The overall goal of this procedure is to isolate hepatic stellate cells from mouse liver. This is accomplished by first performing in C two perfusion of mouse livers with digestive enzymes. After perfusion, the livers are cut into small pieces to increase the efficiency of the in vitro digestion, which is performed next.
As a final step, the hepatic stellate cells are separated from other liver cell types by density gradient centrifugation. Ultimately, hepatic stellate cells are obtained and their purity can be assessed by light and immunofluorescence microscopy Demonstrating the procedure will be Patrick Meyer, a graduate student from a laboratory. In preparation for the NC two perfusion of mouse livers first warm the SC one buffer and enzyme perfusion solutions in a 37 degree Celsius water bath.
Next, set up the peristaltic pump to obtain a laminar flow of 6.5 milliliters per minute and equilibrate the silicone tube of the pump with SC one solution. Now confirm by a loss of reflexes that the anesthetized mouse is deeply sedated. Then fix the mouse in a supine position on a suitable base.
Use scissors and forceps to perform a longitudinal incision in the abdominal skin and expose the peritoneum. Carefully open the peritoneum and move the intestines out of the abdominal cavity to the left side of the animal in order to expose the portal vein. The most difficult aspect of this procedure is the zeto perfusion of the livers with digestive enzymes, which will be demonstrated.
Next, it's important to use a stereo microscope when inserting the cannula and to closely monitor the change in liver structures during the perfusion While working under a stereo microscope, insert the cannula of the infusion set into the portal vein. Start the perfusion of the liver with 30 milliliters of SC one solution. Open the Vanna Cava inferior immediately after initiating the flow.
Successful flushing of the liver is indicated by a loss of color of the liver tissue next, peruse the liver with 30 milliliters of Pronase E solution. The liver lobes will swell and the lobules will appear distinct through the capsule after profusion with pronase E solution perfused with 30 milliliters of collagenase P solution. When the profusion with collagenase P is complete, harvest the liver from the mouse by carefully dissecting it from the diaphragm and surrounding organs and storing it in 70 milliliters of SC two solution on ice.
The liver at this point has lost its shape and looks atonic and amorphous. The procedure for the digestion of mouse livers should be carried out under sterile conditions. In a laminar flow hood.
Use sharp scissors to cut the livers into pieces of approximately two by two by two cubic millimeters. Then combine the suspension of 70 milliliters of SC two and the liver pieces with 50 milliliters of protease E collagenase P solution. Add one milliliter of dna, one solution digest the livers for 20 minutes at 37 degrees Celsius while stirring.
After enzymatic digestion of the liver tissue, the stellate cells are separated from other hepatic cell types by density gradient centrifugation. To begin this procedure, filter the cell suspension through 70 micrometer cell strainers into six 50 milliliter falcon tubes and wash the cells with E two buffer centrifuge for 10 minutes at 600 Gs and four degrees Celsius. After centrifugation, carefully aspirate 40 milliliters of the supernatant from each tube.
Then add 150 microliters of DNA's, one solution to each tube and resuspend the cells. Pool the cell suspensions into four 50 milliliter Falcon tubes and wash with G-B-S-S-P centrifuge for 10 minutes at 600 Gs and four degrees Celsius. Carefully aspirate as much of the supernatant as possible without disturbing the pellet and add 150 microliters of DNAs one solution to each tube.
We suspend the cell pellets in 10 milliliters of G-B-S-S-B per tube. Pool the cells into two 50 milliliter falcon tubes and add G-B-S-S-B to a total volume of 36 milliliters per tube. Add 14 milliliters of den solution to each tube and mix well.
Next, transfer 10 milliliters of the cell suspension into one 12 milliliter gradient centrifugation tube resulting in a total of 10 tubes. Gently overlay the cell suspension with 1.5 milliliters of G-B-S-S-B per tube. Centrifuge the gradients for 15 minutes at 1500 Gs and four degrees Celsius without break.
Subsequently, hepatocytes will be pelleted at the bottom of the tube, whereas stellate cells are found in the interface as a white ring. Using a five milliliter pipette, carefully harvest the interface containing the stellate cells and transfer to a 50 milliliter tube. Wash the cells by adding G-B-S-S-B and centrifusion for 10 minutes at 600 Gs and four degrees Celsius.
Aspirate the supernatant and resuspend the cells in 20 milliliters of DMEM with supplements. After counting the cells, transfer them into tissue culture flasks at a concentration of two times 10 of the fourth cells per square centimeter incubate the cells at 37 degrees Celsius and 5%carbon dioxide. Approximately two hours after their preparation, the hepatic stellate cells should be adherent, change the media to wash off dead cells and cell debris.
Return the cells to the incubator representative results of preparing hepatic stellate cells from mouse livers using this protocol are shown here. Cells on day one and day three of in vitro culture show the characteristic morphology of hepatic stellate cells. They are astro lichen shape and exhibit storage of lipid vesicles.
At per nuclear sites, the purity of the isolated hepatic stellate cells can also be verified by the expression of glial fibrillary acidic protein or GFAP. These images show a representative immunofluorescent staining for GFAP shown in red in hepatic stellate cells, which have been cultured for three days following cell isolation. After watching this video, you should have a good understanding of how to isolate hepatic stellate cells from neuron liver for an adequate yield of pure stellate cells.
It is important to ensure efficient digestion upon liver perfusion. Finally, it is vital to determine the proper entity of isolated cells by assessing the morphology and phenotype of hepatic dal cells.
