Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation

All methods were approved by the institutional ethics review board at the University of Oxford. Adipose tissue was obtained by needle biopsy under local anesthetic from healthy volunteers.

1. Preparation of cell culture medium and buffers

1. Prepare a collagenase digestion buffer by dissolving collagenase H (1 mg/mL) in Hanks Balanced Salt Solution (HBSS) (without calcium chloride and without magnesium chloride) and sterile filter using a 0.2 µm pore syringe filter.
   1. Prepare the collagenase digestion buffer no more than 10 min before use.
2. Prepare growth medium (GM) as follows: Dulbecco's Modified Eagle's Medium/Ham's nutrient mixture F12 (DMEM/F12) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, and 0.5 ng/mL fibroblast growth factor (FGF).
3. Prepare the basic differentiation medium (basic DM) as follows: DMEM/F12 supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, 17 µM pantothenate, 100 nM human insulin, 10 nM triiodo-L-thyronine, 33 µM biotin, 10 µg/mL transferrin, and 1 µM dexamethasone.
4. Prepare complete differentiation medium (complete DM) by supplementing the basic DM from step 1.3 with 0.25 mM 3-isobutyl-1-methylxanthine and 4 µM troglitazone.
5. Prepare 10 mM fatty acid stock solutions complexed to 10% essentially fatty acid free bovine serum albumin (BSA) as follows.
   1. Dissolve 16 g of BSA in 160 mL of DMEM/F12 medium and warm to 37 °C. In three separate 50 mL conical tubes, weigh out 150 mg of sodium oleate, 139 mg of sodium palmitate, and 151 mg of sodium linoleate. Add 50 mL of the warmed BSA solution to each tube and mix well by repeated vortexing.
   2. Return the oleate and linoleate tubes to the 37 °C water bath for 15 min. Mix by vortexing until completely dissolved.
   3. For the palmitate solution, place the tube in a 65 °C water bath for 2-3 min. Mix vigorously by vortexing.
   4. Repeat step 1.5.3 until fully dissolved-approximately 30 min.
      ​NOTE: Some small particles may still be visible.
   5. Sterile filter the fatty acid solutions using a 0.2 µm pore syringe filter.
   6. Confirm the non-esterified fatty acids concentration of each stock solution using an appropriate assay.
      ​NOTE: Fatty acid stocks should be 10 mM (±10%) to give a molar ratio of 6:1 between fatty acid and BSA. If the concentration is outside this range, the stock solution should be remade. Stock solutions can be aliquoted and stored at -30 °C.

2. Digestion of human adipose tissue biopsies

1. Slowly strain the contents of the biopsy syringe through a 200 µm sterile cell strainer attached to a 50 mL conical tube so that the adipose tissue is collected in the strainer.
2. Transfer the strainer to a new 50 mL conical tube and wash the adipose tissue three times with 10 mL of HBSS.
   NOTE: It may be necessary to remove blood clots and fibrous tissue with surgical scissors or to repeat the HBSS wash to remove excess red blood cells.
3. Weigh the washed tissue.
   NOTE: We typically obtain 400-800 mg of tissue by needle biopsy.
4. Put the collagenase digestion buffer in a sterile 50 mL conical tube and add the washed tissue. Surgical scissors can be used at this step to mince the tissue into equal sized pieces.
   NOTE: Use 5 mL of buffer per 0.5 g tissue. For larger tissue, samples can be minced in a Petri dish.
5. Place the tube into a 37 °C shaking water bath and incubate for 35-40 min.
   ​NOTE: Following successful digestion, the solution should appear milky. If small pieces of tissue are still visible, shake by hand for a further 10-20 s.

3. Isolation of preadipocytes

1. To pellet the preadipocyte fraction centrifuge at 1,000 x g for 5 min.
2. Aspirate and discard the floating adipocyte layer and supernatant leaving approximately 1 mL of HBSS covering the cell pellet.
3. Resuspend the pellet in 5 mL of HBSS and pass the cell suspension through a 250 µm pore size mesh followed by a 100 µm mesh to remove any undigested material. Collect the cell suspension in a 15 mL conical tube.
4. Centrifuge at 1,000 x g for 5 min.
   NOTE: After this step, the preadipocyte fraction can be treated with a red blood cell lysis solution if red blood cell contamination is an issue.
5. Aspirate and discard the supernatant.
6. Resuspend the cell pellet in 5 mL of GM (step 1.2) and seed into a 25 cm² adherent tissue culture flask. Place the flask in a cell culture incubator (5% CO₂, 37 °C).

4. Maintenance of preadipocyte cultures

1. Replace the GM every 2 days while the cells are proliferating
   NOTE: Cell proliferation rates may vary between donors.
   1. When the cells reach approximately 80% confluence, transfer them to a 75 cm² adherent tissue culture flask. Remove the GM and wash the cells with phosphate buffered saline (PBS).
   2. Discard the PBS and add 0.5 mL of highly purified cell dissociation enzymes to disassociate the attached cells. Incubate at 37 °C for 5 min.
   3. Tap the flask sharply to release the cells and add 5 mL of GM. Collect the cell suspension in a 15 mL conical tube.
2. Centrifuge at 1,000 x g for 5 min.
3. Aspirate and discard the supernatant. Resuspend the cell pellet in 5 mL of GM and transfer to a 75 cm² flask. Top up the GM to a final volume of 12 mL and replace the flask in the cell culture incubator (5% CO₂, 37 °C).
   NOTE: To maintain the cells, continue to change the GM every 2-3 days. When the cells reach 80% confluence, repeat steps 4.2-4.3 and divide the cells 1:3 into new 75 cm² flasks. This can be repeated for several passages until a sufficient number of cells have been generated for the experimental set up. We would not recommend more than 10 passages as proliferation and differentiation capacity of the preadipocytes declines.

5. Seeding preadipocytes for adipogenic differentiation

1. Count the cells using a hemocytometer and seed 200,000 cells into multiple wells of a 6-well plate in 2 mL of GM. Alternatively, seed 3.5 million cells per 175 cm² flask in 22 mL of GM media.
2. Allow the cells to proliferate for a further 2-4 days until they reach full confluence, changing the GM on the second day of the culture.
3. To begin adipogenic differentiation, remove the GM and replace with complete DM for 4 days (2 mL of complete DM per well of a 6-well plate or 22 mL for a 175 cm² flask).
   NOTE: Replace with fresh complete DM on day 2.
4. On day 4, remove the complete DM and replace with 2 mL basic DM supplemented with 22.5 µM oleate, 15 µM palmitate and 12.5 µM linoleate to give a total fatty acid concentration of 50 µM per well of a 6-well plate or 22 mL for a 175 cm² flask. Replace with media every 2 days for a further 10 days.
   NOTE: From day 7 onward, lipid droplets should be visible in the differentiating preadipocytes.
5. On culture day 14, collect the media from the cells for isolation of adipocyte-derived EVs.

6. Preparation of cell culture supernatant for extracellular vesicle isolation or storage and future extracellular vesicle isolation

1. Remove cell culture supernatants from each 6-well plate and combine. Add to a 15 mL tube or remove all the cell culture supernatants from a 175 cm² flask and add to a 50 mL tube.
2. Centrifuge at 1000 x g for 10 min at 4 °C.
3. Decant supernatant into a new clean 15 mL or 50 mL tube by pouring, respectively.
4. Remove the barrel of a 10 mL syringe and attach a 0.45 µm syringe filter.
5. Pour in the supernatant to the syringe reservoir and apply gentle pressure at the syringe barrel opening with a thumb or palm until the cell culture supernatant freely passes through the filter.
   NOTE: The speed of this filtration step can vary depending on the type of the syringe filter.
6. Collect the filtrate in a clean 50 mL tube.
   NOTE: If required, the conditioned filtered media can be stored at this point at -80 °C for several weeks. When required, defrost the cell culture supernatants at 4 °C and centrifuge at 1000 x g for 10 min at 4 °C. Decant the supernatant into a clean tube by pouring before continuing with the EV isolations.

7. Isolation of extracellular vesicles

1. Label a 13 mL ultracentrifugation tube by drawing a circle at the bottom of the tube, where the expected EV pellet will form and mark a line around the neck of the tube for orientation in the ultracentrifugation tube rotor. Label the tube with a sample identifier.
2. Place tube in the tube holder.
3. Attach a 16 G needle to a 10 mL syringe and remove the syringe barrel.
4. Remove the protective cover from the needle and insert the needle into the neck of the ultracentrifugation tube.
5. Pour the cell culture supernatant directly into the syringe barrel to fill the ultracentrifugation tube.
6. Top up the tube with PBS as necessary until full.
   NOTE: Allow the tube to slightly overflow if necessary, ensuring there are no air spaces in the ultracentrifugation tube and no air bubbles.
7. Seal the ultracentrifugation tube using a soldering iron, ensuring the tube is airtight by squeezing the tube gently.
8. Place the ultracentrifugation tubes in the ultracentrifugation rotor; ensure the line marked at the top of the tube and the circle drawn at the base of the tube are facing outwards, where the EV pellet will form.
9. Ultracentrifuge at 120,000 x g for 2 h at 4 °C.
10. Carefully remove the rotor from the ultracentrifuge.
11. Remove ultracentrifugation tubes from the rotor and place in the tube holder.
12. Attach a 16 G needle to a 10 mL syringe.
13. Pierce the top of the ultracentrifugation tube and insert the needle 2 cm into the top of the tube and aspirate the supernatant into the syringe.
14. Decant this EV-depleted supernatant into a 1.5 mL tube and freeze at -80 °C.
15. Reinsert the needle into the tube and carefully aspirate the remaining supernatant and discard.
16. Cut the top of the tube with a pair of scissors.
17. Pour off the remaining supernatant in one quick action.
18. Allow the tube to hang upside down for 1 min.
19. Pat dry any liquid that forms on the tube with paper towel.
20. Invert the tube and place in the tube holder.
21. Add 100 µL of PBS to the tube.
22. Using the tip of a pipette, gently dislodge the EV pellet at the base of the tube using a circular motion in the area marked in step 7.1.
23. Vortex briefly (1-2 seconds), twice.
24. Label a new 13 mL ultracentrifugation tube by drawing a circle at the bottom of the tube, where the expected EV pellet will form and mark a line around the neck of tube for orientation in the ultracentrifugation tube rotor. Label the tube with a sample identifier.
25. Place the tube in the tube holder.
26. Add 12 mL of PBS to a new ultracentrifugation tube using a clean syringe and needle.
27. Using a syringe and needle, collect the 100 µL EV sample and add to the tube. Carefully mix the EVs and PBS and rinse the syringe and needle by gently collecting PBS and aspirating into the tube.
    NOTE: Avoid creating bubbles.
28. Seal the ultracentrifugation tube using a soldering iron, ensuring the tube is airtight by squeezing the tube gently.
29. Place the ultracentrifugation tubes in the ultracentrifugation rotor; ensure the line marked at the top of the tube and the circle drawn at the base of the tube are facing outwards.
30. Ultracentrifuge at 120,000 x g for 1 h at 4 °C.
31. Carefully remove the rotor from the ultracentrifuge.
32. Attach a 16 G needle to a 12 mL syringe.
33. Pierce the top of the ultracentrifugation tube and insert the needle upto 2 cm into the top of the tube; aspirate the supernatant into the syringe and discard.
34. Cut the top of the tube with a pair of scissors.
35. Pour off the remaining supernatant in one quick action.
36. Allow the tube to hang upside down for 1 min.
37. Pat dry any liquid that forms on the edge of the tube.
38. Place the tube in the tube holder.
39. Add 100 µL of PBS to the tube.
40. Using the tip of a pipette, gently dislodge the EV pellet at the base of the tube using a circular motion in the area marked in step 7.24.
41. Vortex briefly (1-2 seconds), twice.
42. Pipette 100 µL of PBS/EV solution into a clean 1.5 mL tube and keep on wet ice.
    ​NOTE: EV are ready for downstream processing and may be frozen and stored at -80 °C.

8. Determination of EV size and concentration using Nanoparticle Tracking Analysis (NTA)

1. Preparing the system
   ​NOTE: A detailed method for the use of Nanoparticle Tracking Analysis (NTA) for determination of EV size and concentration was reported by Mehdiani et al.¹³.
   1. Defrost samples and keep them at 4 °C.
   2. Start the NTA software by clicking on the software-icon.
   3. The software will open in "cell check" and prompt to fill the flow cell with deionized water. Fill a 10 mL syringe with deionized water and push into the machine, ensuring no air bubbles pass into the loading chamber.
   4. Follow the on-screen instructions to prepare the system through a quality check (QC). The software will perform a cell check and give a measure of cell quality. This should be very good to excellent.
   5. Prepare the quality control consisting of a 100 nanometer polystyrene beads. Pipette 1 µL of the standard into 999 µL of deionized water (diluted stock). Subsequently, add 10 µL of the diluted stock to 2.5 mL of deionized water (QC sample). Mix the solution by gently vortexing for 2-3 s and by pipetting.
      ​NOTE: Quality control samples should be prepared freshly every day, but the initial diluted stock sample is stable for 1 week at 4 °C.
   6. Fill a 1 mL syringe with the 1 mL of the QC sample and remove all air bubbles from the syringe.
   7. Inject, without introducing air bubbles, into the NTA sample loading chamber by gently tilting the tip of the syringe into the injection chamber, while simultaneously pushing the plunger. Inject upto 950 µL of the QC sample into the chamber.
      ​NOTE: Do not introduce air bubbles into the sample loading chamber.
   8. Wait as the software performs an auto alignment (to focus the camera) and checks the voltage for Z-potential readings – the voltage graph should be a smooth U-shaped curve.
      ​NOTE: If there is an error message "voltage too low error", the cell may be wet from cleaning or not secure or there may be an air bubble.
   9. Once the auto alignment/voltage check is complete, use the camera position drop down to check all positions (0.1-0.9) for unusual marks – presence of these indicate the cell may need flushing and/or cleaning. Following QC, the system will display "ready for measurements".
   10. Prime the flow cell with PBS. Open the Pump/Temp tab and under Pump, click on Run for pump 2 (PBS reservoir). This will run PBS through the cell for 1 min, then automatically stop.
       ​NOTE: There may be particles in the chamber, but these can be cleared by pushing 10 mL of PBS through the loading port.
   11. Fill a 10 mL syringe with PBS and load into the chamber with no air bubbles.
   12. Create a protocol for the device to run that will measure multiple positions of the laser and average the particles per frame.
   13. Check the particle count is less than 5 (as close to 0 as possible) before proceeding – flush with more PBS, if necessary.
   14. Prior to dilution, mix the EV sample by pipetting.
   15. Dilute the sample 1:1,000 in PBS and mix by pipetting. Fill a 1 mL syringe with the sample and load into the chamber with no air bubbles.
   16. Check that the particle count is within the acceptable range (the bar above the count value should be in the green region, or close to it). If not, adjust the sample dilution accordingly. In between samples and sample dilutions, load 10 mL of PBS into the front of the machine to clear the chamber, then load 1 mL of the diluted sample.
       ​NOTE: Each sample will have independent dilution based on the concentration of the sample. The particle count of the device should be between 50-200 particles per frame for accurate measurements.
   17. Go to the Measurement tab in the software.
   18. Click on the Run video acquisition button.
   19. Enter a sample name and select a destination folder to save the measurement.
2. Create an EV standard operating procedure (SOP).
   1. Create > Save > Load an EV SOP that will measure the particle number and size at a shutter speed of 100 and camera sensitivity of 80, 11 camera positions for 2 cycles.
      NOTE: Unless the sample has special requirements, there is no need to change these settings.
   2. Enter the dilution of the sample and add any other notes you wish to add.
   3. Click on OK and the software will automatically start recording. Once finished, the software will automatically load a pop-up table showing average particle counts and sizes for each camera position.
      ​NOTE: Do not touch the machine or nearby countertops during this time as vibrations will influence the final readings.
   4. Camera positions with statistically unusual readings will automatically be excluded-to manually exclude any camera positions (or re-include excluded ones for any reason) click the checkbox to the left of the sample details.
   5. Click Weitermachen and the software will create and open a PDF of the sample with the size and concentration results.
3. Statistical analysis
   1. Analyze the NTA data by one-way or two-way ANOVA with post-hoc Tukey correction. Plot data as group means ± standard deviation. A P value of <0.05 was considered significant.