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UNIVERSITY of CALIFORNIA SAN DIEGO Live-Cell in Vitro Aneurysm UNIVERSITY OF CALIFORNIA SAN DIEGO Live-Cell In Vitro Aneurysm System using 3D Printing Athesissubmittedinpartialsatisfactionofthe requirements for the degree Master of Science in Bioengineering by Quanyou Shi Committee in charge: Shu Chien, Chair Alexander Arash Khalessi Geert W Schmid-schoebein Dayu Teng 2021 Copyright Quanyou Shi, 2021 All rights reserved. The thesis of Quanyou Shi is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California San Diego 2021 iii TABLE OF CONTENTS SignaturePage ..................................... iii TableofContents ................................... iv ListofFigures ..................................... v Acknowledgements................................... vi AbstractoftheThesis ................................. vii Introduction ...................................... 1 MaterialsandMethods ................................ 3 Results ......................................... 9 Discussion........................................ 16 Results ......................................... 18 References........................................ 19 iv LIST OF FIGURES Figure 1: 3D design of the aneurysm vessels. 4 Figure 2: Block diagram for the in-vitro perfusion system. 6 Figure 3: Diagram for aneurysm vessel flow chamber. 7 Figure 4: Computational simulation and flow validation for the parallel-plate aneurysm vessel. 10 Figure 5: Computational simulation and flow validation for the half-round aneurysm vessel. 11 Figure 6: Full vessel scan and zoomed-in images at the vessel center region at Day 0, 4, 6, and 8. 13 Figure 7: Zoomed-in images at the Proximal and distal aneurysm neck region at Day 0, 4, 6, and 8. 14 Figure 8: Zoomed-in images at the aneurysm belly region at Day 0, 4, 6, and 8. 15 Figure 9: In vitro live-HUVECs images overlaid with the shear stress simulation. 18 v ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my committee chair, Dr. Chien, and my advisor, Dr. Teng, for their great mentorship, support and training throughout my project. Their guidance, critics, and patience have helped me to finish my thesis and improve myself along the way. I would also like to thank Dr. Khalessi and Dr. Schmid-schoebein for kindly serving as my committee members. I want to thank Dr. Khalessi for supporting and collaborating in this project and providing crucial insights. Dr. Schmid-schoebein has been a wonderful professor of fluid mechanics course, which is the foundation of this project. I would also like to express my gratitude to current Chien lab members, especially Dr. Yi-shuan Li for her guidance on the project, Phu and Jerry for their great help on the project and daily lab works, and Daniel for his assistance. I would also like to thank all other lab members for their great support. vi ABSTRACT OF THE THESIS Live-Cell In Vitro Aneurysm System using 3D Printing by Quanyou Shi Master of Science in Bioengineering University of California San Diego, 2021 Professor Shu Chien, Chair Intracranial aneurysm (IA) rupture is a major health risk that often leads to per- manent neurological damages and even death. Management of IAs has been challenging due to limited understanding of the underlying cellular mechanism of aneurysm progres- sion. The endothelial cells have been known to play an important role in most vascular diseases. However, there has been a lack of understanding on how endothelial cells contribute to the pathogenesis of aneurysms. This study aims to develop an in vitro platform that will enable future studies of the e↵ects of fluid mechanical environment on live endothelial cells by using 3D printing technology. The elements of the flow environment, e.g., the velocity field and shear stress in an aneurysm, were simulated using computational fluid dynamics (CFD). These elements were then compared with those computed by particle image velocimetry (PIV) using the videos of tracers flown through a 3D printed aneurysm model. For live-cell experiments, the human umbilical vein endothelial cells (HUVECs) were cultured on the substrate in the area enclosed by the 3D printed aneurysms perfused at di↵erent flow rates. The results showed a region-specific pattern in HUVECs density that can be correlated to the fluid mechanical elements such as velocity field and shear stress. The cell density decreased significantly at the aneurysm proximal neck and belly regions, where shear vii stress is low with non-directional flow, and increased at the distal neck region, where shear stress is higher. This study demonstrates the feasibility of a live-cell in vitro aneurysm model created using 3D printing. The development of this system with long-term live-cell culture in 3D printed aneurysm structures will enable future investigations to study the e↵ects of fluid mechanical elements on endothelial cells. Such studies will contribute to better device design and clinical management for the aneurysm patients. viii Introduction Intracranial aneurysm (IA) is an abnormal focal dilation of a cerebral artery with attenuation of the vessel wall.[1] Major classifications of IAs based on their geometry include saccular IA, fusiform IA, microaneurysm, and giant IA.[1, 2] Among these, saccular IA, which shapes as a round thin-walled sac, with well-defined aneurysmal dome and neck connecting to the parent vessels, is the most common type of aneurysm and accounts for approximately 90% of the IAs.[1, 2, 3] IAs have a low risk of rupture of approximately 1%.[4] However, IA ruptures often lead to devastating results, causing subarachnoid hemorrhage and are associated with high rates of fatality and permanent disability.[5] Therefore, appropriate management of the unruptured IAs is crucial. The IA treatments include surgical method of aneurysm clipping, and endovascular methods such as coiling and deployment of flow diverters.[5, 6, 7] The flow diverter is a cylin- drical mesh stent that is placed in the parent vessel across the aneurysm dome. The device alters hemodynamic parameters such as flow velocity and shear stress inside the aneurysm dome, thus inducing intra-aneurysmal thrombosis that can close o↵the aneurysm.[8, 9, 10] In addition, the stent also serves as a sca↵old for endothelization that promote formation of neointima to permanently close o↵the aneurysm.[11, 12] Flow diverter is the latest innovation, with two devices (Pipeline Embolization Device from Medtronic and Surpass Streamline Flow Diverter from Stryker) approved in the last decade.[13] Flow diverters have rapidly become the treatment of choice because of its high cure rates and low complication rates.[8] However, despite the significant progress in the development of flow diverters and exponential increase in their usage in recent years, flow diverters are still associated with a non-negligible rate of complications, including delayed intraparenchymal hemorrhage, thromboembolism, and spontaneous rupture of previously unruptured IAs after flow diverter treatment. Little is known about the causes leading to these complication events.[9, 14, 15] In addition, among all the available treatment techniques, the preferred therapeutical strategy for the unruptured IAs remains uncertain due to a lack of clinical data.[2, 6] Many of these difficulties stem from a lack of fundamental understanding of the cellular mechanism associated with aneurysm. The fundamental hemodynamic and cel- lular mechanisms underpinning the e↵ect of flow diverters are still poorly understood.[9] Hence, a deeper understanding of the cellular mechanism of aneurysm formation, growth, and rupture is required for better management of the unruptured IAs in patients. En- 1 dothelial cell (EC) dysfunction is known to play a major role in the pathogenesis and development of IAs. The hemodynamic factor of wall shear stress (WSS) exerted on ECs by blood flow has been demonstrated to be a key regulator.[16, 17] It is gener- ally accepted that the formation of aneurysms is associated with abnormally high WSS. However, there is no consensus on the WSS pattern leading to aneurysm growth and rupture, and controversial results of both high and low WSS have been reported to be responsible.[18, 19, 20] The lack of fundamental understanding of the cellular mechanism underlying the aneurysm growth and rupture is largely due to the limited available research methods. The complexity of individual IA in terms of both its shape and location poses great challenges for conducting studies on the aneurysms. Most of the in vivo studies for EC responses in aneurysms have been conducted in animal models such as rat or rabbit. Even though these in vivo studies provide a physiological condition closer to human body, there are still significant di↵erence in anatomy, physiology and pathologies that could lead to uncertainty.[21] In addition, as costly and time consuming as it, the in vivo studies would not provide an exact control over the WSS and flow pattern over the ECs inside the aneurysm. On the other hand, with the improvement in computational power, computational fluid dynamics (CFD) simulation of patient aneurysm models have also been done extensively to identify risk factor for aneurysm rupture in an attempt for better prediction.[22] CFD analysis has also been performed for the assessment of FD stents by observing the modulation in hemodynamic factors inside aneurysm.[18, 23] However, its applicability in clinical settings is still limited due to several limitations that make it difficult to produce consistent predictions and reliable results. CFD is often
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