Cardiovascular device-induced blood cell trauma investigated using a hemostable flow loop

In this doctoral dissertation, a series of experimental and biological models were developed to improve the hemomechanical compatibility of blood-contact biomedical devices and bring in vitro test conditions closer to clinical reality. The studies encompass both engineering solutions to reduce device-induced blood damage and innovative approaches to preserving the biological integrity of blood under experimental conditions. In the first phase, considering the inadequacy of conventional flow-loop systems commonly used in in vitro testing to reflect clinical conditions, a metabolically sustainable system integrating nutrition, oxygenation, and dialysis modules was designed. In this system, human blood was circulated for 12 and 48 hours, and biomarkers such as hemolysis (NIH, pfHb, bilirubin, haptoglobin), inflammation (IL-8, TNF-α, C3a), coagulation (fibrinogen, TAT), platelet and endothelial activation (β-TG, vWF), and ferritin were monitored. While pH decreases, lactate increases, and inflammatory/coagulatory parameters increased in the control circuit, these parameters remained stable or decreased in the dialyzed circuits, and hemolysis was found to be 77–81% lower. These results demonstrate that long-term testing systems that can preserve the biochemical integrity of blood can provide a more reliable platform for predicting the clinical performance of devices. In second study, a new centrifugal pump design was developed that reduces blood damage by approximately 30% compared to the FDA-recommended reference pump, and its performance was evaluated in flow-loop experiments with fresh human blood. Comparisons with commercial pumps showed that while hemolysis and biomarker levels exceeded tolerance limits in the FDA reference pump, the optimized design reduced hemolysis by 68% and performed similarly to commercial devices in terms of inflammation, coagulation, and platelet activation. These findings demonstrate that improvements in device design can provide clinically meaningful biological benefits. In third study, a new methodology based on an organ culture approach is presented, treating blood not as a passive fluid but as a metabolically active and living system. Viability was maintained with blood gas analyses and nutritional solutions in a flow system constructed using standardized cardiovascular bypass components, and oxygenator and pump prototypes were tested. Throughout the experiments, erythrocyte deformability, hemolysis indices, enzyme activities (glutathione peroxidase, superoxide dismutase, catalase), and 2,3-diphosphoglycerate levels were maintained, and minimal submicron particle release was observed. This approach reduced biological variation in blood injury experiments, resulting in more realistic results. In the last work, the necessary infrastructure for genetic modulations to increase mechanical stress resistance in erythrocytes differentiated from hematopoietic stem cells was investigated. The differentiation mechanism of cells was investigated using the CD235a surface marker, the fluorescently labeled siRNA uptake system was validated using electroporation, and the membrane elasticity of control erythrocytes differentiated from stem cells was measured by micropipette aspiration. Findings showed that cells in suspension had a higher differentiation rate, but this rate decreased as time and passage number increased. Furthermore, a system for shaping erythrocyte biomechanics through genetic manipulations was developed, and it is envisioned that this method could create a new research platform that could be used both in basic biology studies and in hemocompatibility testing of blood-contact devices. Overall, this thesis aims to improve hemomechanical stability through design optimizations of blood-contact devices, while also reducing blood damage through metabolically sustainable test systems and genetically modified cell models. The obtained results contribute to the development of new standards in the preclinical evaluation of cardiovascular devices, providing important scientific data aimed at improving both patient safety and the effectiveness of biomedical devices.