The overall goal of this protocol is to present an optimized method for hepatocyte isolation in mice after partial hepatectomy without the need for density gradient centrifugation. We usually see a large percentage of lipid-laden hepatocytes during early regeneration. Those steatotic hepatocytes usually lost upon density gradient centrifugation due to the low density are retained with this protocol.
To prepare the perfusion equipment, connect a 26 gauge IUI cannula to the outlet end of the tubing using a Luer lock connector. Then flush the tubing and insert the inlet end of the tube into the pre-warmed perfusion buffer tube in the water bath. Prime it with warm perfusion buffer at a pump speed of three milliliters per minute.
Before proceeding with the surgery, ensure that the mouse is adequately anesthetized. Then clean the abdomen with a surgical scrub solution and 70%ethanol. Then cut the sutures to reopen the midline incision previously made for the partial hepatectomy and gently pull the wound edges apart.
If the hepatectomy is older than 24 to 48 hours, remove the suture and cut the skin with scissors or a scalpel. Use a retractor or simple clips to keep the abdomen open. The abdominal cavity should be exposed as much as possible to optimize access and visualization.
Fix a 5-0 polypropylene suture to the sternum. Pull it cranially and fix it in this position. Move the intestines to the right using sterile cotton swabs to reveal the portal vein and the vena cava.
Use a wet cloth to retain the intestines. Place a heavy object of approximately two centimeter height such as a metal weight ring adjacent to the mouse's hind legs. Then place the tubing with the connected 26 gauge IUI cannula on the object and position the needle carefully on top of the vena cava.
Adjust the length of the tubing as required. Transfer the collagenase stock solution into the pre-warmed digestion buffer tube. Prepare 10 to 20 milliliters of digestion buffer per animal.
Turn on the pump after adjusting the pump speed to three milliliters per minute. Discard the first two to three milliliters of the pre-warmed perfusion buffer once the buffer reaches the needle. To perform cannulation of the inferior vena cava, use a sterile cotton swab to gently pull the vena cava caudally below the puncture so that the provided tension facilitates the insertion of the cannula into the vein.
Insert the 26 gauge IUI cannula at a shallow angle into the inferior vena cava below the kidney while the buffer runs through the needle. Ensure that the needle bevel points upward. Look for blood in the flash chamber of the catheter when the needle enters the lumen.
Advance the needle an additional two to three millimeters to ensure that the catheter's tip enters the vein. Slide the plastic catheter over the needle and into the vena cava another five millimeters. Remove the needle slowly and very carefully.
After two to three seconds, look for white spots forming in the liver or swelling of the portal vein, which indicates that the perfusion buffer is flowing through the liver and entering the liver lobules from the central vein. Wait for the portal vein to visibly swell within one to two seconds of white spots forming on the liver surface. Cut the portal vein with scissors as distally as possible from the liver hilus.
Increase the flow rate to four to seven milliliters per minute depending on the animal's weight, liver size, and the extent of the initial hepatectomy. Clamp the portal vein with tweezers or a vascular clamp for seven to 10 seconds. Make sure no fluid is passing through.
Perform a second clamp after approximately 30 seconds and ensure that the liver swells and relaxes. Continue flushing the animal for three to four minutes and observe the clear buffer flowing out of the portal vein. Clamp the portal vein for three to four seconds before the digestion buffer reaches the liver.
Ensure that the liver relaxes upon release of the clamp and that the perfusion fluid remains clear. Once the digestion buffer reaches the liver, clamp the portal vein once again. The liver should not relax after the release of the clamping.
Digest for approximately four minutes at a flow rate of five milliliters per minute. As digestion progresses, look for signs of the liver beginning to swell and small transparent sections on the surface of the liver. Observe that the liver takes on the texture of a wet piece of cloth and appears almost soggy.
Probe the consistency by carefully touching it with a damp, sterile cotton swab. Continue with the perfusion until a marked difference in the surface texture of the liver can be observed. Observe that the liver assumes a very light color and a bubbly appearance indicating the Glisson's capsule separating from the parenchyma.
Stop digestion as soon as the liver has acquired these properties. Remove the needle before the air gets into the liver. Grasp the central connective tissue between the lobes using forceps and slightly lift it upward using it as an anchor point.
Cut all the connections of the liver to other organs and remove the gallbladder. Remove the liver gently from the abdominal cavity. Be careful as it will be flimsy and fragile at this stage.
Place the liver in the ice cold preservation buffer. Transfer the liver to a 10 centimeter Petri dish and add 10 milliliters of ice cold Williams'Medium E.Rupture the Glisson's capsule with fine tip tweezers in a few locations along the liver surface. Grasp a central part with two pairs of tweezers.
Slowly pull them apart, allowing the capsule to tear without damaging the hepatocytes and release the cells by gently shaking the capsule. Filter five milliliters of liver pulp through a 100 micrometer cell strainer into a 50 milliliter tube. Rinse the filter with 10 milliliters of fresh ice cold medium and filter the remaining five milliliters of pulp through the cell strainer.
Spin the extract at 50 G for five minutes at four degrees Celsius. Aspirate most of the supernatant, leaving one milliliter to resuspend the cells by gently swirling the tube. Add 40 milliliters of cold Williams'E Medium and repeat the step three times.
Proceed with the isolation of hepatocytes as described in the text manuscript. After 70%hepatectomy in mice, the final hepatocyte yield was approximately 10 to 15 x 10 to the 6th with 78%mean final viability. While after extended 86%hepatectomy, the hepatocyte yield was four to nine x 10 to the 6th with 65%mean viability.
After partial hepatectomies, hepatocytes show an increased cell size compared to normal livers corresponding to increased lipid content. Increased lipid content was observed in murine hepatocytes 24 hours after partial hepatectomy, seen as an increase in granularity or directly observed by the presence of lipid vesicles inside enlarged hepatocytes. While using the classic density gradient purification method, large fatty hepatocytes were lost in the supernatant and only smaller lean hepatocytes are collected in the cell pellet.
With the improved protocol, the lipid-filled hepatocytes were not lost and all hepatocytes were pelleted. The repetitive clamping facilitates the flushing process. With that, the liver is optimally cleared from remaining blood components that could inhibit the digestion enzymes.
