The overall goal of this procedure is to isolate high quality human endothelial cells and to use them to examine the mechanisms regulating neutrophil trans migration, underflow conditions. This is accomplished by first isolating and plating human umbilical vein endothelial cells. The second step is to split the cells into IBD chambers.
Next freshly isolated human neutrophils are profused across activated endothelial cells, resulting in neutrophil rolling firm adhesion and transmigration. The final step is to quantify transmigration using video microscopy. Ultimately fluorescent and brightfield microscopy can be used to evaluate neutrophil transmigration across activated endothelial cells.
Endothelial cell isolation is easy to describe, but difficult to learn in part because key steps require visualization of the umbilical cord. For this reason, visual demonstration of this method is critical. This method will help us to understand key questions in the inflammation field, such as how leukocyte recruitment happens under flow condition, and how endothelial cell behaves under flow condition will be better.Understood.
Though this method can provide insight into neutrophil recruitment during inflammation. It can also be applied to leukocyte rare population such as natural killer cells and eosinophil. Demonstrating this procedure along with Ritu Sharma is Hong Jang, a technician in my laboratory At least one hour before the procedure preco, and then incubate a T 75 flask with gelatin at 37 degrees Celsius.
Then begin the endothelial cell isolation procedure in the laminar flow hood by using a pair of scissors to cut an umbilical cord from a placenta. After changing into sterile gloves, closely examine the cord and discard any portions that contain blood clots or are damage from cord clamping during delivery. Then identify the two arteries and the single vein present in the cord.
Next, gently insert a cannula with a two-way stop cock attached to it into one end of the vein, and then place a clamp around the cannula to hold it firmly in position. One of the trickiest parts of the procedure is perfusing the umbilical cord. To ensure this is done successfully, perfuse the cord with excess buffer and watch the flow through until it runs clear.
Perfuse the vein with cord buffer until the flow through is clear and then clamp the end of the cord. Occasionally during perfusion, a small hole is revealed because physicians have removed cord blood from the umbilical cord. In these situations, hemostats can be used to block the hole and perfusion can continue while isolating endothelial cells.
It's critically important to precisely time the collagenase treatment. Too little time and not enough endothelial cells will be harvested too long and contaminating smooth muscle cells or damage to the endothelial cells can occur. Then perfuse the vein with freshly prepared warm collagenase.
Close the stop cock and incubate the cord in a beaker containing warm cord buffer. After 10 minutes, remove and then gently massage the cord to loosen the endothelial cells from the lumen of the vein. Drain the solution into a tube containing five milliliters of endothelial cell media or ECM flushing the cord with cord buffer twice to remove any remaining cells.
Now spin down the cells for 10 minutes at three 50 times G.At room temperature, remove the supernatant and then resuspend the pellet. In 10 milliliters of endothelial cell media, transfer the cells to the gelatin coated T 75 flask and culture, the endothelial cells overnight at 37 degrees Celsius and 5%CO2. The next day, gently shake the flask to dislodge any red blood cells.
Then remove the supernatant and wash the endothelial cell culture with warm HBSS. After removing the HBSS feed the cells with 10 milliliters of warm ECM. Then examine the cells under the microscope to ensure that the red blood cells were removed and to evaluate the degree of confluence and morphology of the endothelial cells.
Continue to change the media every three days until the cells reach co fluency. At that point, split the cells using a trypsin and EDTA solution. Then harvest the detached cells and resuspend them in ECM.
Finally, coat each well of an IBD chamber with gelatin. Remove the excess gelatin and then add 1.5 times 10 to the six per milliliter of the endothelial cell suspension into each well of the chamber. Begin by inducing the upregulation of adhesion and activation molecule cell surface expression by incubating the endothelial cells with an appropriate stimulant while the endothelial cells are being activated.
Isolate neutrophils from the peripheral blood of healthy human volunteers by density centrifugation as previously published, and then suspend the cells at a concentration of one times 10 to the six per milliliter in HBSS plus human albumin. After setting up the microscope, attach the IBD chamber containing the stimulated endothelial cells to the tubing of the microscope. Select the 10 x phase contrast objective, and then set the syringe pump to withdraw and begin the flow of HBSS at the desired shear stress.
Briefly stop the flow by turning the three-way stop cock on the outlet side to the off position and switching the inlet line from HBSS to isolated neutrophils. Start the flow again by turning the stop cock back to the on position. Then begin recording using a CCD camera connected to A DVD recorder.
Start the timer. When neutrophils enter the chamber after four minutes, switch back to HBSS to prevent the attachment of new neutrophils. If data for total interactions rolling and firm adhesion are desired, use a 10 x phase contrast.
Objective to image six random fields for 10 seconds each between minutes four and five. Finally switch to a 40 x objective and record a single field of view at the time of interest. After the neutrophil perfusion at 10 minutes, collect between five and 10 random fields of view.
Unstimulated endothelial cells do not support neutrophil recruitment. Therefore, the following figures show neutrophils interacting with TNF alpha stimulated endothelial cells. Note the adherent neutrophil as indicated by the yellow arrowhead and the trans migrating neutrophil as denoted by the white arrowhead.
Neutrophil transmigration can occur at cell junctions or through the endothelial cells themselves. To differentiate between these two conditions, endothelial cells were labeled with an anti VE coherent antibody. As seen here in red transmigration is scored as occurring paracellular or trans cellularly.
In this model, virtually all transmigration is paracellular as demonstrated by this figure showing the overlay of the previous two figures. In this first of three graphs, neutrophil interactions were examined and measured using image J software. Note that significantly more neutrophil endothelial cell interactions occurred between neutrophils and TNF alpha activated endothelial cells than with the control unstimulated endothelial cells.
The total interacting cells were further characterized as rolling or firmly adherent or trans migrating as before. Significantly more neutrophil transmigration occurred in the wells containing activated endothelial cells Following isolation of the endothelial cells. The other methods like western blotting real time PCR and endothelial resistance can be performed in order to answer additional questions related to endothelial cell biology After development.
This method will help the researchers in the field of inflammation and vascular biology to understand how leukocyte recruitment happens under flow condition in a human model system. After watching this video, you should have a good understanding of how to isolate human umbilical vein endothelial cells and use them to study neutrophil transmigration under flow conditions using an ity chamber.
