Isolation of Tissue Extracellular Vesicles from the Liver

All studies involving animals were performed in accordance with a protocol that was approved by the Mayo Clinic Institutional Animal Care and Use Committee.

1. Bench Preparation

1. Prepare a water bath and set it to 37 °C. The other necessary equipment and set-up are shown in Figure 1.
2. Measure 100 mg of collagenase type IV and add it to a 125 mL flask containing 100 mL of HBSS in a water bath (40 °C). Ensure that the collagenase has been dissolved by swirling the liquid in the flask, and also make sure that the flask is submerged completely in the water bath. For greater efficiency, allow the collagenase to dissolve for at least 30 min even though it may appear to dissolve instantly.
3. Submerge a 125 mL flask containing 50 mL of HBSS in a water bath at 40 °C. This will be used for initial flushing.
4. Spray down the surface of all instruments such as the scissors and forceps with 70% ethanol. Prepare a few clean cotton swabs.
5. Rinse out the tubing of the pump by running 70% ethanol through the pump and remove any residual ethanol by rinsing it twice with running water.
6. Lay an absorbent bench pad on the lab bench and place a hard box container on top. This will be needed to contain any excess liquids during the mouse perfusion. Wrap a polystyrene foam pad with aluminum foil and place it inside the container.
7. Place a sterile 10 cm culture dish close to the bench. This will be used to hold the digested liver after the perfusion is completed.
8. Pour 10 mL of collagenase medium (step 1.2) into a sterile culture dish in advance.
9. Using a winged blood collection set, connect the 23-gauge to the free end of the pump tubing (cutting the wings off the butterfly cannula can lead to better handling). Pass until the tip of the cannula of blood collection is filled.

2. Animal Preparation

1. Before starting anesthesia using isoflurane, make sure that an adequate amount of supply gas is available for the duration of the procedure. Turn on the oxygen (O₂) supply to the induction chamber at 1-2 L, then turn on isoflurane between 2-4% using a flowmeter.
2. Put the mouse into the induction chamber and close the top door. Monitor the mouse until it is recumbent.
   NOTE: The gases in the chamber will keep the mice anesthetized for several minutes.
3. Place the mouse on a polystyrene foam pad wrapped with aluminum foil. Switch the flow from the induction chamber to a nosecone. Ensure that anesthesia is adequate. If the mouse has started responding, gently restrain it in a nose cone until the it is fully anesthetized.
4. Ensure anesthesia by monitoring respiration and response to stimulation during the procedure. Adjust the rate flowmeter as needed to ensure adequate anesthesia. The paws must be nonresponsive to the pinch test under anesthesia. Additional details may be obtained from the laboratory guide for the care of animals.
5. Tape or pin down all four limbs of the mouse.
6. Clean the skin over the abdomen by spraying with 70% ethanol and wiping it off with gauze and an alcohol pad. This step is critical to avoid contamination from the mouse fur.
7. Make a wide-open cut through the skin from the anterior to the pelvis bone using a set of sterile scissors. Be careful not to cut any internal organs . Displace the intestines to the animal's left side by using a cotton tipped applicator to gently to expose the portal vein (PV) and inferior vena cava (IVC).

3. Cannulation and Perfusion (0.5 h)

1. Using curved forceps, place a thread underneath the portal vein and tie a knot loosely to prepare cinching tightly after cannulation.
2. Insert the cannula (23G from the blood collection set) into the portal vein 5-10 mm below the ligature. Do not insert the cannula past the first portal branch, otherwise the right anterior lobe can be inadequately perfused.The cannula can be fixed or fastened with thread using a stopper knot.
3. Start the pump to infuse in HBSS at a low flow rate (1-2 mL/min). Once the cannulation is confirmed to be successful, the liver will begin to blanch.
4. Cut the IVC to relieve pressure and allow excessive fluid within the liver to drain. This is best performed using the operators other hand so the cannula is not moved.
5. Slowly increase the flow rate to 8 mL/min and complete the perfusion using the entire 50 mL volume of HBSS through the liver, over the next 5 min.
6. Change the collagenase containing medium (step 1.2) into the beaker just before the HBSS is starting to run out. Ensure that air bubbles are not present and do not flow into the liver when changing the medium.
7. Apply transient pressure to the IVC at 5 s intervals by clamping with forceps. This will cause the liver to swell and help with tissue digestion and dissociation (7-8 min). As digestion progresses, the liver will swell and become white. The liver can swell uniformly to approximately twice its original size.
8. Turn off the pump and remove the cannula once digestion is completed. The completion of liver digestion will depend on the size of the mouse and condition of the liver. A dent in the liver can be observed if a cotton-tipped applicator is used to gently probe the liver.
9. Remove the gallbladder from the liver, being careful not to tear it. Using a washed pair of scissors and forceps, extract the liver from the mouse into a sterile 10 cm culture dish containing phosphate-buffered saline (PBS) for surface washing. Transfer the liver carefully into the sterile 10 cm culture dish containing collagenase medium (from step 1.8). This is a critical step for avoiding contamination from the mouse blood and bile.
10. Grab and tear apart the liver with two clean forceps while gently shaking the cells from the liver. As this happens, the medium will become clouded. All hepatocytes can be shaken away, leaving behind the connective tissue and vascular tissue.
11. Triturate the cell solution many times using a 3 mL syringe until the undigested parts of the liver are shaken off. Pour it off into a 50 mL conical tube with 70 µm nylon cell strainers to filter any undigested connective tissue. Wash the dish with HBSS to collect the remaining cells and fill up the 50 mL conical tube.
12. Centrifuge softly the 50 mL conical tube in a swinging bucket rotor at 50 x g for 10 min at 4 °C.
13. Transfer the supernatant to a new 50 mL conical tube.

4. Isolation of EVs (5 h)

1. Centrifuge the supernatant at 300 x g for 10 min at 4 °C. Transfer the supernatant into a new 50 mL conical tube.
2. Centrifuge the supernatant at 2000 x g for 20 min at 4 °C to remove cell debris and aggregates.
3. Transfer the supernatant to a round-bottom tube and centrifuge the supernatant at 10,000 x g for 70 min at 4°C.
4. Collect the supernatant and place into a polycarbonate ultracentrifuge tube and centrifuge at 100,000 x g for 70 min at 4 °C.
5. Collect the pellet in an ultracentrifuge tube which is then washed by re-suspending in PBS. Centrifuge the supernatant further at 100,000 x g for 70 min at 4 °C.
6. The final pellet comprising of cellular nanovesicles can be directly used for experiments or re-suspended with 1000 µL of PBS and stored at -80 °C.

5. Assessment of Quality and Yield of Isolations

1. Assess the size distribution and concentration using nanoparticle tracking analysis or tunable resistive pulse sensing following the instrument manufacturer's protocols.
2. Perform further isolation and purification of specific vesicle populations by various approaches such as the addition of a sucrose gradient or cushion, immunoaffinity techniques, or size exclusion chromatography, based on specific experimental needs.