Mice

This protocol has been standardized for muscle derived-EVs isolation with mice from different backgrounds, C57BL6 and C57BL/10ScSN. In our particular case, we have not found differences in terms of yield with different strains for these backgrounds. However, we cannot anticipate that changes in mouse strain could lead to differences in isolation yield. We recommend using mice aged 8–12 weeks independently of their sex, given that very young or old mice could have differences in EVs secretion.

Reagents

All reagents were prepared and stored according to the manufacturer’s instructions.

The use of alternative reagents from other companies may not guarantee the anticipated results.

• Chlorohexidine, 2%.

• Eye lubricator.

• PBS without calcium and magnesium, pH 7.4 (Wisent, cat. no. 811-012-FL).

• FBS (Wisent, cat. no.098–150).

• Penicilyn/Streptomycin (Wisent, cat. no. 450-201-EL).

• DMEM media (Wisent, cat. no. 319-016-EL).

• Cardiotoxin, citotoxin I (Latoxan, cat. no. L81 02).

• NaOH (Fisher Scientific, cat. No. S318-500).

• Sodium Chloride, 0.9% (Baxter, cat. no. JB1301).

• MilliQ water.

• Ice.

FBS EVs-depleted for coating

Centrifuge FBS under sterile conditions at 100,000 g for 90 min using Beckman Coulter Optima L-100XP Ultracentrifuge, Rotor 45Ti (Beckman Coulter, cat. no. 339160) and tubes (Beckman Coulter, cat. no. 355622). Upon centrifugation carefully transfer the EVs-depleted FBS and make 50 mL aliquots. Storage them until use at -20 °C. Note, FBS for use in conditioned media supplementation should be centrifuged at 120,000 g for 18 h.

PBS microparticles-depleted

Centrifuge PBS 1x at 100,000 g for 90 min using Beckman Coulter Optima L-100XP Ultracentrifuge, Rotor 45Ti (Beckman Coulter, cat. no. 339160) and tubes (Beckman Coulter, cat. no. 355622). Upon centrifugation filter the PBS using a Stericup under sterile conditions and storage it 1 L sterile glass bottles.

Sample collection

• 5

  Four days after injury euthanize mice, accordingly to the animal procedure protocol of the institution.

Critical step. Do not proceed to step 6 until there are no signs of reactivity upon gentle pressure on the toes with a surgical forceps.

• 6

  Immobilized the euthanized mice over a Styrofoam lid using pins covered in aluminum foil.

• 7

  Using operating scissors cut the skin perpendicular to the midline around the abdomen and de-glove the skin (see Fig. 4B).

• 8

  Collect all hindlimb muscles from both legs using sterile surgical tools and place them on ice in a 50 mL Falcon tube full of sterile cold PBS. See anatomical images to aid dissection in Fig. 4C.

Critical step. Each mouse should be treated as a separate single sample to minimize potential contaminations.

• 9

  Transfer samples into the TC hood.

• 10

  Collect the muscle tissue with a forceps and place all the muscles in a 150 mm petri dish.

• 11

  With the help of surgical scissors and forceps remove all fat depots and tendons to avoid cross-contamination with other sources of EVs.

Critical step. Ensure that the large fat pad within the gastrocnemius muscle is removed (Fig. 4D).

• 12

  Once the muscle is clean, cut into small pieces of about 1 mm³ (Fig. 4E).

• 13

  Add 50 mL of pre-warmed PBS and transfer the minced muscle with PBS into a 50 mL Falcon tube using a 50 mL pipette.

• 14

  Centrifuge at 300 g for 5 min room temperature in a Benchtop centrifuge using a TX-1000 rotor. Alternatively, another rotor compatible with a 300 g centrifugation step can be used.

• 15

  While tissue is centrifugating, rinse 150 mm Petri dishes, one per mouse, with pre-warmed 37 °C FBS EVs-free (to wet the dishes).

• 16

  Aspirate the excess of FBS but do not let the dishes to fully dry.

Critical step. Removal of serum is crucial to allow attachment of tissue fragments to culture dish.

• 17

  Once the muscle is pellet down remove PBS carefully with a glass fine pipette connected to the aspirator to avoid muscle loss for aspiration.

• 18

  Rapidly resuspend explants with 10 mL of pre-warmed 37 C°C FBS EVs-free.

• 19

  Using a 1000 pipette connected to a tip, spot drops of this mixture on the surface of the wet dishes (Fig. 4F).

• 20

  Lift the side of the dish to remove FBS in excess by with a glass fine pipette connected to the aspirator to avoid muscle loss for aspiration as in step 16.

• 21

  Incubate the dishes on a cell incubator at 37 °C, 5% CO₂ for 2 h to allow explants to adhere to the dish.

• 22

  After 2 h, gently add 30 mL pre-warmed 37 °C DMEM medium containing antibiotics with a 25 mL pipette, covering all the muscle tissue.

• 23

  Incubate the dishes on a cell incubator at 37 °C, 5% CO₂ for 2 days.

• 24

  After 2 days, collect medium with a 25 mL pipette into 50 mL Falcon tubes. Discard the muscle tissue into the corresponding biological disposable container.

Pause point. At this point, medium can be stored at -80 °C prior to proceeding with EV isolation. However, is optimal to continue with the isolation protocol to avoid any loss of samples.

EVs pre-clarification

• 25

  Centrifuge conditioned medium, obtained in point 24, in a 50 mL Falcon tube at 300 g for 10 min at room temperature to remove floating cells from the mixture. Use a benchtop centrifuge with a swing bucket rotor TX-1000 Thermo Scientific. Alternatively, another rotor compatible with a 300 g centrifugation can be used. If media has been previously frozen, thaw in a water bath at 37 °C prior to centrifugation.

Critical step. After centrifugation, handle tubes gently to avoid pellet detachment and cross contamination with supernatant.

• 26

  Using a 50 mL pipette, transfer supernatant carefully into a new 50 mL Falcon tube.

Critical step. Leave 3 mL of supernatant in the bottom of the Falcon tube to avoid potential aspiration of the pellet and consequent cross-contamination with cells fraction.

• 27

  If pellet is not required as a control, avoid steps 28–29 and go directly to point 30.

• 28

  Resuspend pellet with PBS microparticles-free and spin it once again using same conditions than point 25. This step does not need to be performed under sterile conditions.

• 29

  Aspirate PBS and resuspend pellet in the appropriate buffer (p.e. RIPA buffer for immunoblot) and store accordingly. This step does not need to be performed under sterile conditions.

• 30

  Centrifuge conditioned medium obtained in point 26 at 2500 g for 10 min at room temperature to remove apoptotic bodies from the mixture. Use a benchtop centrifuge with a swing bucket rotor TX-1000 Thermo Scientific. Alternatively, another rotor compatible with a 2500 g centrifugation step can be used.

• 31

  Using a 25 mL pipette transfer supernatant into a sterile 50 mL polycarbonate tube.

• 32

  If the pellet containing apoptotic bodies pellet is required, repeat steps 28–29 but using conditions from step 30. Otherwise continue with point 33.

Critical step. Leave 3 mL of supernatant in the bottom of the Falcon tube to avoid potential aspiration of the pellet and consequent cross-contamination with apoptotic bodies fraction.

Critical step. Ensure tubes are filled accordingly to manufacture´s instructions with the minimal and maximal volume recommended to avoid spills and damage to the tubes.

• 33

  Centrifuge supernatant obtained in point 31 at 20.000 g for 20 min at 4 °C to remove microparticles. For this step use a High-speed centrifuge. Different combinations of tubes rotors can be used depending on user availability:

  • A)

    Rotor SS34 with Beckman Coulter cat. no. 344,058 30 mL tubes.

  • B)

    Rotor JA25.50 with Beckman Coulter, cat. no. 357,002 50 mL tubes. This is the best option in terms of volume capacity as 8 samples can be centrifuged in one step with bigger capacity tubes.

Critical step. Centrifugation conditions are calculated based on the JA25.50 rotor. If a different rotor is used, recalculate the pelleting time taking into account the K factor specific for each rotor [42]. Pelleting time depends on the k factor, a parameter that is specific for each rotor. K factor measures rotor pelleting efficiency at maximum speed. The lower the K factor the shorter running time is going to be at the same speed. Smaller rotors have a lower K factor, as the pathlength of the sample to be pellet down is smaller. This is a critical point as longer running times could compromise the integrity of the vesicle.

• 34

  Using a 25 mL pipette transfer supernatant into a 50 mL FlexyBoy bag (Fig. 3D).

• 35

  If the pellet containing microparticles is required for control purposes, please repeat steps 28–29 but using conditions from step 33. Otherwise continue with point 36.

Pause point. At this point, medium can be frozen in the Flexboy bag at -80 °C prior to conducting the TFF procedure. However, it is recommended to continue with the isolation protocol for optimal EV recovery.

Tangential Flow Filtration

• 36

  Connect to the TFF machine the set up circuit (Fig. 3D), using the previously prepared cartridge.

• 37

  Connect the sterile PBS bottle to the auxiliary pump washing port (Fig. 3A).

• 38

  Repeat steps 4–6 from New cartridge rinsing and sanitation procedure.

• 39

  Start main pump at 37 mL/min. Make sure there are no bubbles, and all pressures are stable, P_f ~4–6 psi and P_r ~1–2 psi. Run for about 5 min.

• 40

  Once pressure is stable, start the automatic backpressure valve at 3 psi to get a TMP of 3 psi.

Critical step. Adjust the permeate tubing with a manual clamp (Fig. 3E), to get a positive permeate pressure~0.2 psi to dimmish loss of EV material through the cartridge pores due to the TMP.

• 41

  Ensure that shear rate is ~3000 in the software display, based on the parameters previously setup in points 39–40.

• 42

  Filter until approximately one inch of media remains in the Flexboy bag and stop the pump.

• 43

  Start the step of washing isolated EVs sample in continuous mode as we did in point 11 of New cartridge rinsing and sanitation procedure. This means that while washing with PBS, sample is permeating through the cartridge and the volume in the sample bag remains steady.

• 44

  Open the auxiliary pump T-valve and started the auxiliary pump to run at 1.5 mL/min.

• 45

  Re-start main pump at 37 mL/min with TMP valve at 3 psi.

Critical step. Maintain the volume in the bag low without allowing it to empty. Flow rate may need to be adjusted (0.5 mL/min-3.0 mL/min depending on sample type). Continue to adjust so that volume is steady, meaning same volume of PBS gets permeated than gets into the sample bag from the auxiliary pump. If the sample is getting too diluted, decrease the flow rate.

• 46

  Tare volume on scale and set up an alarm with the desired amount of washing volume to permeate (use Perm HI STOP for pump to stop at certain volume).

Critical step. For muscle explant derived EVs, use double the amount of the starting volume sample due to the high volume of undesired free proteins (e.g. if starting sample is 20 mL, use 40 mL of PBS). However, for Wnt7a HEK293T cell-derived EVs, the same volume of PBS as the sample volume is used to get wash out of the Wnt7a free protein. Adjust the PBS volume, depending on the origin and application of the EVs.

• 47

  When washed volume is reached, stop the pumps, and close the T-valve.

• 48

  Concentrate the sample by re-starting the main pump with a flow of 37 mL/min and permeate the entire volume of the Flexboy bag until bubbles appear in the sample loading tube.

Critical step. To have a better control of this step, do not restart the back-pressure valve to set up the TMP at 3 when permeating the sample. The TMP generated by the main pump feed is enough to permeate the remaining volume of the sample. Otherwise, it would be too fast, and sample can be lost.

• 49

  Once all the volume in the Flexboy bag is permeated, stop the main pump, and clamp the Flexboy bag port attached to retentate (left side) using the clamp provided with the Flexboy bag. Then, disconnect the retentate tubing from the clamped Flexboy bag.

• 50

  Take a 15 mL tube to drain the dead volume of the tubing set.

• 51

  Place the retentate tubing into the new tube. Set the flow rate of main pump at the maximum flow, 132 mL/min, and make sure the automatic backpressure valve is open and there is no clamp in the permeate tubing.

• 52

  Start main pump and collect all the retentate fraction into the new tube. This fraction is the EVs fraction ready to use. Volume should be around 5–7 mL (Fig. 3E).

Pause point. At this point EVs can be frozen in a Falcon tube at -20 °C.

• 53

  For cartridge cleaning, please proceed directly to point 59.

Size exclusion chromatography

• 54

  Once the retentate fraction, that contains the purified EVs, is collected proceed to pellet it down by ultracentrifugation. To reduce the pelleting time is better to use a benchtop ultracentrifuge instead of a regular ultracentrifuge. Transfer the contents of the sample using 1mL pipette to an appropriate tube using:

• A)

  Ultracentrifuge L:90 with rotor 70.1Ti and 10.4 mL polycarbonate bottles with cap (Beckman Coulter, cat. number tubes ref. 355603). Run for 70 min at 100,000 g (32,900 rpm for 70.1Ti rotor) at 4 °C.

• B)

  Bench ultracentrifuge with rotor TLA.110 and 3.2mL polycarbonate tubes (Beckman Coulter, cat. no. 362305). Run for 30 min at 100,000 g (49,000 rpm for TLA.110 rotor) at 4 °C.

Critical step. If EVs are for in vivo or in vitro experiments, is very important to maintain the sterility working in a laminar flow hood and with autoclaved material.

Critical step. Depending on the volume, use several smaller 1.5 mL eppendorf tubes with the adaptors instead of 3.2 mL open tubes. The pelleting time is the same.

• 55

  Remove supernatant with a 1000 pipette. Resuspend the pellet in the minimal amount of buffer required to be load into the SEC column and proceed accordingly to manufacturer’s instructions.

• 56

  Collect the different eluted fractions and proceed to quantification to verify the fraction or fractions that contain EVs of interest, based on the number of particles with a NTA equipment accordingly to manufacturer’s instructions (Fig. 5A).

  

  Open in a separate window

  Fig. 5

  Purified muscle-derived EVs with Tangential Flow Filtration and SEC. AUpper panel. Quantification of EVs concentration on the different fractions collected upon SEC. Lower panel. EVs size distribution graphs for F3 to F7 upon SEC, showing how fraction F4 to F6 correlate with small EVs size (fraction of interest) and not before (F3) or after (F7). B Immunoblot analysis showing the expression enrichment of EVs markers on muscle-derived EVs. C Representative TEM pictures of isolated EVs derived from new regenerating hindlimb muscles using post-embedding techniques. Representative of 3 independent experiments. Size Exclusion Chromatography (SEC), Fraction (F), Transmission Electron Microscopy (TEM)

• 57

  Fractions of interest can be combined and pellet down as in point 54 or used directly. If they are required for in vivo or in vitro bioactivity assays is better to filter them using a syringe microfilter of 0.22 μm.

• 58

  Analyze and quantify concentration following MISEV 2023 guidelines (Fig. 5):

• A)

  Perform immunoblot analysis with different positive and negative EVs markers (Fig. 5B).

• B)

  Perform Transmission Electron Microscopy (Fig. 5C).

Flush cartridge

• 59

  Change the PBS bottle of the auxiliary pump for the sterile Milli-Q water bottle used in the previous cartridge activation. Screw a plastic cap on the T-valve to cover the open end of the retentate port. Change permeate collection for waste bucket and connect both permeate and retentate into this bucket. Open auxiliary pump and T-valve so circuit is open to Milli-Q water and tubing is not obstructed. Unclamp the retentate port of the Flexboy bag. Make sure that back pressure valve is still opened and main pump is set up at 132 mL/min and there is no clamp in the permeate tubing (Fig. 3E).

• 60.

  Start main pump and flush 25 mL of MilliQ water through the system.

• 61.

  Stop the main pump. Close the auxiliary pump. Remove the plastic cap on the T-valve and reconnect retentate tubing to Flexboy bag port.

• 62.

  Start the auxiliary pump at flow rate of 10 mL/min. Start the main pump at 132 mL/min and flush another 25 mL of MilliQ water with the backpressure valve opened.

• 63.

  Stop the pumps.

• 64.

  To purify another sample, proceed with step 36. Otherwise continue with point 65.

• 65.

  Upon complete of purifications, rinse with NaOH to sanitize the cartridge.

• 66.

  Change the auxiliary pump bottle for the 0.5 M NaOH used in the previous cartridge activation.

• 67.

  Set up the auxiliary pump for 1–2 mL/min to wash the cartridge in continuous washing mode as in steps 43–46. Rinse with 0.5 M NaOH at main pump flow rate 37 mL/min and with backpressure valve opened. Keep settings until permeate 25–50 mL is complete.

Critical step. If media contains red phenol, make sure that the colorant is all washed off and the cartridge is not reddish anymore.

• 68.

  Stop the flow and connect retentate to waste. Screw a plastic cap on the T-valve to cover the open end of the retentate port. Connect the sterile PBS bottle to the auxiliary pump. Wash with 50 mL of PBS at maximum speed, 132 mL/min with the back pressure valve open, to flush the remaining NaOH.

• 69.

  Stop the flow. Remove the plastic cap on the T-valve and connect retentate flow for recirculation, back to the continued circuit. Initiate the flux with a speed rate of 37 mL/min and TMP 3psi.

Critical step. Recirculate 50 mL of PBS and check with pH paper strips that pH is 7 approximately, meaning non traces of NaOH remain inside the circuit that could damage the upcoming sample purification. Otherwise keep recirculating PBS until optimal neutral pH 7 has been reached.

• 70.

  Column is ready for use again.

Critical step. Keep a record of the number of times the filter has been used and how much volume was filtered. Cartridges can be reused multiple times depending on the type of sample and how concentrated it is. With EVs from HEK293T cells, up to 1 L of filtrate can be prepared as long as the cartridge has been washed and stored properly. However, this volume can change depending on the origin of the conditioned media and the conditions of its use.

• 71.

  Save and close worksheet, choosing to “End Collection”. Clamp and detach sample bag from circuit, closing tubing ends with plastic caps. Detach other components (e.g. PBS tubing) and close off with plastic caps. It is better to storage the cartridge separately from the tubing and sensors in a plastic bag in a drawer protected from light and high temperatures.

Conditioned media production for cell derived EVs

Equal numbers of HEK293T cells, 20 millions cells per 150 mm dish, were seeded and pcDNA3_Wnt7a-HA plasmid was transfected with linear polyethylenimine (Polysciences), accordingly to manufacturer’s instructions. Later, transfected cells were cultured with 10% FBS EV-depleted [44] in DMEM (Gibco) and maintained at 37 °C in a humidified incubator equilibrated with 5% CO2. After 48 h of secretion conditioned media was collected for EVs isolation and samples were purified following the protocol described in this manuscript.

Pre-embedding immunogold labeling for EVs

TEM on EVs was performed following our previous published protocol [33]. Briefly, EVs specimens were fixed in Karnovsky´s fixative for 2 weeks. Fixed EVs pellets were treated with 0.1% sodium borohydride in PBS. EVs were permeabilized with 0.1% Triton X-100 for 10 min. After, EVs were blocked in blocking buffer (10% donkey serum+0.6% gelatin from cold water fish skin in PBS) for 2 h. EVs were incubated with the primary anti-HA antibody (Benthly) for 48 h. Pellets were incubated overnight with the secondary antibody (Jackson ImmunoResearch). Immunogold-labelled EVs were fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer and enhancement was performed with a silver enhancement kit on the immunogold-labelled cells. All samples were post-fixed with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer. Then, samples were dehydrated in increasing concentration of ethanol and infiltrated in Spurr resin overnight at 70 °C. Ultrathin transversal Sect. (80 nm) were collected onto 200-mesh copper grids and counterstained with 2% aqueous uranyl acetate and with Reynold’s lead citrate. Finally, specimens were observed under a transmission electron microscope (Hitachi 7100, Gatan digital camera). For our analysis, approximately 50 immunoelectron micrographs were examined per sample at at 100,000X and 150,000X magnifications. Importantly, the same protocol was applied to muscle-derived EVs but without immunolabeling in this case. For detailed information about iTEM muscle-derived EVs please refer to [33].

Immunoblot analysis

Immunoblot analysis was performed as described previously [46] with the following modifications. The lysates from EVs were not clarified by centrifugation. The immunoblot transferring was performed onto PVDF membranes.

Pre-embedding immunogold labeling for muscle-tissue iTEM

iTEM was performed following our previous published protocol [33]. Briefly, Tibialis Anterior specimens were fixed in Karnovsky´s fixative for 2 weeks. After fixation all segments were subsequently washed with 0.1 M sodium cacodylate, and treated with 0.1% sodium borohydride in PBS. Samples were permeabilized with 0.1% triton X-100 and blocked with 10% donkey serum+0.6% fish gelatin. TA samples were incubated with Wnt7a antibody (R&D). After 48 h incubation, segments were rinsed thoroughly with PBS and incubated overnight with the secondary antibody Ultra small (0.8 nm) Gold conjugated (EMS) in blocking buffer at RT. Later, samples were rinsed with 0.1 M sodium cacodylate and post-fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate. Pre-embedding enhancement was realized with silver enhancement kit (AURION R-Gent SE-EM, EMS) according to the manufacturer’s instructions. After enhancement, samples were secondly post-fixed with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer. Then, samples were dehydrated in increasing concentration of ethanol and infiltrated in Spurr resin. Ultrathin transversal Sect. (80 nm) were collected onto 200-mesh copper grids and counterstained with 2% aqueous uranyl acetate and with Reynold’s lead citrate. Finally, specimens were observed under a transmission electron microscope (Hitachi 7100, Gatan digital camera). For our analysis, approximately 50 immunoelectron micrographs were examined per muscle at different magnifications.

Nanoparticle tracking analysis equipment

Size distribution of EVs samples was defined measuring the Brownian motion with a NanoSight LM10 system equipped with a fast video capture and particle-tracking software (Malvern, UK). Each video was analyzed to acquire particle size and an estimate of the particle concentration. Each sample was acquired 3 times and average curve was calculated for each sample.

Size exclusion chromatography

Size exclusion chromatography (SEC) was performed with the centrifuged retentate fraction. The pellet was resuspended in 0.5 mL of filtered PBS and purified with a 35 mm column using Automatic Fraction Collector V2, following manufacturer’s instructions. Briefly, the column was pre-washed with 17 mL of filtered PBS and then the sample was loaded, followed by 12 mL of filtered PBS to allow the sample to flow gently through the column. From each SEC column, 12 eluted 800 µL fractions were collected sequentially. These EV samples were stored at −80 °C until further characterization.

The authors thank Jennifer Richie for the mice colony management, Maria Teresa Rojas (Centro Severo Ochoa, Spain) for technical help with Transmission Electron Microscopy, and Juan Anguita (CIC bioGUNE) for the legal advice with animal experimentation.