Isolation of Extracellular Vesicles Derived from Mesenchymal Stromal Cells by Ultracentrifugation

Materials and Reagents

1. 22 G needle

2. 15 ml conical tubes (Sarstedt, catalog number: 62.554.502 )

3. 50 ml conical tubes (Sarstedt, catalog number: 62.548.004 )

4. Serological pipettes 10 ml (Sarstedt, catalog number: 86.1254.001 )

5. Serological pipettes 25 ml (Sarstedt, catalog number: 86.1685.001 )

6. Tissue Culture Flask, 150 cm² filter (TPP, catalog number: 90151 )

7. Tissue Culture Flask, 75 cm² filter (TPP, catalog number: 90076 )

8. 0.22 μm sterile syringe filters (Sarstedt, catalog number: 831.826.001 )

9. 0.1 μm sterile syringe filters (Pall Life Sciences, catalog number: 4668 )

10. 20 ml Sterile syringes (BD Plastipak, catalog number: 10569215 )

11. Glass Pasteur pipettes (Deltalab, catalog number: 702 )

12. Countess cell counting chamber slides (Thermo Fisher Scientific, Invitrogen, catalog number: C10228 )

13. Open-Top Thinwall Ultra-Clear Tube, 38.5 ml capacity (Beckman Coulter, catalog number: 344058 )

14. Lewis and Fisher rats (200 g)

15. Trypan blue (Thermo Fisher Scientific, Invitrogen, catalog number: C10228 )

16. Alpha MEM (Lonza Bioscience, catalog number: BE02-002F )

17. RPMI 1640 Medium with L-Glutamine (Lonza Bioscience, catalog number: BE12- 702 F)

18. Fetal bovine serum (FBS) (EuroClone, catalog number: ECS0180L )

19. Penicillin-Streptomycin (10,000 U/ml) (Thermo Fisher Scientific, Invitrogen, catalog number: 15140122 )

20. Trypsin-EDTA (0.25%), phenol red (Thermo Fisher Scientific, Invitrogen, catalog number: 25200072 )

21. Dulbecco’s phosphate buffered saline (PBS) 10x, Without Ca++ and Mg++ (Cultek, catalog number: BE17-517Q )

22. Sterile water for irrigation, Serrasol solution (Serra Pamies, catalog number: 374561 )

23. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 276855 )

24. L-glutamine solution (Sigma-Aldrich, catalog number: G7513 )

25. Complete alpha-MEM medium (see Recipes)

26. Freezing medium (see Recipes)

Equipment

1. -80 °C freezer

2. Water bath set to 37 °C (Thermo Fisher Scientific, model: JP Selecta^TM Precisterm 6000141 )

3. Refrigerated benchtop centrifuge (Thermo Fisher Scientific, model: Heraeus^TM Multifuge^TM X1)

4. Countess automated cell counter (Thermo Fisher Scientific, Invitrogen, catalog number: C10281 )

5. DHD Air-Jacketed Automatic CO₂ Incubator (NuAire, catalog number: NU-5510E )

6. Inverted Modulation Contrast Microscope (Leica, model: DMIRB )

7. C2200 CBC Complete Balance (Cobos, 200-20018 )

8. Preparative ultracentrifuge (Beckman Coulter, model: Optima L100XP )

9. SW32 Ti Swinging-Bucket Rotor Package (Beckman Coulter, catalog number: 369694)

10. NanoDrop Microvolume Spectrophotometers (Thermo Fisher Scientific, model: ND1000)

11. NanoSight instrument equipped with a 638 nm laser and CCD camera (model F-033) (Malvern, model: LM10)

12. Cryo-electronmicroscope (Jeol, model: JEM 2011)

13. EMR Carbon support film on copper 400 square mesh (EMR, catalog number: 22-1MC040-100)

14. Flow cytometer (Becton Dickinson, Biosciences, model: FACS Canto II)

Software

1. FlowJo v10 software (BD Bioscience, https://www.flowjo.com/solutions/flowjo/downloads)

2. Nanosight NTA Software version 3.1 (build 3.1.46) (Malvern Panalytical, https://www.malvernpanalytical.com/en/learn/events-and-training/webinars/W150326NanoSightSoftwareRelease)

Procedure

1. Mesenchymal stem cell preparation

   Bone marrow-MSCs (BM-MSCs) were isolated from femurs of Lewis and Fisher rats (200 g). In brief, the bone shaft was extracted inserting a 22 G needle and flushed out with alpha-MEM supplemented with 10% FBS and 2 mM EDTA and cell clumps desegregated. The cell suspension was centrifuged at 400 x g for 20 min at room temperature. Rat BM-MSCs were seeded and expanded in alpha-MEM supplemented with 10% FBS (Ramírez-Bajo et al., 2020a). Therefore, BM-MSCs were characterized according to standardized criteria defined by International Society for Cellular Therapy (ISCT) (see Note 1; Figure 1).

   1. Culture MSCs in T75 cm² flask using 7 ml of complete alpha-MEM medium. Trypsinization is performed at approximately 80% confluence.

   2. Wash cell culture with pre-warmed PBS, add 3 ml trypsin and incubate at 37 °C for 4 min.

   3. Stop the reaction with 9 ml of complete medium and spin down at 600 x g for 5 min at room temperature to count cells and check viability of culture.

   4. Count MSC’s number and their viability with an automated cell counter following manufacturer’s instructions (see Note 2).

   5. Plate the cells at a density of 5 x 10⁵ cells in a flasks T150 cm² with 15 ml of complete alpha-MEM medium.

   6. Incubate at 37 °C in 5% CO₂ environment.

   7. Replace with fresh complete alpha-MEM medium (pre-warmed to 37 °C) every two days thereafter.

      
      

      
      Figure 1. Representative images of MSCs characterization by flow cytometry and multi-lineage differentiation potential. A. Flow cytometry analysis of MSC. Cells were positive for surface stem cell markers (CD44, CD29 and CD90), and negatives for endothelial (CD31), and hematopoietic (CD45) markers. B. Adipogenic differentiation (x200 magnification) as demonstrated showing positivity for oil red staining. C. Osteogenic differentiation (x100 magnification) as demonstrated showing positivity for alkaline phosphatase activity, and was revealed by SigmaFast BCIP/NBT chromogen staining.

      
      

2. EVs isolation by ultracentrifugation

   1. When the cells are approximately 80% confluence, aspirate the supernatant and add 6 ml of PBS (pre-warmed to 37 °C) to wash the residual FBS. Repeat washing step one more time.

   2. Replace PBS with 15 ml of RPMI1640 deprived of FBS (pre-warmed to 37 °C) and incubate for 16 h.

   3. Harvest the MSC conditioned serum free medium and transfer to 50 ml conical tube and keep at 4 °C.

   4. Trypsinize MSCs and determine cell number and viability in an automated cell counter following manufacturer’s instructions (repeat Steps A1-A3).

      Our estimation at time of collection was ± 1.5 x 10⁶ MSCs and ± 95% of live cells.

   5. Centrifuge MSC conditioned serum free medium at 3,000 x g for 20 min at 4 °C and micro filtrate with 0.22 μm pore filter membranes to remove cell debris and apoptotic bodies.

   6. Transfer cell-free supernatants into a 38.5 ml capacity open-top thin wall ultra-clear tube. Balance opposite tubes within 10 mg of each other (see Notes 3-4).

   7. Ultracentrifuge at 100,000 x g for 1 h, 4 °C, using SW32 Ti rotor.

   8. Carefully remove tubes from the rotor.

   9. Remove supernatant by aspiring carefully with a Pasteur pipette.

   10. Resuspend EVs pellets with a 200 µl of freezing medium and froze them at -80 °C for later use (see Notes 5-6).

       
       

3. EVs characterization

   See Note 7.

   1. EVs quantification:

      1. Nanosight Tracking Analysis (NTA)

         Particle size distribution and concentration measurement:

         1. Take 1 ml of EVs solution (dilution factor 1:40 in PBS 0.1 µm filtered) and vortex.

         2. With a syringe without needle, inject EVs solution slowly (avoiding bubbles) into the chamber of particle size analyzer for visualization. It is important to have approximately 20-60 particles in the field of view, to obtain an accurate concentration and size values (Video 1).

            
            
            
            Video 1. EVs moving under Brownian motion recorded by NTA. This video visualizes the expected Brownian motion of the EVs in liquid suspension illuminated by a laser light source. The EVs size distribution and concentration are measured using the combination of the light scattering properties with Brownian motion. The real time monitoring helps to determine changes in the characteristics of vesicle populations.

            
            

         3. Take triplicate readings during 60 s at 30 frames per second (fps), camera level at 16 and manual monitoring of temperature. Figure 2A shows representative results obtained by NanoSight from EVs produced by MSCs.

      2. Protein quantification:

         Determination of protein concentration (A_280nm) was checked by NanoDrop using 2 µl of EVs solution following the manufacturer's instructions.

   2. Assessment of absence of contaminants by Electron Microscopy

      1. Use a holey carbon support film on a 400-mesh copper grid. Glow discharge the grid and place 3 µl of the EVs sample (from Step B10) on a plunger (Leica EM GP) and blot it with Whatman No. 1 filter paper.

      2. Vitrify the suspension by rapid immersion in liquid ethane (-179 °C) and mount the grid on a Gatan 626 cryo-transfer system and insert it into the microscope.

      3. Take images using cryo-electron microscope operates at 200 kV, record on a GatanUltrascan US1000 CCD camera and analyze with a Digital Micrograph 1.8 (n = 3 per group) (Figures 2B-2C).

         
         

      
      Figure 2. Characterization of EVs by electron microscopy and Nanoparticle Tracking Analysis (NTA). A. NTA measurement shows the concentration and size distribution. The mean EVs size was 201.9 ± 101.4 nm. B-C. Representative cryo-electron microscopy images of EVs (scale bars 0.5 and 0.2 µm, respectively). Cryo-electron microscopy allows to visualize the grid with an irregular distribution of hole sizes and shapes, and inside of them EVs of various sizes with lipid bilayers. The white arrow points a vesicle with a double membrane (white arrow). Most vesicles have an intact membrane and a round shape. Some vesicles are single, double and multilayer, in the case that contains inside another smaller or two and more (black, blue and red arrow, respectively).

Data analysis

1. MSCs characterization by flow cytometry: Flow cytometric data obtained from BD FACS Canto II cytometer were analyzed with FlowJo software following the gating strategy described by Ramírez-Bajo et al., 2020b.

2. Nanosight tracking analysis: Data were analyzed with the Nanosight NTA Software version 3.1 (build 3.1.46), with detection threshold set to 5, and blur, min track length, and max jump distance set to auto.

Notes

1. According to standardized criteria defined by ISCT’s guidelines, MSCs must a) be plastic adherent when kept under standard culture conditions, b) positively expressed MSC markers (CD29, CD44, and CD90) and negatively endothelial (CD31) and hematopoietic lineage (CD45) markers, and c) retain a multipotent phenotype with the ability to differentiate in adipogenic and osteogenic lineage under the standard differentiation conditions (Figures 1B-1C).

2. In 2018, the International Society for Extracellular Vesicles (ISEV) published and update of the MISEV2014 guidelines with different recommendations to improve the reproducibility of published EVs results. The ISEV checklist summarizes the major aspects to follow in EV science (Lotvall et al., 2014).

   The estimation of number of cells/ml or /surface area and % of live/dead cells at time of collection (or at time of seeding with estimation at time of collection) is a mandatory requirement of ISEV Checklist.

3. To maintain sterility of EVs: Firstly, buckets must be cleaned with ethanol and let dry in the hood. Secondly, insertion or removal of the tubes from buckets has to be done also in the hood.

4. Balance the weight within the buckets. Thinwall tube needs to be full to provide tube wall support in the bucket. If the volume is too low it may collapse. For this reason, it is important to fill the tube with supernatant or adding PBS 0.1 μm sterile filtered.

   Using SW32 Ti requires balancing the weight of the tubes with the buckets following Beckman’s recommendation’s: https://www.beckman.com/resources/fundamentals/principles-of-centrifugation/balancing-your-rotor.

5. In order to resuspend the EVs pellets, you have to pipette them up and down with a 200 μl pipette but not too roughly, because it is better to avoid producing air bubbles.

6. EVs can be stored for 2 days at 4 °C. Storing samples at -80 °C is recommended for extended period.

7. Following ISEV recommendations it requires quantification by at least 2 methods. In our case, we determined particle number by Nanosight and protein count by NanoDrop Spectrophotometer. These results have to be expressed per volume of initial fluid or number of producing cells.

Recipes

1. Complete alpha-MEM medium

   Alpha-MEM

   10% FBS

   1x L-Glutamine

   1x P/S

   Filter sterilize the solution and store at 4 °C

2. Freezing medium

   RPMI 1640

   1% DMSO

   Filter sterilize the solution and store at 4 °C

Note: Filter all the solutions using a 20 ml syringe and a 0.22 μm filter into a new sterile tube.

Acknowledgments

This study has been partially funded by the research grant from Sociedad Española de Nefrología (2014, JC), Redes Temáticas de Investigación Cooperativa En Salud, REDINREN (RD12/0021/0028 and RD16/0009/0023) co-funded by ISCIIISubdirección General de Evaluación and Fondo Europeo de Desarrollo Regional (FEDER) “Una manera de hacer Europa,” and Secretaria d’Universitats i Recerca and CERCA Programme del Departament d’Economia i Coneixement de la Generalitat de Catalunya (2017-SGR-1331). This work has been developed in the context of AdvanceCat with the support of ACCIÓ (Catalonia Trade and Investment; Generalitat de Catalunya) under the Catalonian ERDF operational program (European Regional Development Fund) 2014-2020. This work has been developed at the Centre de Recerca Biomèdica Cellex, Barcelona, Spain. Our protocol has been based on a previous protocol described by Herrera et al. (2010).

Competing interests

The authors declare no conflicts of interests.

Ethics

Bone marrow-MSCs were isolated from femurs of Lewis and Fisher rats (200g) (Ramírez-Bajo et al., 2020b). The study was approved by and conducted according to the guidelines of the local animal ethics committee (Comitè Ètic d’Experimentació Animal, CEEA, Decret 214/97, Catalonia, Spain).

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