Presented here is a protocol for a standardized in vitro hemodynamic loop model. This model allows to test the hemocompatibility of perfusion tubes or vascular stents to be in accordance with ISO (International Organization for Standardization) standard 10993-4.
This hemodynamic loop model allows researchers to test the in-vitro hemo compatibility of vascular devices such as perfusion tubings or stents. under standardized conditions. So this technique does not exert any impact on the blood by mechanical force and thereby avoid the misleading results by intrinsic activation of the blood components.
Before attempting an experiment, proper mechanical part assembly, perfect loop closure system construction and slow blood loading into the loops must be practiced. To prepare the loop assembly, use a tube cutter to cut 250 centimeter long, five millimeter inner diameter pieces of tubing on a flat surface. And plug the open endings of the tubes into a short piece of Silicon tube fitting the outer diameter of the investigative tube to generate a loop shape.
Carefully tighten the locking screw of the tension band connector and adjust the closing force so that no gap remains between the two bendings. If the locking screw is fully tightened and the tension of the polycarbonate band seems too low to close the gap between the two bendings, open the locking system and cut a few millimeters of the tension band. To prepare a stent loop assembly, open two of the loops and take the tube out of the tension band system.
Then insert the stent into the middle of the tube as instructed by the manufacturer. When the appropriate number of loops for the experiment have been generated secure the loops in the loop cradle of the rotation unit, outside of a 37 degree water bath and attach the loop cradle to the rotation unit inside the water bath. Next, fill one 10 milliliter syringe for every two loops of blood to be collected with 150 microliters or freshly prepared heparin stock solution and use a 21 gauge butterfly to gently collect blood into the syringes, taking care to avoid homolysis or cell activation due to excessive vacuum.
When all of the blood has been collected transfer the blood to a glass beaker and use a five milliliter serological pipette, to gently mix the blood and heparin. When a homogeneous solution has been obtained with the pipette tip not fully inserted into the tube, carefully, fill each loop with five milliliters of blood. Close each loop after it has been filled and confirm that the loop, tension band, and rack are properly secured.
Then rotate the loops for three hours at 30 revolutions per minute. At the end of the rotation, allow the loops to stand in the rack for two minutes to let the blood accumulate at the loop bottoms before carefully opening the connectors and pulling the blood from duplicates into individual 10 milliliter glass beakers. When all of the blood has been collected rinse each tube with 10 milliliters of PBS and use a scalpel to carefully cut a one centimeter sample from the end of each tube.
Incubate the samples overnight in 2%glutaraldehyde at four degrees celsius, followed by a 15 minute incubation and 1%osmium tetroxide at room temperature. At the end of the osmium tetroxide incubation, dehydrate the samples in an ascending ethanol series for 20 minutes per concentration, followed by a 30 minute dehydration in 100%ethanol. At the end of the 100%ethanol incubation, let the samples dry overnight at room temperature before imaging the samples by scanning electron microscopy at an acceleration voltage of five kilovolts, using an x-ray micro CT scanner.
For flow cytometric analysis of the blood samples. Transfer 100 microliters of blood from each sample into each of nine five milliliter FACS tubes and fix the samples with the addition of 100 microliters of 4%power formaldehyde per tube for 15 minutes at room temperature protected from light. After washing, re-suspend each pellet in one milliliter of red blood cell lysis buffer per tube, for a five minute incubation at room temperature, protected from light.
At the end of the incubation, wash the samples by centrifugation in one milliliter of PBS per tube and place the samples without their supernatants on ice. To prepare single stain compensation beads add four drops of positive and four drops of negative beads to individual one milliliter volumes of FACS buffer, and vortex the solutions to obtain homogeneous bead suspensions. Add 100 microliters of each solution to each of four or five milliliter FACS tubes labeled as indicated and wash the beads with one milliliter of FACS buffer per tube.
Next add 100 microliters of the appropriate vortex antibody cocktail to each experimental and fluorescence minus one tube with gentle mixing. For a 30 minute incubation at room temperature, protected from light. At the end of the incubation, wash the samples with one milliliter of fresh FACS buffer per sample and resuspend pellets in 250 microliters of FACS buffer per tube.
Before analyzing the beads on cell samples by flow cytometry, according to standard protocols. Blood cell distributions can be visualized over the entire surface of spliced two bloops by micro CT and scanning electron microscope imaging while no clots are observed in closed loops without gaps. Analysis of the whole blood cell count, does not show significant differences in erythrocyte numbers between any of the tested conditions, platelet and leukocyte numbers however, are drastically reduced in the latex loop group and free hemoglobin numbers are dramatically increased, indicating a very poor biocompatibility of latex.
While all of the tested vascular devices induce an increased activation of the coagulation system and compliment component. The heparin coated PVC loops exhibit a trend for decreased levels of both types of activations compared to polymer coated, PVC loops. Latex loops exhibit the highest levels of TNF IL-6 and PMN elastase, highlighting the potent activation of leukocytes by latex.
CD41 positive platelets recovered from latex tubes, exhibit an exceedingly high median fluorescence intensity for CD62P, while integrated expression is drastically reduced on granulocytes and lymphocytes. Of interest, integral levels are higher in monocytes from latex tubes, suggesting monocyte activated platelet interactions. Indeed staining for monocyte platelet aggregates reveals an increased tendency for aggregation in latex and stent loops.
Despite the reduced frequency of monocytes within latex loops. To avoid intrinsic blood component activation it is important to ensure a proper loop closure and to handle the blood with care. So following this procedure, it is possible to analyze the interaction between allogeneic proteins or TRUCKs and blood components that are used in inflammation or pharmaceutical research.
