STRESSED AND STRUNG OUT: THE DEVELOPMENT AND TESTING OF AN IN VIVO LIKE BENCH-TOP BIOREACTOR FOR THE OBSERVATION OF CELLS UNDER SHEAR STRESS Thesis Submitted to The School of Engineering of the UNIVERSITY OF DAYTON In Partial Fulfillment of the Requirements for The Degree of Master of Science in Bioengineering By Andrea Marie Chambers UNIVERSITY OF DAYTON Dayton, Ohio August, 2015 STRESSED AND STRUNG OUT: THE DEVELOPMENT AND TESTING OF AN IN VIVO LIKE BENCH-TOP BIOREACTOR FOR THE OBSERVATION OF CELLS UNDER SHEAR STRESS Name: Chambers, Andrea Marie APPROVED BY: Robert J. Wilkens, Ph.D., P.E. Carissa M. Krane, Ph.D. Advisory Committee Chair Member Advisory Committee Chair Member Professor, Director of Bioengineering Associate Professor Department of Chemical Department of Biology and Materials Engineering Kristen Comfort, Ph.D. Committee Chair Member Assistant Professor Department of Chemical and Materials Engineering John G. Weber, Ph.D. Eddy M. Rojas, Ph.D., M.A., P.E. Associate Dean Dean School of Engineering School of Engineering ii ABSTRACT STRESSED AND STRUNG OUT: THE DEVELOPMENT AND TESTING OF AN IN VIVO LIKE BENCH-TOP BIOREACTOR FOR THE OBSERVATION OF CELLS UNDER SHEAR STRESS Name: Chambers, Andrea Marie University of Dayton Advisors: Dr. Robert Wilkens and Dr. Carissa Krane Bioreactor systems used for tissue engineering applications are an essential component of understanding the development of new tissues and studying the biochemical interactions between cells and their environment. A bioreactor is typically designed to mimic physiological, environmental, and mechanical stimuli that occur in vivo, and bioreactors are generally created for a specific application, such as for studying 3-dimensional tissues or dynamic fluid flow in 1-dimensional cell monolayers. The leading cause of death in the United States is coronary artery disease, which is treated with bypass graft surgery using a left internal mammary artery or human saphenous vein as the graft. Since human saphenous vein grafts often fail, investigating vascular function as a whole will help to understand more about the method of graft failure. A bioreactor system to study vascular function was successfully developed using the application of endothelial cells under shear stress in a microfluidic slide. The iii temperature control and diffusion rate of CO2 were recorded inside the bioreactor to confirm the system could stay within a temperature range of 37ºC +/- 0.5ºC and a CO2 concentration between 56,000 ppm and 45,000 ppm. Also, a physiological level of shear stress was determined to be feasible with the peristaltic pump. The performance characteristics of the bioreactor were analyzed, and the apparatus was determined to be successful in generating physiological relevant conditions. Then, human umbilical vein endothelial cells were exposed to both static conditions and venous shear stress conditions for up to four days in an IBIDI® microfluidic chamber. The cell morphology, alignment, and elongation were also evaluated. The cells stayed viable during the duration of all of the dynamic flow experiments, and the cells showed evidence of cell division. The cells were also more aligned and elongated towards the direction of flow for the 48 a nd 72 hour flow experiments compared to the 48 and 72 hour static experiments (P-value < 0.05). The 96 hour flow experiment cells were also more aligned than the cells exposed to static conditions (P-value < 0.05). The 48 hour, 72 hour, and 96 hour dynamic flow experiments had a statistically significant difference in cell alignment compared to the 24 hour flow test, and the 72 hour dynamic flow experiment also had a statistically significant difference in cell alignment compared to the 48 and 96 hour flow experiments (P-value < 0.05). The 72 hour flow experiment was more elongated than the 24, 48, and 96 hour flow experiments (P-value < 0.05). Overall, the lab setup and bioreactor system yielded desirable results and provided a system that was fully capable of studying endothelial cells under venous shear stress conditions for studies up to 4 days. iv Dedicated to my family and friends v ACKNOWLEDGEMENTS First, I would like to thank my advisors, Dr. Robert Wilkens and Dr. Carissa Krane for providing me the opportunity to work on such a challenging and stimulating project. I would also like to thank my other committee member, Dr. Kristen Comfort for her guidance and support, not only with my thesis, but also as a professor for many of my graduate school classes. I am grateful to Mike Green who helped with the beginning stages of assembling my bioreactor and for the equipment I was able to use, and Justin DelMar for using his tools during the bioreactor construction. I also would like to thank Dr. John Sternick for his valuable troubleshooting advice and support. I also owe a debt of gratitude to both Kyle McGrail and Emily Breitner who provided assistance throughout my thesis. I am indebted to Street Barnett for his tremendous support and assistance through almost the entire process of completing my thesis. Lastly, I am thankful for the continuous encouragement from both of my parents and my brother, and for them listening to me whenever I needed support. vi TABLE OF CONTENTS ABSTRACT ....................................................................................................................... iii DEDICATION…………………………………………………………………………….v ACKNOWLEDGEMENTS ............................................................................................... vi LIST OF FIGURES ........................................................................................................... ix LIST OF TABLES ........................................................................................................... xiii LIST OF ABBREVIATIONS AND NOTATIONS ........................................................ xiv CHAPTER 1: INTRODUCTION ....................................................................................... 1 CHAPTER 2: BACKGROUND ......................................................................................... 4 2.1 Assessment of Fluid Flow Dynamics In Vitro ......................................................... 4 2.1.1 Bioreactors ........................................................................................................ 4 2.1.2 Fluid Flow Systems ........................................................................................ 15 2.1.3 Applications for Bioreactors for Vascular Tissues ......................................... 27 2.2 Coronary Artery Disease, Bypass Graft Treatment, and Graft Failure.................. 40 2.2.1 Coronary Artery Disease and Bypass Graft Treatment .................................. 40 2.2.2 Role of the Endothelium in Vascular Graft Failure ........................................ 41 CHAPTER 3: EXPERIMENTAL APPARATUS ............................................................ 44 3.1 Test Apparatus ....................................................................................................... 44 3.1.1 Materials and Hardware .................................................................................. 46 vii 3.2 Procedure ............................................................................................................... 58 3.2.1 Testing CO2 Diffusion .................................................................................... 59 3.2.2 Testing Cell Morphology................................................................................ 62 CHAPTER 4: RESULTS .................................................................................................. 66 4.1 CO2 Diffusion ....................................................................................................... 66 4.2 Bioreactor Control and Cell Morphology ............................................................. 70 CHAPTER 5: CONCLUSIONS AND FUTURE RECOMMENDATIONS ................... 83 REFERENCES ................................................................................................................. 87 APPENDIX A: Bioreactor Drawings ............................................................................... 95 APPENDIX B: Bioreactor Calculations ......................................................................... 104 APPENDIX C: Laboratory Protocols ............................................................................. 118 viii LIST OF FIGURES Figure 1: Typical advanced bioreactor illustration (reproduced from Massai et al., 2013) 6 Figure 2: Spinner-flask bioreactor (reproduced from Plunkett and O'Brien, 2011) ........... 8 Figure 3: Parallel-plate flow chamber constructed from polycarbonate (reproduced from Lawrence, et al., 1987) ...................................................................................................... 10 Figure 4: Harvard Apparatus ORCA™ Bioreactor System ("Harvard Apparatus", 2015) ........................................................................................................................................... 12 Figure 5: Schematic of a cone-and-plate flow system (reproduced from Cheresh, 2008) 16 Figure 6: Circular parallel-plate flow chamber (reproduced from Kaur, et al., 2011) ..... 20 Figure 7: Microfluidic chamber with three culture units (reproduced from Hattori, et al., 2014) ................................................................................................................................. 24 Figure 8: Novel hemodynamically-equivalent pulsatile bioreactor system