The present protocol may serve as a new platform for the study and development of drug carriers to target disease sites within the human vascular system. The main advantage of our technique is that it allows the study of drug carriers'accumulation inside human-replicated 3d arterial model. This is done under physiological conditions, including blood flow.
This approach can be used to optimize drug carriers for the diagnosis and treatment of a wide range of vascular diseases, including a atherosclerotic arteries. Begin by selecting images from patients or previously studied geometries of the human carotid artery bifurcation and using the images to create a computer-aided design model of the mold. Use a 3d printer to print the design, remove the temporary printing supports and rinse the model with acetone before using sandpaper to polish and smooth the molds, especially the areas from which the supports were cut.
After sanding, rinse the model with isopropyl alcohol to remove any plastic dust and allow the model to dry in a chemical hood for two to three hours. To facilitate easy disillusion of the plastic, spray the model with transparent lacquer three times, allowing the lacquer to air-dry for one hour between applications. After the last application, use a paint brush and lacquer to glue transparent, rectangular strips of smooth plastic to each side of the frame, such that the model will be sealed at the bottom and open at the top.
After drying, place the mold in a desiccator and slowly poor freshly-prepared silicone rubber solution into the opening. Remove air bubbles until the mixture is clear and leave the mold inside the desiccator overnight. When the mixture is fully dry, remove the transparent slides and immerse the model in absolute acetone for 48 hours in a chemical hood until the plastic is fully dissolved.
To evaporate the trapped acetone before cell seeding, incubate the model for at least four days at 60 degrees Celsius. To seed the model with cells, first sterilize the model and two inlet and outlet connectors by ultraviolet light or radiation. After 20 minutes, using a syringe to coat the model with four milliliters of 100 microgram per milliliter fibronectin for two hours at 37 degrees Celsius.
At the end of the incubation, remove the fibronectin solution through the outlet and wash the model with endothelial cell medium. Use a syringe to fill the rinsed model with four milliliters of a 2.5 times 10 to the sixth endothelial cells per milliliter of endothelial cell medium cell suspension and secure the model to a rotator inside a cell culture incubator 48 hours at one revolution per minute, to ensure homogenous distribution of the cells. At the end of the incubation, use a 10 milliliter plastic syringe to wash the model with PBS.
Fix the cells for 15 minutes, with four milliliters of 4%paraformaldehyde, then wash the model three times with PBS as demonstrated before storing the model at four degrees Celsius in four milliliters of fresh PBS. Before beginning an experiment, use the outlet tubing to connect an oscillation damper connected to a peristaltic pump to the inlet of the cultured carotid model and use a piece of tubing to merge the two outlets. Then split the outlet tubing to two outlet tubes and add a plastic clamp to each tube.
To set up a closed circuit configuration, add 300 milliliters of PBS to a closed circuit container and place one inlet and one outlet tube inside the PBS filled container with the B and C clamps open, respectively, then place the other inlet tube into a one-liter washing container filled with distilled water, and the other outlet tube into an empty one-liter waste container with the A and D clamps closed, respectively. Place the carotid model under a stereo microscope, set the pump to a starting flow rate of 10 revolutions per minute, with five revolution per minute increment increases every four to five minutes. When the flow rate reaches 100 revolutions per minute, add 1.6 micrograms of fluorescent carboxylated polystyrene particles per milliliter of PBS to the closed circuit container and image the region of interest every 10 seconds for one and a half hours.
To set up an open circuit configuration, open the A and D clamps and immediately close the B and C clamps. Let most of the water flow from the washing container to the waste container at 100 revolutions per minute. Before the water has been completely transferred, stop the pump and close the tube clamps before the inlet and after the outlet of the carotid model.
Using the appropriate filters, capture images of the model at the region of interest to show the deposition and adhesion of the particles to the cells. Upon completion of the experiment, use a customized software code to analyze the images captured at the region of interest, enter the name of the image and set the estimated threshold. Then run the code.
Inspect the resulting image and the counted number of particles in the image. In this representative analysis, photo micrographs of cultured endothelial cells seeded in a 3d carotid artery model can be observed by brightfield and fluorescence confocal microscopic imaging. Fluorescent 10 micron diameter glass beads, seeded into the 3d model exhibited a recirculation pattern, suggesting a successful mimicking of physiological conditions within the model.
Imaging of the particle deposition revealed that a higher level of adhesion of the particles to the cells was observed outside of the recirculation area where the wall shear stress was high. Following this protocol, functionalized particles with specific binding kinetics and a core culture of cells within different human artery models may be explored to improve the model and to study specific formulations. This technique provides a new platform for studying the vascular targeting of drug carriers.
We have recently used it to show how the additivity of particles can be tailored to target different diseased vessel regions.
