The overall goal of this procedure is to study mechanisms of leukocyte recruitment across human liver endothelial cells in the presence of physiological levels of sheer stress. This is accomplished by first preparing a confluent monolayer of human hepatic sinusoidal endothelial cells in a micro slide, which is subsequently stimulated with inflammatory cytokines. The second step is to isolate lymphocytes from peripheral blood.
Next, the micro slide is connected to a flow assay system and perfused with lymphocytes in the presence of physiologically relevant sheer stress. The final step is to visualize lymphocyte interactions under conditions of flow with a phase contrast microscope. Ultimately, the offline analysis of phase contrast images acquired during the flow assay are used to compare the effect of various inhibitors on lymphocyte recruitment across liver endothelial cells.
This method can help answer key questions in the hepatology field, such as which adhesion protein and chemokine receptor combinations are important for the recruitment of diverse immune cell populations across the sinusoidal endothelium and into the hepatic parenchyma. The implications of this technique extend to the therapy of chronic inflammatory liver disease, the majority of which are driven by lymphocyte recruitment into liver tissue. Understanding the molecular mechanisms could lead to new anti-inflammatory therapies.
Though this method can provide insight into the recruitment of inflammatory cells to the human liver. It can also be applied to other cell types such as endothelial cells, isolated from umbilical vein or other vascular beds. To begin, prepare a working solution of 200 micrograms per milliliter rat tail collagen, type one by diluting it from a stock solution into sterile PBS.
Then inject 30 microliters of the diluted collagen into each channel of a six channel micro slide and incubate for two hours at 37 degrees Celsius. Next, prepare complete media by supplementing human endothelial basal media with L glutamine penicillin and streptomycin heat inactivated ab, human serum vascular endothelial growth factor and hepatocyte growth factor. Then using trypsin EDTA harvest cultured human hepatic sinusoidal endothelial cells from a confluent T 75 flask, pellet the cells wash them in PBS and then re suspend them at three times 10 of the sixth cells per milliliter in complete media.
Next, wash the micro slide three times with PBS and then add 30 microliters of the cell suspension into each channel of the collagen coated micro slide. Leave cells to adhere for one hour in a humidified incubator at 37 degrees Celsius with a 5%CO2 atmosphere. After the cells have adhered, fill the ports on either side of each channel with complete medium and place them back in the incubator for 24 hours.
After 24 hours, use an inverted phase contrast microscope to assess that the cell growth has reached 80%co fluency. Next, prepare the cytokines for cell stimulation by adding 10 nanograms per milliliter TNF alpha and 10 nanograms per milliliter, interferon gamma to one milliliter of complete media. Then 24 hours prior to performing the adhesion assay replaced the cell growth media with 0.1 milliliter of the cytokine supplemented media to stimulate the cells, purify the mononuclear fraction from whole blood by density gradient centrifugation over an appropriate cell separation, centrifugation media such as lympho light for 25 minutes at 800 times gravity.
Then transfer the mononuclear fraction to a new tube and top it off with PBS containing 0.1%volume per volume BSA centrifuge the tube for 10 minutes at 125 times gravity to deplete the solution of platelets. Next, wash the pellet with PBS containing 0.1%volume per volume BSA, and then centrifuge for 10 minutes at 800 times gravity. Resus suspend the pellet in 10 milliliters of RPMI containing 0.1%volume per volume BSA place the cells in a tissue culture treated T 75 flask and incubate for one hour to allow for adherence of the monocytes in the fraction.
Next, carefully remove the lymphocyte enriched supernatant and pellet the cells at 800 times gravity for 10 minutes. Then pretreat the isolated lymphocytes by resus, suspending them in RPMI solution containing 0.1%volume per volume BSA and either 200 nanograms per milliliter of pertussis toxin specific function blocking antibodies or small molecule inhibitors of chemokine receptors. Incubate lymphocytes in the inhibitor solution for 30 minutes at 37 degrees Celsius, and then pellet the cells and wash them in PBS with 0.1%volume per volume BSA.
Next pellet the cells again and then resuspend them in flow medium at a concentration of one times 10 to the sixth cells per milliliter. Prewarm a thermostatically controlled transparent environmental chamber to 37 degrees Celsius. The chamber should contain ports for the insertion of silicone tubing and an electronic supply for a solenoid valve.
Fill a 50 milliliter glass syringe with a lure lock with 10 milliliters of sterile distilled water. Then attach a length of 25 centimeter silicone tubing to the syringe port and insert the syringe into a syringe pump. Adjust the pump's withdrawal rate according to the micro slide manufacturer's instructions to maintain a sheer stress of 0.05 pascals or 0.5 dines per square centimeter.
Discard the plungers from two five milliliter syringes and attach the barrels to the two inflow ports of an electronic solenoid valve using one centimeter of silicone tubing. Then attach 12 centimeters of silicone tubing to the outflow valve. Insert cell-free wash buffer into both syringe valves and flush the electronic solenoid valve.
Make sure buffer is flowing from one barrel through the valve and into the outflow silicone tubing. Next, turn the valve switch to the other barrel and ensure buffer is still flowing. Remove all bubbles from the system.
Then replace the wash buffer in one of the barrels with the lymphocyte suspension containing one times 10 to the sixth cells per milliliter. Connect the silicone tubing from the syringe pump to one port of a chosen micro slide channel using a micros slide adapter. Then connect the silicon tubing from the outflow valve to the opposite port on the same micro slide channel also with a micro slide adapter.
Next, secure the micro slide on the microscope stage with clips or tape to prevent movement. Set the microscope to the 10 x objective with the appropriate phase setting prior to starting the perfusion. Ensure that a camera is properly attached to the microscope and ready to relay images to a monitor for recording.
Ensure that the endothelial monolayer is in focus using a syringe pump perfuse the endothelial layer with cell-free wash buffer for two minutes by commencing withdrawal of the syringe pump to remove any debris or unbound blocking antibody. Then switch the valve to allow a five minute bolus of lymphocyte solution at a constant wall. Sheer stress 0.05 pascals during the last two minutes of the lymphocyte bolus.
Record 10 random fields along the length of the micros slide for 10 seconds. Each movements between recordings should be made against the direction of flow. To avoid recording this same rolling cell twice After the lymphocyte bolus, return the valve to its start position to follow with a five minute bolus of cell-free wash buffer.
Record a second pass of 10 randomly selected fields for five seconds each. During the last two minutes of this bolus, the rolling motion of the lymphocytes can be easily visualized using this flow assay technique. Rolling cells are identified by the reduced velocity over the endothelial surface as compared to flowing cells.
In this flow assay. Fewer than 10%of adherent lymphocytes persistently rolled over stimulated endothelial cells, which confirms that the flow assay reflects the environment of the hepatic sinusoids. The recording from the wash buffer bolus is used to evaluate total lymphocyte adherence.
Firmly adherence cells are defined as cells that are stationary or shape changed with slow crawling behavior. Shown here are representative fields from a control slide and a slide pretreated with intercellular adhesion molecule, one blocking antibody. The extent of lymphocyte adherence in each condition is expressed as adherence cells per square millimeter per million cells perfused.
Following this procedure, other methods can be performed such as confocal microscopy to answer additional questions such as the exact root of trans endothelial migration, or cytoskeletal changes that take place during recruitment under sheer stress After its development. This technique paved the way for researchers studying inflammation in the liver or other organs to explore how organ specific endothelial cell populations govern the process of leukocyte recruitment. After watching this video, you should have a good understanding of how to evaluate the recruitment of lymphocytes across human liver endothelial cells, and enumerate immune cells at different stages of the adhesion cascade.
