This method can help to answer key questions in the field of hepatology and toxicology and offer us the opportunity for development of new in vitro co-culture and tissue engineered liver models. The advantage of this new technique is to isolate all liver cell populations from the same donor and the same small piece of liver tissue. To begin this procedure, weigh the freshly dissected liver tissue under stile conditions and place the liver tissue sample in the laminar airflow hood.
Then clean the blood away from the surface of the tissue sample. Next, flush the cannulas using 1X perfusion solution one. Use the tissue glue to fix the olives of the cannulas in the larger blood vessels.
Then, test the perfusion and check for leakages of the cannulas. Subsequently, close all blood vessels leaking clear 1X perfusion solution one with tissue glue. After that, place the cannulated liver tissue sample into the Buchner funnel.
Then, set the flow rate of the peristaltic pump between 7.5 and 14.6 milliters per minute, depending on the number of cannulas used and the resistance of the liver tissue. Perfuse the tissue until the whole blood is flushed out for at least 20 minutes and for a maximum of 30 minutes. In some cases, it may be necessary to clamp one of the cannulas with plastic clamps or to increase the inner pressure of an area by pressing softly against the liver capsule with a spatula in order to optimize the perfusion.
A complete color change from brownish red to light brown or light yellow indicates a good perfusion. Afterward, change the perfusion fluid to digestion solution containing collagenase P.Then, rearrange the setup for the digestion step. Subsequently, perform a circular flow of digestion solution for up to 15 minutes.
Periodically stop the perfusion and check the degree of digestion. It is essential that the perfusion with digestion solution is stopped immediately when the liver tissue sample is digested sufficiently. Take the liver out of the device when ready and put it in a glass dish.
Rinse the outside of the tissue sample with ice cold Stop Solution. Remove the cannulas from the liver tissue sample. Then, use a scalpel to open the liver tissue sample by incising the middle of the area where the cannulas were attached and be sure the Glisson's capsule stays intact.
Rinse the inside of the tissue sample and then cover the whole tissue sample with Stop Solution. Next, shake the tissue gently in order to release the cells out of it. Then, collect the cell suspension and filter it through a gauze funnel into 10 50-milliliter plastic tubes.
Add more Stop Solution to the liver tissue sample until a final volume of 500 milliliters is consumed. Afterward, centrifuge the cell suspension, collect the supernatant for NPC isolation later, and pool the cell pellets into 250-milliliters plastic tubes. Subsequently, wash the cell pellets with PBS.
For that, centrifuge the cell suspension. Collect the supernatant, pool the pellets, and resuspend the pellet in hepatocyte incubation medium. Then, determine the cell number and viability in the resulting cell suspension using Trypan blue staining.
To isolate the non-parenchymal liver cells, centrifuge the collected supernatant to eliminate the remaining erythrocytes and hepatocytes. Collect the supernatants and centrifuge them twice to gain the cell pellets for the sedimentation of HCS, LEC, partly KC, and the remaining KC.Next, re-suspend the pellets in HBSS. Then, prepare a 25%and a 50%density gradient by mixing the density-gradient solution and PBS for density-gradient centrifugation.
Place the 25%density gradient solution carefully on top of the 50%density gradient solution layer. Subsequently, put the NPC suspension carefully and slowly on top of the 25%density gradient solution layer to achieve a clear separation of both layers. Centrifuge the cell suspension on the density gradient without break.
The NPC are located in the interphase between the 25%and 50%density gradient layers. Aspirate the dead cells and cell debris from the uppermost layer and collect the interphase carefully. After dual centrifugation, perform a cell count for the KC and the MPC fraction.
Then, centrifuge the NPC fraction with the previously described dual centrifugation step and resuspend the NPC in Kupffer Cell seeding medium. Seed the KC-containing fraction on the plastic cell culture vessels at a density of five times 10 to the 5th KC per square centimeter. Incubate the KC cultures for 20 minutes in a humidified incubator at 37 degrees Celsius 5%carbon dioxide.
Primary KC should adhere on the cell culture plastics within a short period of time. Then, collect the supernatant containing the non-adhered NPC consisting mainly of LEC and HSC. To separate LEC from HSC, start with a centrifugation of the supernatant.
Then, wash the pellet with PBS. After the second centrifugation, re-suspend the cells in stellate cell endothelial cell separation medium and perform a cell count for all remaining cells. Next, re-suspend 10 million cells in one milliliter of stellate cell endothelial cell separation medium.
Then, add 20 microliters of blocking solution from the MAX kit and 20 microliters of the CD31 microbeads for immuno-labeling and incubate the resulting suspension for 15 minutes at four degrees Celsius temperature. Subsequently, separate LEC from HSC as described in the manufacturer's protocol for the magnetically activated cell sorting system max and collect the HSC fraction. Elute the magnetically retained CD31 positive LEC and suspend them in stellate cell endothelial cell culture medium.
Here, these images show the different isolate liver cell populations directly after the isolation process in phase contrast microscopy view. This set of images presents the isolated and cultivated cells after 24 hours of cultivation. The immunofluorescence-based characterization of different cell fractions is shown here.
PHH showed positive signals for the hepatocyte marker CK18 24 hours after isolation, KC were positive for the marker CD68 24 hours after isolation, LEC showed positive signals for vimentin 72 hours after isolation, and HSC were positive for GFAP 72 hours after isolation. While attempting this procedure, it's important to remember that a successful isolation depends on optimal digestion of the liver tissue sample. After watching this video, you should have a good understanding of how to control the digestion and how to perform the density gradient separation, which are the critical steps of this procedure.
After its development, this technique paved the way for researchers in the field of medical research to explore hepatic functions and diseases in newly-created in vitro cell cultures and tissue engineered liver models.
