The overall goal of this procedure is to isolate a relatively pure population of rat primary portal fibroblasts without the need for passage in culture. This is accomplished by first perfusing the rat liver in situ with a solution of collagenase. The second step is to separate the biliary tree from the liver parenchyma.
Next, the biliary tree is further digested with enzymatic solutions. The final step is to pass the digested biliary tree through a fine mesh in order to isolate portal fibroblasts by size selection, ultimately immunofluorescence microscopy for elastin and alpha smooth muscle. Actin can be used to show the relatively pure population of portal fibroblasts recovered from the rat liver.
The may advent of this technique over existing methods like the outgrowth technique, is that other methods require portal fibroblasts to be pathogen culture before they can be studied With this method, a relatively pure population of primary cells can be directly recovered from the liver without the need for pathogen cell culture. Before beginning the procedure, prime the perfusion system with warm HBSS minus calcium and magnesium. Then when sedation has been confirmed by toe pinch, placed the adult male anesthetized rat in a supine position on a plastic tray and fix the limbs to the tray with tape.
After cleaning the abdomen with 70%ethanol, make a small incision in the skin at the abdominal midline with sterile scissors. Make a large U-shaped cut on the abdomen to dissect the skin away from the facia, and then cut the abdominal muscle tissue in the same way to expose the peritoneal cavity. Next, use two cotton swabs to gently move the abdominal viscera to the right side, exposing the portal vein.
Then pass two sterile 2.0 silk sutures underneath the portal vein and tie them loosely. Now cannulate the portal vein with a 16 to 18 gauge IV catheter and secure the catheter in place by tightening the sutures. Inject diluted heparin through the IV catheter.
Note that the liver will blanch. When properly perfused. Connect the IV catheter to the perfusion system and perfuse the liver with HBSS minus calcium and magnesium.
Transect the inferior vena caver below the liver to allow drainage of blood and PERFUSE eight, aspirating the blood as it pulls in the abdominal cavity. Continue perfusion at a rate of 20 milliliters per minute For 10 minutes after the HBSS perfusion has been completed, change the perfusion fluid to a 0.3%collagenase solution. Cover the liver with the previously dissected abdominal flap to keep it moist during the collagenase perfusion.
Maintain the intraabdominal temperature at approximately 37 degrees Celsius by placing a Petri dish cover over the abdominal flap and positioning a heat lamp over the area. Begin by removing the perfused liver from the animal and placing it in a sterile tissue culture dish filled with cold leibovitz's. L 15 media working in the tissue culture hood.
Peel off the liver capsule and gently tease the liver parenchyma apart with forceps. Move the liver through several dishes filled with levy's L 15 media while continuing to tease away the parenchyma until the biliary tree is completely isolated. Note that the liver parenchyma is tan colored while the biliary tree is white.
Place the biliary tree into a 50 milliliter conical tube filled with 10 milliliters of penicillin streptomycin, and then place the tube on ice for 15 minutes. Now, place the biliary tree into a clean 50 milliliter tube and mince it with sterile scissors. Then add about five milliliters of enzyme solution number one to the tube, and continue to mince the biliary tree until it reaches the consistency of a slurry.
Pour the slurry into a sterile glass bottle. Wash the tube with the remaining solution, number one, and then pour the solution into the glass bottle. Incubate the tissue slurry at 37 degrees Celsius with shaking at 100 RPM for 30 minutes, and then filter the slurry into a beaker covered with a single layer of 30 micron.
Pour nylon mesh. Next, pour about 25 milliliters of solution number two into a sterile 15 centimeter Petri dish. And then carefully remove the mesh from the beaker by holding it at the edges.
Now invert the mesh and dip it into the solution in the Petri dish. To remove any remaining tissue, transfer the solution in the Petri dish into a clean glass bottle. Then rinse the Petri dish with the remaining 25 milliliters of solution number two, and transfer the rinse to the same glass bottle.
Incubate the tissue solution in the glass bottle of 37 degrees Celsius with shaking at 100 RPM for 30 minutes. In the meantime, pour the filtrate from the beaker into a 50 milliliter tube. After centrifuging the filtrate at 460 times G for five minutes at room temperature, aspirate the media and resuspend the pellet in 25 milliliters of solution Number three.
Now filter the tissue solution from the glass bottle into a second clean beaker covered with a new 30 micron mesh. And then after centrifuging the tissue solution, re suspend this pellet with 25 milliliters of solution number three, as well consolidate the two tissue slurry fractions, and then after centrifuging the combined solution, re suspend the pellet in 10 milliliters of portal fibroblasts culture media. Finally, after counting the cells for viability plate, the cells at about 0.5 to five times 10 to the six cells per 10 centimeter tissue culture plate.
Here, representative primary portal fibroblasts on days one, three, and seven after isolation are shown. Note that the cells are elongated with a typical fibroblast morphology. Cells can be stained with anti elastin antibody shown here in green to confirm purity.
Also, note how portal fibroblasts undergo myofibroblastic differentiation in culture demonstrated in this figure by the red immuno staining for alpha smooth muscle actin. After watching this video, you should have a good understanding of how to isolate primary portal fibroblasts from a red liver by inci your liver perfusion.
