Platelets from individuals with diabetes, cystic fibrosis, multiple sclerosis, hypertension, and sickle cell anemia are hyperactive or more likely to aggregate and form a blood clot or thrombus. Furthermore, each of these diseases exhibits abnormal red blood cell (RBC) adenosine triphosphate (ATP) release, an agonist of platelet activation. RBC-derived ATP release is also a proven factor in the regulation of vascular tone through a signal cascade that results in nitric oxide release from endothelial cells and relaxation of smooth muscle cells. This relaxation leads to vessel dilation and a localized increase in blood flow. The presence of hyperactive platelets in conjunction with altered RBC-derived ATP release results in an impaired ability to dilate local resistance vessels, and ultimately puts individuals with these diseases at higher risk for deleterious thrombus formation. The work detailed in this dissertation outlines the development of microfluidic and 3D printed in vitro models of in vivo circulation, capable of inducing an injury to a localized region of the endothelium. Specifically, chemical and electrical lysis of endothelial cells will be demonstrated through the use of embedded or removable electrodes or with laser irradiation of a photochemical dye. The fabricated devices mimic flow seen in blood vessels, facilitate the study of platelet adhesion to sub-endothelial collagen, and allow for the study of thrombus formation in stored blood samples showing altered ATP release from RBCs. With a more rapid fabrication process, reusability of the final device, and possibility of standardization via open software sharing, 3D printing offers a more attractive method to develop and utilize an in vitro thrombus mimic compared to more widely employed soft lithographic techniques. Channels of 3D printed devices featured a stenosis region (0.8 mm height, 2 mm length, and 1 mm width) and wide regions (device 1: 5; device 2: 3.83 mm width). Surface modification of channels with either polydimethylsiloxane (PDMS) or polystyrene (PS) was necessary to promote endothelial cell adherence. Thicknesses of PDMS and PS channel coatings were determined using scanning electron microscopy. The PDMS coating varied in thickness from 3 μm to 100 μm. Multiple PS coatings were required to form a 100 μm thick coating. Cells remained viable on the devices for five days (98% viable), though cell coverage decreased after day four with static media delivery. Optimal lysis conditions (applied electrical potential and duration) were determined for the two different geometries of the 3D printed devices to ensure localized endothelial cell clearance. Selective cell lysis was achieved with efficiencies of 94% (device 1) and 96% (device 2). FDA approved blood storage solutions expose RBCs to hyperglycemic amounts of glucose. As seen with RBCs from individuals with diabetes, ATP release from stored RBCs is significantly decreased compared to control RBCs. The effect of this decreased ATP release on thrombus formation was evaluated when hyper and normoglycemic stored RBCs were reincorporated with platelet rich plasma by measuring percent transmittance through the device, where increased thrombus coverage corresponded to a decrease in transmittance. Within the same storage period, RBCs stored in hyper and normoglycemic conditions showed no significant difference in hemoglobin absorption indicative of cell adherence. However, a significant decrease in cell adherence between day 1 and week 3 hyper and normoglycemic samples was observed (normo: p = 0.005, n ≥ 3; hyper: p ˂ 0.003, n ≥ 3).
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