Setting a Successful Sorting for Extracellular Vesicle Isolation

So the main focus of our research is to study mesenchymal stromal cells, extracellular vesicle for tissue regeneration. An issue and a goal for our research is to translate the cell-free approach into clinical application alongside cell therapies. Characterization is crucial and today to have pure vesicle for a deep characterization is very difficult because there are not protocols to achieve high purity.

Therefore, we wanted to implement the cell sorter technology to study, characterize, and eventually, translate into clinical practice this new approach based on extracellular vesicle, Our protocol consists in four parts, the sample preparation, extracellular vesicles characterization, sorting, and post-sorting analysis. We will focus on the sorting part. As you will see, some commands will be performed on the sorter software while others on the touch screen.

Our protocol offers significant advantages compared to other method of EV separation. Firstly, the use of 70 micro nozzle at 35 psi preserve the integrity of EV post-sorting. Then it is possible to isolate a specific population of EV and then finally, the instrument allows to save sort setting in order to make the process reproducible.

In our lab, we are working to develop new multicolor panel for extracellular vesicles analysis. In particular, we are focusing on the instrument setup and on the study of fluorochromes. We believe that being able to identify and to separate rare population of vesicles, could provide good improvement to their characterization and also, for the study of their role in biological processes.

To begin, open the pressure line and vacuum system of the cell sorter. Switch on the instrument, open the biosafety cabinet, and launch the sorter software. Pressurize the fluidic system and then switch on the fluidic system.

Activate drop drive the piezoelectric crystal in the nozzle that vibrates to generate droplets. Perform the de-bubbling procedure to eliminate bubbles in the system. Then run a five-milliliter tube of cleaning solution for five minutes at high differential pressure, followed by a five-milliliter tube of deionized water for five minutes.

To perform the manual startup procedure, go to the laser control tab and turn on the laser power. Examine the vertical alignment of the stream. For laser spot determination, press the laser stream intercept tab.

Click the green arrow button and follow the instructions on the touchscreen monitor. Initialize IntelliSort, and set the drop drive frequency along with the default amplitude value which causes the stream to form a droplet. For the fine laser alignment procedure, load a five-milliliter tube of QC alignment beads into the sampler and run it in the QC protocol.

On the fine alignment tab, select the desired parameters on the X and Y axes to visualize well compacted and collumated beads. Once the manual alignment is checked, perform the automatic QC with the diameter of the QC beads set to three micrometers. Save the QC acquisition on the software alignment protocol.

To finalize the IntelliSort, locate the deflection plates in the correct position in the sorter, and turn on the voltage to about 3, 000 volts. Choose the six-tube holder sort output. Select the streams on the stream indicator to enable them and perform the test stream.

Enable the IntelliSort automatic drop-delay determination button to set the correct drop-delay and verify the streams again. Then activate IntelliSort maintain mode and verify the drop-delay manually. Load the manual, drop-delay protocol in the sorter software and acquire fluorescent control beads.

After inserting the correct slide holder, select sort, followed by drop-delay wizard and sort logic. Verify with a fluorescent microscope that 97%of fluorescent beads are present on the fifth puddle. To begin, set up the cell sorter and activate IntelliSort.

After performing scatter calibration, perform the sample acquisition steps for each sample. To create a new protocol for sample sorting, select file followed by protocol and new on the toolbar. Click data acquisition setting on the sorter software.

In the acquisition parameter window, select the channels of interest and disable the others. Then place a five-milliliter tube of the sample in the sampler. On the touch screen, click the load button.

On the sorter software toolbar, select acquisition, and press start. When the sample properties window appears, insert the sample name and click Okay. For creating the gating strategy, select histogram and then create histogram.

Create three dot plots, the first one with 488-FSC1-Height log on the X axis and 488-SSC-Height log on the Y axis. The second one with 488-FSC-2 Height log on the X axis and 488-SSC-Height log on the Y axis, and the third one with 488 513 X 26 CFSE Height log on the X axis and 488-SSC Height log on the Y axis. Click acquisition followed by start and insert the name of the sample.

During acquisition, draw region identifying the CFSE negative events, and one identifying the CFSE positive events. Then on the sort tab, identify the region to be sorted. When the flow rate is stable, select acquisition followed by stop.

Start sorting by selecting sort on the toolbar of the sorter software. To save the sort settings, select sort in the toolbar and then click save sort settings. To check the purity of the sorted population, first acquire a five-milliliter tube of cleaning solution for 10 minutes at high-differential pressure followed by a five-milliliter tube of deionized water for 10 minutes.

Acquire 0.22-micrometer, filtered PBS and check that no CFSE positive events are present. Then dilute five microliters of the sorted sample in 100 microliters of 0.22-micrometer, filtered PBS. Acquire the sorted samples and record the volumes Expression of CD9, CD63, CD81, and CD44, in the adipose-derived extracellular vesicles did not change after CFSE sorting.

Nanoparticle tracking analysis showed that the size distribution of the vesicles remained consistent pre and post-sorting. Transmission electron microscopy analysis indicated that the morphology of the vesicles was not significantly altered by the sorting process. Additionally, treatment was 0.1%Triton X-100 reduced the number of CFSE positive vesicles confirming their vesicular origin.