This method can help us to address key questions regarding the role of extracellular vesicles in vivo, by providing us a source of vesicles that are representative of the local tissue microenvironment. Studies of extracellular vesicles within the tissue milieu can further our understanding of the physiological roles of extracellular vesicles and their alterations in pathophysiological processes in vivo. A visual demonstration of the portal vein cannulation is helpful as the small size of the vein makes it difficult to turn manipulate.
Demonstrating the procedure with Doctor Kaori Ishiguro will be Irene Yan, a research technologist from my lab. Before beginning the procedure, secure the limbs of an anesthetized mouse and connect the 23 gauge needle of a winged blood collection set to the free end of a piece of syringe pump tubing. Clean the abdominal skin of the mouse with 70%ethanol and an alcohol pad and use scissors to open the mouse from the anterior of the chest to the pelvic bone, taking care not to damage any internal organs.
Use a cotton tipped applicator to gently move the intestines to the right side of the abdominal cavity to expose the portal vein and inferior vena cava and use curved forceps to place a thread under the portal vein. Tie a knot loosely in the suture to prepare for cinching after the cannulation and fill the cannula with 40 degrees Celsius HBSS until the solution reaches the cannula needle tip. Insert the cannula into the portal vein five to 10 millimeters below the ligature and secure the cannula with the suture.
Start the infusion at a one to two milliliters per minute flow rate. If the cannula was correctly placed, the liver will begin to blanche. Cut the inferior vena cava and allow the excessive fluid within the liver to drain.
Slowly increase the flow rate to eight milliliters per minute until a full 50 milliliters of HBSS has been perfused through the liver over the next five minutes. Just before the HBSS runs out, change freshly prepared 40 degrees Celsius collagenase four solution to the perfusate beaker and use forceps to apply transient pressure to the inferior vena cava at five second intervals to cause the liver to swell and to help with tissue digestion and dissociation. As the digestion progresses, the liver will swell and become white.
When the remains depressed after gentle probing with a cotton tipped applicator, stop the pump and remove the cannula. Remove the gallbladder from the liver, being careful not to tear the gallbladder tissue, and use clean scissors and forceps to harvest the liver into a sterile 10 centimeter culture dish of PBS. After gentle washing, transfer the liver into a new 10 centimeter culture dish containing fresh collagenase four solution and use two clean forceps to tear the liver while gently shaking to dissociate the cells from the liver tissue.
When the entire organ has been fragmented, use a three milliliter syringe to triturate the tissue slurry until the undigested pieces of liver are shaken off. Pour the resulting cell solution through a 70 micron nylon mesh strainer into a 50 milliliter conical tube, washing the dish with HBSS to collect any remaining cells. Pool the wash with the single cell suspension and remove the hepatocytes by centrifugation.
Then transfer the supernatant to a new 50 milliliter tube. To remove any other cells, centrifuge the supernatant before transfer into a new 50 milliliter conical tube. Remove the dead cells with another centrifugation and transfer the supernatant into a round bottomed tube to remove any cell debris and aggregates.
Now transfer the supernatant into a polycarbonate ultra centrifuge tube and resuspend the pellet in about 20 milliliters of PBS for a second and final ultra centrifugation. Then resuspend the extracellular nanovesicle pellet in one milliliter of PBS for minus 80 degrees Celsius storage if the vesicles will not be immediately analyzed. From a single mouse liver, this method yields a tissue extracellular vesicle concentration that ranges from 1.74 to four times 10 to the twelfth with a mean of 3.46 times 10 to the twelfth particles per milliliter as determined by nanoparticle tracking analysis.
The mean size of the isolated liver tissue extracellular vesicles is 157.7 nanometers with a mode size of 144.5 nanometers and extracellular vesicles sizes ranging from 100 to 600 nanometers by nanoparticle tracking analysis. The extracellular vesicles that are isolated using this approach are suitable for downstream applications and the further characterization may include morphological assessments by electromicroscopy or analysis of their surface markers or content.
