The overall goal of this procedure is to perform a successful Mouse Liver Perfusion to facilitate the purification of specific liver cell populations of interest. This method can help answer key questions in the hepatology field about how to assist the functions of specific liver cell populations within in vitro or in vivo assays. The main advantage of this technique is the high amount of hepatocytes in sinusoidal endothelial cells that can be obtained from each liver.
Visual demonstration of this method is critical as it is essential to see the color and shape of an optimally digested liver to preserve cell viability. To place the portal vein catheter, first use the back of a forceps to move the intestines to the right side of the mouse abdominal cavity, exposing the portal vein, and use curved forceps to slide a 20 centimeter piece of polyester sewing thread under the portal vein between the liver and the superior pancreato duodenal vein. Use the forceps to carefully pull the thread to the other side of the animal, so that it is centered under the portal vein.
And tie a loose overhead knot around the portal vein. Place the curved forceps under the vein, and gently pull all tissue material toward the tail to straighten out the vein. Next, place the bevel of a catheter needle facing up and parallel with the lower part of the portal vein near the forceps, and gently puncture the vein with the needle.
When the bevel is in the lumen of the vein, retract the spring loaded needle, and continue to push the palmer catheter through the vein, until the bevel is near the venus branched area. After securing the knot around the catheter, immediately set the peristaltic pump at four millimeters per minute and connect the male end of the tubing to the female end of the catheter, at which point you should start to see the liver branch. Cut the aorta abdominalis for drainage.
Use masking tape to immobilize the tubing onto the under pad. Use the scissors to cut the abdominal skin vertically, to allow drainage of blood and perfusion liquid. Then squeeze the effluent blood vessel a few times with the straight forceps to inflate the liver, ensuring that all of the blood has drained out, and perfuse the liver with PBS until the liver tissue blanches.
To harvest the hepatocytes, first change the outflow tubing from the one liter flask of PBS to a 125 milliliter flask, containing buffer two, supplemented with collagenase-4. When the collagenase reaches the liver, briefly but tightly squeeze the effluent blood vessel to build up pressure and allow the digestion buffer to fill all the lobes of the liver. Release the vessel before the buffer bursts through the connective tissue surrounding the liver.
And allow the buffer to perfuse through the liver until the entire volume has flowed through the tissue. Next, place a crystallizing dish containing 10 milliliters of buffer one next to the mouse, and use straight forceps and scissors to transfer the liver to the dish. Transfer the dish to a sterile tissue culture hood, and using sterile technique, grab the liver to peel back the capsule.
Gently shake the cells from the liver tissue, and pour the resulting cell solution through a 100-micron filter into a 50 milliliter conical tube. Then rinse the liver remnants from the filter with more buffer one. When the liver appears to be devoid of cells, strain the pulled filtrate through a 40-micron filter into a second 50-milliliter tube and collect the cells by centrifugation.
Pour off the non-parenchymal cell and dead hepatocyte containing supernatant into a new 50-milliliter tube on ice in one motion, to preserve the pellet and re-suspend the pellet in 40 milliliters of fresh buffer one for the next centrifugation. To isolate the sinusoidal endothelial cells, centrifuge the supernatants containing non-parenchymal cells. Pull the cell suspensions into a single new 50-milliliter conical tube, and bring the total volume up to 35 milliliters.
Collect the pulled cells by centrifugation, and use a 25 milliliter pipette to carefully transfer the top 25 milliliters of the non-parenchymal cell containing supernatant into a new 50-milliliter conical tube on ice. Add 25 milliliters of fresh medium to the tube, and re-suspend the pellet for a second centrifugation and supernatant removal. Then centrifuge the supernatant and add 15 milliliters of a 50%Percoll solution to a new 50-milliliter tube.
Using a pipetter set to the lowest ejection speed, overlay 20 milliliters of 25%Percoll onto the 50%Percoll solution, and re-suspend the non-parenchymal cells in 10 milliliters of four degree Celsius medium. Carefully layer the cells onto the Percoll solution. And separate the cells by density gradient centrifugation.
Next, aspirate the first 30 milliliters of solution, and use a plastic five-milliliter transfer pipette to collect the sinusoidal endothelial and Kupffer cells located at the 25 to 50%density gradient solution interphase into a new 50-milliliter conical tube. Wash the cells with 50 milliliters of medium and re-suspend the pellet while rinsing the sides of the tube bottom in 12 milliliters of warm medium. To separate the sinusoidal endothelial cells from the Kupffer cells, transfer the cell suspension into a polystyrene Petri dish in a humidified tissue culture incubator for eight minutes.
At the end of the incubation, use a 25-milliliter pipette to aspirate the supernatant and use the supernatant to rinse the dish with the pipetter set at the lowest speed to collect the remaining sinusoidal endothelial cells. Then transfer the sinusoidal endothelial cells into a new 50-milliliter tube. Then collect the cells by centrifugation for their re-suspension and plating in collagen coated cell culture dishes.
Scanning electron microscopy of a mouse liver after PBS infixitive perfusion in a manner similar to as just demonstrated, reveals a clear view of the liver micro-anatomy and the microvilli between the hepatocytes. Hepatocytes harvested during the low speed spins early in the procedure, demonstrate a high purity after four washes in bovine serum albumin containing buffers. Separation of the sinusoidal endothelial cells by selective adhesion on a collagen-coated polystyrene Petri dish results in an 83 to 90%purity that largely depends on the overall effectiveness of the collagenase digestion.
Quantitative measurements using light microscopy from a representative separation procedure, as just demonstrated, reveals typical isolation results of a nearly 100%pure hepatocyte population. And a just over 89%pure sinusoidal endothelial cell population. After watching this video, you should have a good understanding of how to cathetize a mouse portal vein and of how to purify both hepatocytes and sinusoidal endothelial cells from the mouse liver.
This procedure may also be used to purify satellite cells from the non-parenchymal fraction after hepatocyte isolation using different density gradients and centrifugation speeds.
