start under the acquisition menu of summit software and record 20,000 events.
NOTE: The number of events is optional. No less than 5,000 events are recommended to meet statistical significance. - Right-click on the FSC vs. SSC dot plot to create rectangles and drag to resize and reposition the regions of electronic noise and 100 nm microsphere population. Right-click on the regions to rename them as R7 and R4, respectively. Repeat the above steps to frame the 200 nm and 300 nm microspheres with rectangles successively and rename them as R5 and R6, respectively.
- Finally, determine the 50-200 nm sorting region based on the electronic noise and the position of 200 nm particles defined as R8.
NOTE: The lower detection limit of 50 nm is placed just above the electronic noise of the flow cell sorter. The region between the noise and the 200 nm microsphere population can therefore be defined to be between 50-200 nm. - Edit sort decisions in the sort logic and statistics panel. Double-click on the blank field of the left one (L1) stream. Select the region named R8 to create the sort logic. Choose the Abort mode of purity and select a droplet envelope of 1 drop for the L1 stream.
- Load 500 µL sample of EVs mixture from step 2.3. Press the Start button under the acquisition menu in Summit to acquire data, and then press Start in the sort menu to collect 50-200 nm sEV into a 15 mL centrifuge tube.
NOTE: A sterile environment is necessary to minimize contamination during any procedure. - Observe the presence of sEV by transmission electron microscopy (TEM) and verify the particle size of sEV using NTA. Perform western blot (WB) analysis to further confirm the expression of CD9, CD63, and CD81 markers18.
Representative Results
The flow chart diagram for the experimental protocol is shown in Figure 1. In this method, standard sized polystyrene microspheres were used as reference standards for particle size distribution. Under the specific instrumental parameter condition, the particle signal could be clearly distinguished from the background noise in the FSC vs. SSC plot using the logarithmic form. Gating strategies are shown in Figure 2. R4, R5, and R6 refer to the positions of 100 nm, 200 nm, and 300 nm microspheres, respectively. R7 is the detection limit of electronic noise below 50 nm, and R8 is the position range of 50-200 nm particles.
The particle size distribution of the EVs mixture derived from PANC-1 cells was in the wide range of 40-400 nm after ultracentrifugation (Figure 3). To isolate and purify the 50-200 nm sEV, flow cytometric sorting was performed to obtain the specific size sEV according to the location of standard microspheres (Figure 4). The quality of isolation was verified by NTA, and it was found that the particle size range of sEV after sorting was between 50-200 nm (as shown in Figure 5). The presence of sEV was further observed by TEM, indicated by red arrows in Figure 6A, and the isolated sEVs were confirmed to contain markers of CD9, CD63, and CD81 by WB analysis (Figure 6B).
Figure 1. Flow chart diagram for the experimental protocol. PANC-1 cell culture medium was collected and ultracentrifuged to obtain the EVs mixture. Sorting parameters were optimized for isolation and purification of 50-200 nm sEV which was verified by NTA. Please click here to view a larger version of this figure.
Figure 2. Flow cytometry analysis of standard-sized polystyrene microspheres to locate the 50-200 nm size range in the FSC vs. SSC plot. Diluted suspensions of 100 nm, 200 nm, and 300 nm microspheres were loaded and analyzed by a flow cytometer as shown in the FSC vs. SSC dot plot. R4, R5, and R6 refer to the positions of 100 nm, 200 nm, and 300 nm particles, respectively. R7 is the electronic noise as the detection limit for 50 nm particles, and R8 represents the position range of 50-200 nm particles. Please click here to view a larger version of this figure.
Figure 3. NTA analysis of particle size distribution of EVs obtained by ultracentrifugation. The frequency distribution of different particle sizes is represented by the data as particle percentage vs. size. Please click here to view a larger version of this figure.
Figure 4. Flow cytometry analysis of the collected EV sample. The FSC vs. SSC dot plot shows a sample of the collected EV mixture loaded and analyzed by a flow cytometer. The sEV in the size range of 50-200 nm within the R8 region were sorted. Please click here to view a larger version of this figure.
Figure 5. Verification of particle size distribution of sEV after sorting using NTA. Representative particle percentage vs. size histogram shows the size distribution of the sorted sEV. Please click here to view a larger version of this figure.
Figure 6. Characterization of sEV isolated by sorting. (A) A representative TEM image of sorted sEV (indicated by red arrows). Scale bar = 200 nm. (B) Western blot analysis of CD9, CD81, and CD63 markers in the sorted sEV. Please click here to view a larger version of this figure.
Discussion
This protocol outlines an optimized method to isolate and purify sEV with the specified particle size of 50-200 nm using a flow cell sorter, which was validated by NTA. The method solved the bottleneck problem of obtaining sEV with uniform particle size and high purity, avoiding interference from unrelated biological molecules wrapped in large-sized EVs22. Fast, high-throughput analyses are possible with flow cytometry, which can capture 100,000 particles per second and make 70,000 sorting decisions per second17, greatly reducing the time and reflecting the heterogeneity of sEV particles. Moreover, this flow cytometry-based protocol can be customized based on individual interests in subpopulations of EVs of specific sizes. Alternative gate ranges can be used to identify and isolate the populations of interest with the same instrument parameters set as the above, such as air spray nozzle, sheath fluid pressure, sample flow pressure, and triggering threshold parameter.
The technical difficulty of this method lies in the separation of populations with different size ranges, particularly distinguishing from background noise. We identified a panel of critical sort settings. First, the size of the nozzle affects the sorting rate. In order to improve the concentration of sEV obtained by sorting, a 50 µm nozzle is used in this method, and 80 psi of sheath fluid pressure is required to stabilize the side stream. With higher sheath fluid pressure, drop frequencies increase, resulting in higher event rates and shorter sorting time23. However, increasing sheath fluid pressure attenuates the sensitivity of FSC and fluorescence signals due to the reduced time it takes to pass through the laser and the number of photons reaching the detector24. Notably, it is difficult to maintain the stability of the side stream under this pressure, which requires high cleanliness of the nozzle and pipeline. Second, ultrasonic cleaning of the nozzle and flushing of flow cytometry pipes are strongly recommended to increase the likelihood of experimental success. Lastly, voltage and gain are critical for population separation, especially for small size particles. Here, a voltage of 250 V and gain of 0.6 can effectively separate the sEV at 50-200 nm with FSC triggering and plot in logarithmic form.
One caveat of the approach is that in some cases, the concentration of the sEV is too high, or the sEV aggregate, during the sorting process, making it difficult to achieve a single-particle suspension. The aggregated sEV will be discarded due to the over-amplified signal.
Collectively, with the advantages of high-throughput analysis of flow cytometry, this method optimized nozzle size, sheath fluid pressure, sample flow pressure, and triggering threshold parameter leading to successful isolation of 50-200 nm sEV. Besides, not only does this method allow for the separation of specific size particles, but it also allows for synchronous classification or proteome analysis of sEV based on antigen expression, opening up numerous downstream research applications.
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Scientific Research Fund of Zhejiang Chinese Medicine University (2020ZG29), the Basic Public Welfare Research Project of Zhejiang Province (LGF19H150006, LTGY23B070001), the Project of Zhejiang Provincial Department of Education (Y202147028) and the Project of Experimental Technology of Zhejiang University Laboratory Department (SJS201712, SYB202130).
Materials
| Centrifuge tube | Beckman Coulter | 344058 | |
| Culture flasks | Corning | 430641 | |
| Dulbecco’s modified eagle medium | Corning Cellgro | 10-013-CV | |
| Fetal bovine serum | SUER | SUER050QY | |
| Flow cell sorter | Beckman Coulter | Moflo Astrios EQ | |
| Human pancreatic cancer cell, PANC-1 | NA | NA | PANC-1 cells were donated by Professor Weijun Yang, College of Life Sciences, Zhejiang University |
| Laser particle size and zeta potential analyzer | Malvern | Zetasizer Nano ZS 90 | |
| Phosphate buffer saline | Gibco | C20012500BT | |
| Polystyrene fluorescent microspheres | Beckman Coulter | 6602336 | |
| Transmission electron microscopy | JEOL | JEM-1200EX | |
| Trypsin-EDTA solution | Gibco | 1713949 | |
| Ultra rainbow fluorescent particles | Beckman Coulter | B28479 | |
| Ultracentrifuge | Beckman Coulter | Optima-L80XP | |
| Ultracentrifuge rotor | Beckman Coulter | SW32TI |
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