The overall goals of this procedure are to assess endocytic activity of liver cells, followed by isolation of a pure population of viable liver, sinusoidal endothelial cells that can be cultured in plastic dishes. This is accomplished by cannulating and excising the liver of a live animal and flushing the liver with a salt buffered solution to clear out the blood. The cells of the liver are then exposed to labeled ligands for internalization.
The next step is to digest the liver with collagenase to produce a mixture of liver cells in suspension. Next differential centrifugation with perol gradients is used to separate secs from stellate cells, small hepatocytes, remnant blood cells and cell debris. The final step is to separate secs from cooper cells by adhesion on glass.
In the end, viable purified secs are obtained that can be assayed for ligand uptake and or be cultured for downstream applications. This method can help answer the key questions in the field of liver biology, such as the role of the liver endothelial cells and the clearance of macromolecules in blood, or the responses to different physiological conditions such as liver injury, fatty liver disease, ssis, fibrosis and aging. Unlike other liver cells immortalized, differentiated liver sinusoidal endothelial cell lines do not exist.
So these primary cells must be derived from the animal directly. The procedure that we're gonna show you today involves the purification of liver sinusoidal endothelial cells without the use of capital equipment like centrifugation and lution. The procedure that we'll show you involves the harvesting of cells on a short term culture basis, where we're not gonna use sterile technique throughout the whole procedure.
However, if you wanna use do long-term cultures, you would use sterile technique and use a biosafety cabinet. Visual demonstration of this procedure is, is critical because placement of the catheter along with tightness of the sutures, as well as taking the liver from the animal without cutting the liver, as well as disengaging the catheter is critical. Prepare for the experiment by weighing out the collagenase and setting it on ice.
Then warm and oxygenate the TBS and prepare the required tools. Next, select a 200 gram rat and deeply anesthetize it in an isof fluorine chamber. Confirm the anesthesia by wetting the ventral abdomen with 70%ethanol.
If there is no response, proceed with opening the abdominal cavity using scissors and forceps for best results. The following steps through blanching of the liver should only take a few minutes. Locate the portal vein and place two sutures beneath the vein, just above the mesenteric branch.
Now cannulate the vein with an 18 gauge catheter. The needle plugging the catheter should go several millimeters beyond the loop of the thread, trigger the needle's release and tighten the suture with two overhand knots, also known as either granny or square knots. Begin flushing the blood from the liver with TBS for no more than 10 minutes.
Sever one of the major arteries while the liver is flushing. Excise the liver by cutting away the GI tract and connective tissue. Place the excised liver over a funnel that allows recirculation of fluids but do not yet recirculate the fluids to 50 milliliters.
RPMI Media in a small beaker. Add to a final concentration, 0.05%BSA and 0.01 millicuries radio labeled heparin or other appropriately labeled ligand. For endocytosis, attach this be to the apparatus to allow for oxygenation.
Turn off the pump, switch the inlet tubing, and then turn on the pump at 20 milliliters per minute. As the labeled RPMI approaches the liver, turn off the pump and allow excess fluid in the funnel and tubing to drain. Place the drain tubing into the media beaker to allow recirculation of labeled media and then restart the pump.
Allow fluids to recirculate through the liver for no more than one hour. During this internalization step, be sure the temperature in the liver is stable by covering it and prepare the collagenase for digestion to terminate endocytosis of the ligand. The liver is re flushed with TBS for two minutes to clear the liver sinusoids of unbound ligand in preparation for the collagenase digestion.
During the two minutes of re flushing with TBS dissolve the pre weighed collagenase A in buffer two to a concentration of 0.45 micromolar and filter the solution immediately after flushing the liver, turn off the pump and switch the fluids to flush in the collagenase. A begin flushing with recirculation at the same flow. Rate for up to minutes after the collagenase.
A perfusion. Remove the catheter and transfer the liver to a dish. Contain buffer one using forceps, peel back the capsule of the liver and shake the cells out in the liquid.
As the liquid becomes opaque, pass the cells through a 100 micron mesh followed by a 30 micron mesh and then into a 50 milliliter conical cylinder. At room temperature, add more buffer one to the dish and mechanically dissociate more liver cells. Then transfer them to the filter.
Continue this process until no cells can be dislodged with reasonable shaking. Begin by washing the cells, centrifuge the cells at 150 times G until they're paled. Then transfer the S natin containing non parenchymal cells to a 50 milliliter conical on ice and resuspend the pellet in 40 milliliters.
Buffer one. Repeat the wash process twice in buffer three to make pellets that are at least 97%hepatocytes to obtain a population of S secs that is at least 95%pure centrifuge, the conical of super natin at 200 times G for 10 minutes at four degrees Celsius. Then discard the liquid and resus, suspend all of the cell pellets in five milliliters of R-P-M-I-B-S-A at four degrees Celsius.
Pull the cells into two tubes and fill the tubes with R-P-M-I-B-S-A to 35 milliliters. Now centrifuge the cells at 100 times G for three minutes and transfer the top 25 milliliters of supernatant containing the secs to a 50 milliliter conical on ice. Then resuspend the pellet in the remaining 10 milliliters of media and add 25 milliliters of cold R-P-M-I-B-S-A solution.
Repeat the wash step once to make two more collections of S natin with secs. Now centrifuge the supernatant collections at 200 times G for 10 minutes at four degrees Celsius during the centrifugation. Fill three conical with 20 milliliters of 25%per call in PBS.
Put them on ice and set up a gradient by underlaying. 15 milliliters of 50%per call in each tube. Resuspend the cells in a total of 30 milliliters, RPMI with BSA, combine them and then carefully overlay each gradient with 10 milliliters of the combined cell suspension.
Centrifuge the gradients at 900 times G for 20 minutes at four degrees Celsius because secs and cofer cells are at the 25 50%interface aspirate from the top down to near the 25 50%interface. This removes the stellate cells now collect the cells in the 25 50%interfaces and transfer them to a conical with cold RPMI to a final volume of about 40 milliliters. This dilutes out the per call centrifuge, the secs and fer cells at 350 times G for 10 minutes at four degrees Celsius.
Resuspend the pellet in about 10 milliliters of pre-warned unmodified. RPMI transfer the suspension to a Petri dish or crystallizing dish and incubate the dish for 15 minutes at 37 degrees Celsius. After the incubation, gently agitate the dish and collect the secs in the supernatant.
The cooper cells will have adhered to the glass and the secs can be cultured on fibronectin coated plates with modified RPMI media cell lysates from cells that adhered to the dish and those that remained in suspension were separated by SDS page and probed with anti CD 1 63 antibody Cooper cells, which contained the CD 1 63 marker were separated from CCS by attachment to glass cells that did not adhere to the glass were plated on fibronectin coated plastic. Six well dishes and incubated overnight. With RPMI supplemented with 0.1%BSA and pen strep.
Under phase contrast like microscopy secs exhibit a very flat cytoplasmic morphology reminiscent of their in vivo architecture. A monoclonal antibody against both isoforms of S Stalin two receptor. A marker of SEC differentiation was used to probe hepatocyte, lysate and CCS collected using the outline protocol.
As expected, the secs were positive for stepin.Two. In prior experiments injected heparin labeled with a biotin tag on the carboxylate group of the heparin. Disaccharide was conjugated to iodinated streptavidin during the 30 minutes post-injection.
The liver was the primary organ that cleared the labeled heparin using the procedures. In this video, we show that the vast majority of labeled heparin is internalized by secs. In contrast to hepatocytes, these internalization events occurred within an intact liver in which cells are in their native state.
So after watching this video, you should have a good understanding of how to purify liver sinusoidal endothelial cells from a live animal. And it's important to use these cells within 24 to 48 hours. As liver sinusoidal endothelial cells lose their instinctive characteristics in a very short time in culture.
