The overall goal of this assay is to assess the anticoagulant properties of endothelial cells, using whole non-anticoagulated blood. This method can be used to answer research questions which are related to cardiovascular research, transportation and surgery related research. Essentially, can be used for all questions which have to do with anti-immunity and the activation of endothelial cells.
The main advantage of this technique is that whole blood, without anticoagulants can be used as anticoagulation is guaranteed by endothelial cells. To begin the protocol, mix seven milliliters of microcarrier beads with 42 milliliters of coagulant solution in a 50 milliliter tube and incubate the beads for one hour at room temperature. After incubation, wash the beads with 25 milliliters of phosphate buffered saline or PBS and rinse the beads by pipetting up and down.
Wait until the beads settle at the bottom of the tube, before discarding the supernatant and repeating. Next, wash the beads one time with 25 milliliters of Dulbecco's Modified Eagle's Medium, DMEM. Then, cover the beads with 10 milliliters of medium 199 and allow them to equilibrate for 10 minutes before further use.
Remove the cell culture media from the T175 flask containing PAEC and add five milliliters of PBS. Then, remove PBS from the T175 flask and replace it with five milliliters of trypsin with 0.05%EDTA. Incubate the cells for three to four minutes at 37 degree celsius.
Collect the cells by rinsing the flask with 15 milliliters of cell culture media and transfer the suspension to a 50 milliliter tube. Centrifuge the cells, after centrifugation, remove excess media and re-suspend the pellet in five milliliters of cell culture media. Add 20 milliliters of cell culture media to the cell suspension and re-suspend the cells.
Add 20 milliliters of cell culture media without cells into a 500 milliliter magnetic stirrer flask. Add the cells to the washed micro carrier beads and mix the solution carefully with a 25 milliliter serological pipette. Transfer the bead cell mixture into the magnetic spinner flask.
Rinse the tube with 10 milliliters of cell culture media to collect the remaining cells. Add an additional 85 milliliters of cell culture media into the spinner flask. Place the flask into the incubator overnight, at 37 degree celsius on a shaker.
Add 50 milliliters of cell culture media for a total volume of 200 milliliters. Continue stirring for an additional 24 hours on a shaker. Add colorless rockwell park memorial institute or RPMI media, until 320 milliliters of the total volume is reached.
Remove 100 milliliters of the old media and add 100 milliliters of fresh supplemented colorless RPMI, every 48 hours. Culture the cells for five to seven days depending on the confluent state of the cell coated beads. Using a 10 milliliter serological pipette, remove the cell coated beads from the magnetic stirrer flask and transfer them into 12 milliliter round bottom polypropylene tubes.
After one to two minutes, when the beads have settled down, remove the excess media. Add more beads to the tubes until each tube contains exactly two millimeters of beads. Next, add five milliliters of clear RPMI to each tube and mix the solution carefully with a 10 milliliter serological pipette.
Let the beads settle down and remove excess media. Repeat the washing procedure one more time with RPMI and remove all excess medium. Carefully and slowly draw blood from a healthy volunteer and collect it in a nine milliliter neutral polypropylene tube, with no anticoagulant.
Slowly transfer eight milliliters of blood into each of the polypropylene tubes containing two milliliters of cell coated beads, to make a total volume of 10 milliliters. Be sure to carefully handle the blood and the beads to avoid premature EC activation. Carefully tilt the blood bead mixture to ensure equal mixing and seal the cap with paraffin film.
Place the tubes on a horizontal tilting table with gentle settings, inside a 37 degree celsius incubator and record the clotting times. At set time intervals, remove at least 1.5 to two milliliters of the blood bead mixture for serum or plasma analysis. For serum collection, leave the blood to coagulate.
Finally, centrifuge the tubes at 2, 500 G for 10 minutes at four degree celsius. Store the serum or plasma at 80 degree celsius until use. Microcarrier beads stained for CD31 and nuclei clearly show confluency.
Visual observation determined that prolongation of the clotting time could be observed if a monolayer of EC was present on the surface of the microcarrier beads. A significant increase in clotting time was observed when porcine aortic endothelial cells, PAEC GalTKO/hCD46/hTBM were present on microcarrier beads, suggesting a successful modulation of both the complement and coagulation systems. Once mastered, the incubation of endothelial cell coated beads with non-anticoagulated human blood can be done in about six hours.
The actual duration of the experiment depends mainly on the clotting time, which is seen in the experiment. While attempting this procedure, it's important to remember to ensure complete coverage of the micro beads with endothelial cells and prevent pre-mature activation of human blood.
