A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

This is a simple and time effective protocol to isolate and concentrate small extracellular vesicles from mesenchymal stem cells. This technique is easy to be adopted in most labs as it is a benchtop scale and only requires simple instruments for operations. To isolate a of small extracellular vesicles, we recommend growing cells on a large scale to obtain a minimum of hundred million cells.

Although it sounds simple, certain critical steps of this technique are better informed through visual demonstration. We hope researchers could learn from our experience and master the skills confidently. Our PhD student, Mr.Chan, Hong Hao, will guide you through our experience in isolation and characterizations of small extracellular vesicles from mesenchymal stem cells.

To begin, centrifuge the conditioned medium at 200g for five minutes at four degrees Celsius to remove cell debris. Collect and filter the supernatant through a 0.22 micrometer filter to remove particles larger than 220 nanometers. Filled a centrifugal filter unit with 30 milliliters of 0.2 micrometer filtered phosphate-buffered saline.

Then centrifuge the centrifugal filter unit at 3, 500g for five minutes at four degrees Celsius. Discard the solution in the filtrate solution cup. Add 70 milliliters of filtered conditioned medium into the centrifugal filter unit and centrifuge the unit at 3, 500g at four degrees Celsius.

Discard the solution in the filtrate solution cup. Add 30 milliliters of filtered phosphate-buffered saline and centrifuge the unit at 3, 500g at four degrees Celsius. Install a concentrate collection cup on the filter unit and reverse centrifuge at 1, 000g for two minutes at four degrees Celsius to obtain the purified small extracellular vesicles.

Filter the small extracellular vesicles through a 0.22 micrometer syringe filter. Transfer the samples to new tubes and store them at 80 degrees Celsius for further analysis. To begin nanoparticle tracking analysis, dilute the isolated human umbilical cord-derived mesenchymal stem cells small extracellular vesicles in filtered phosphate-buffered saline to 20 to 100 particles per frame.

Introduce one milliliter of the diluted sample into the anti-A chamber using one milliliter disposable syringes. Set the measurement settings accordingly. Adjust the Camera Level to level 14.

For each measurement, click the standard measurement and set the dilution accordingly. Next, click Capture, Start Camera, and set the Camera Level to 14 to visualize the small EVs. To initiate the measurement, click Create, then click Run Script, and select the location where the files need to be saved.

Key in the necessary details of the sample and click OK.Click the setting OK to continue the script and click OK to initiate the measurement. Once the measurement has been recorded, analyze the results with a detection threshold of five to include 10 to 100 red crosses per frame. And the blue cross count is limited to five.

Click Setting OK to continue the script and initiate the analysis. Click OK on the script complete notification and export to export the data to the set location. Lyse the human umbilical cord-derived mesenchymal stem cells small extracellular vesicles with ice cold radioimmunoprecipitation assay lysis buffer and incubate for 30 minutes at four degrees Celsius.

Collect the supernatant after centrifuging at 200g for five minutes at four degrees Celsius. Quantify the protein using a BCA assay. Assemble the gel electrophoresis set accordingly and perform gel electrophoresis at 90 volts until the protein reaches the stacking gel's end.

Change the voltage to 200 volts to separate the proteins until the end of the resolving gel. Perform semi-dry transfer to transfer the proteins from the gel to a polyvinylidene difluoride membrane at 15 volts for one hour. Block the PVDF membrane with 3%bovine serum albumin in tris-buffered saline 0.1%Tween 20 for one hour on a shaker at room temperature.

Incubate the PVDF membrane with primary antibodies at four degrees Celsius overnight with constant shaking. The next day, wash the PVDF membrane five times and five minutes each with TBST and further incubate with a secondary antibody for one hour with agitation at room temperature. Again, wash the PVDF membrane with TBST five times and visualize the membrane in a charge-coupled imager using a chemiluminescent detection reagent.

Analyze the expression level of the proteins using ImageJ software. First, open the image file in ImageJ. Enhance the quality of the image by adjusting the brightness and contrast.

Adjust the image until the blots are clearly visible. Click on the Apply button and save the images in TIFF format. For transmission electron microscope based analysis, dilute one part of the human umbilical cord-derived mesenchymal stem cells small extracellular vesicles sample with four parts of filtered phosphate-buffered saline to a total volume of 10 microliters and incubate on a carbon-coated copper grid for 15 minutes.

Remove the excess sample using a laboratory wipe and allow it to air dry for three minutes. Incubate 10 microliters of 1%phosphotungstic acid solution to stain the sample for three minutes. Remove the excess 1%phosphotungstic acid solution using a laboratory wipe and allow it to air dry for three minutes to image under a transmission electron microscope.

Human umbilical cord-derived mesenchymal stem cells small extracellular vesicles have a particle size mode of 53 nanometers, while other significant peaks of particle size were 96 and 115 nanometers. The concentration of human umbilical cord-derived MSC small extracellular vesicles measured by NTA was 7.75 times 10 to the 10th particles per milliliter. The protein concentration of human umbilical cord-derived MSC small extracellular vesicles measured with the BCA assay was approximately 80 micrograms per milliliter.

In western blotting analysis, human umbilical cord-derived MSC small extracellular vesicles demonstrated positive bands for exosomal markers CD9, CD81, and TSG101, but were negative for GRP94. The isolated human umbilical cord-derived MSC small extracellular vesicles were visualized under TEM to determine their size and morphology. Human umbilical cord-derived mesenchymal stem cells small extracellular vesicles sized approximately 100 nanometers and showed a cup-like bilayer membrane structure.

With the successful development of the protocol, we can now uncover the therapeutic potentials of mesenchymal stem cells derived small extracellular vesicles in regenerative medicines.