This In Vitro Disease Model, can be used to study the interaction of whole blood and patient derived cells, specifically the endothelial, to evaluate thrombogenicity and the effectiveness of anticoagulant agents. This is endothelium cells can be used on a various circumstances and can be modified by introducing custom-made microfluidics or by adjusting blood flow parameters. In addition, this model can be used to study thrombus dynamics in inflammatory conditions, or to assess the effects of anticoagulant and antiplatelet therapy.
Ultimately, these approaches can be used in a method of personalized medicine. After obtaining a surgical human pulmonary artery tissue sample, coat high-affinity cell binding 60 milliliter cell culture dish with two milliliters of five micrograms per milliliter fiboronectin and incubate the dish at 37 degrees Celsius for at least 15 minutes. At the end of the incubation, use sterile forceps in a laminar flow cabinet under sterile conditions, to transfer the tissue into a Petri dish of PBS.
If the tissue is still in a ring, cut the artery open with scissors, taking care not to touch the innermost layer of the vessel, as the endothelium is easily damaged and removed. After removing the fibronectin, add four milliliters of complete endothelial cell medium to the dish and use forceps to transfer the tissue sample into the medium, use a scalpel to carefully scrape the inner layer of the vessel into the medium, taking care to avoid any lipid accumulations that are observed in the vessel wall. After the cells have been harvested, place the plate into the cell culture incubator.
If fibroblasts contaminate the culture, use magnetic affinity cell separation for a CD144, according to the manufacturer's instructions to purify the cells. Generally, culture is defined as pure, when flow cytometry detects 10%or fewer contaminating cells. When the sufficient number of endothelial cells have been obtained for experimental use, the primary endothelial cells can be characterized for the presence of endothelial cell specific markers and for the absence of smooth muscle and epithelial cell markers.
For flow chamber preparation, code one channel of a six well flow slide with 30 microliters of 0.1%gelatin and incubate this slide for at least 15 minutes at 37 degrees Celsius. In addition to commercial flow chambers, custom made flow chambers with specific dimensions can be used to study the influence of the vascular geometry on clot dynamics. While the slide is incubating, collect the cells from a confluent pulmonary artery endothelial cell culture with EDTA and trypsin and sediment that cells by centrifugation, re suspend the pellet in 600 microliters of complete endothelial cell medium and forcefully but gently, add 100 microliters of cells into each channel, add an additional 50 microliters of complete endothelial cell medium to the channel, to provide sufficient medium for overnight incubation at 37 degrees Celsius and 5%carbon dioxide, change the medium the next day to wash away the unbound cells.
After six days in culture, the cells should have formed a stable confluent monolayer. On day seven of pulmonary artery endothelial cell culture, acquire a venous blood samples from subjects who do not receive anticoagulation treatment into 109 monosodium citrate anticoagulant tubes and transfer the blood into a 50 milliliter tube. Blood can contain viruses and other infectious agents.
Therefore it is important to always wear gloves and to follow the safety guidance for working with human material. Fluorescently labeled the blood cells with 1 to 10, 000 concentration of Calcein AM Red and 50 micrograms per milliliter and add Alexa 488 fibrinogen, then incubate the blood at 37 degrees Celsius for 15 minutes to allow complete absorption. 30 minutes before the perfusion, treat the pulmonary artery endothelial cells with 100 microliters of one micromolar histamine in endothelial cell medium and 1%fetal bovine serum without any other additives.
And use a 20 milliliter syringe filled with wash buffer to rinse the flow system tubes, load a third to 20 milliliter syringe with two milliliters of wash buffer and load the syringe onto a syringe pump. Use a second 20 milliliter syringe to fill the flow tubes with hippies plus buffer containing 1%bovine serum albumin and use an elbow shape Laura connector to carefully connect the tubes to the micro slide, connect the outlet of the slide to the syringe, repair from microscopy by placing the cell containing slide onto the stage and assembling the setup for the experiment, adjust the microscope and set the parameters for imaging. Recalcification of emphatical blood.
And this is a happy co-sponsor of coagulation, furthermore try to avoid formation of air bubbles because this will damaged endothelial cell layer and influence your results. Immediately before the perfusion, dilute the blood at two times with recalcification buffer and place the inlet tube of the micro slide into the tube of blood, press start on the syringe pump. As soon as the blood starts flowing over the endothelium, press start on the microscope to acquire images using the preselected active channels and region of interest positions, every 15 seconds for five minutes.
At the end of the analysis, stop the recording and the syringe pump and clean up properly. Labeling platelets with Calcein AM Red and the addition of Alexa 488 conjugated fibrinogen, allows the investigation of thrombus formation by platelet adhesion and fiber and deposition. Under non stimulated conditions, there is no binding of platelets or fibrin to the endothelium.
Stimulation with histamine results in an immediate increase in platelet adhesion, that reaches a plateau after 2.5 minutes, at this time point, the platelets begin to secrete autocrine factors that induce a platelet aggregation and fibrinogen cleavage to fibrin, resulting in fiber and deposition at three minutes and the formation of a stable aggregate with the platelets after four minutes. Treatment with a direct anticoagulant, inhibits both clot formation and platelet adhesion compared to untreated blood. Immunofluorescent analysis of pulmonary artery endothelial cell platelet interactions after five minutes of perfusion, reveals maintenance of the endothelial cell, cell contacts, as confirmed by vascular endothelial cadherin staining, indicating that the blood clots form on endothelial model layers rather than on the underlying matrix between endothelial gaps.
human umbilical vein endothelial cells demonstrate less platelet adhesion and fibrin deposition compared to pulmonary artery endothelium. Notably, diseased primary pulmonary artery endothelial cells from chronic thromboembolic pulmonary hypertension patients, exhibit an increased platelet adhesion and more fibrin deposition compared to healthy cells. Thrombogenicity of the endothelium is dependent on multiple factors like the origin of the vascular bed or the parts of biological background.
Using this protocol, intrinsic dysfunctions in vascular and blood components, as well as the effectiveness of anti coagulated therapy can be tested during different phases of thrombosis hemostasis. This protocol can be used for understanding blood and Theo interactions, in various thromboembolic diseases with the ultimate potential to predict patient-specific responses to anticoagulation therapy.
