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Tissue Engineering a Blood Vessel Mimic While Monitoring Contamination Through Sterility Assurance Testing

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Tissue Engineering a Blood Vessel Mimic While Monitoring Contamination Through Sterility Assurance Testing Tissue Engineering A Blood Vessel Mimic While Monitoring Contamination Through Sterility Assurance Testing A Thesis Presented to the Faculty of California Polytechnic State University, San Luis Obispo In Partial Fulfillment of the Requirements for the Degree Master of Science in Engineering with Specialization in Biomedical Engineering By Navid Aliakbar Djassemi July 2012 © 2012 Navid Djassemi ALL RIGHTS RESERVED 2ii COMMITTEE MEMBERSHIP TITLE: Tissue Engineering A Blood Vessel Mimic While Monitoring Contamination Through Sterility Assurance Testing AUTHOR: Navid A. Djassemi DATE SUBMITTED: June 07, 2012 COMMITTEE CHAIR: Kristen O’Halloran Cardinal, PhD COMMITTEE MEMBER: David Clague, PhD COMMITTEE MEMBER: Trevor Cardinal, PhD 3iii Abstract Tissue Engineering A Blood Vessel Mimic While Monitoring Contamination Through Sterility Assurance Testing Navid Djassemi Tissue engineering blood vessel mimics has been proposed as a method to analyze the endothelial cell response to intravascular devices that are used in today’s clinical settings for the treatment of cardiovascular disease. Thus, the development of in vitro blood vessel mimics (BVMs) in Cal Poly’s Tissue Engineering Lab has introduced the possibility of assessing the characteristics of cellular response to past, present, and future intravascular devices that aim at treating coronary artery disease. This thesis aimed at improving the methods and procedures utilized in the BVM model. Initial aspects of this project focused on using an expanded polytetrafluoroethylene (ePTFE) scaffold in conjunction with human endothelial cells to replicate the innermost intimal layer of a blood vessel. Human umbilical vein endothelial cells (HUVECs) were pressure sodded onto ePTFE scaffolds through cell sodding techniques in an attempt to effectively and consistently replicate and assess the intimal layer. Through each study ePTFE grafts were subjected to different culture times and steady flow rates to observe and compare the differences in the endothelial cell deposition. Results were inconsistent, although moderate cell adhesion was noted 4iv throughout each of the BVM setups. Each study exhibited a range of cell sodding density rates. In the second phase of the thesis, contamination assessment protocols were implemented in the BVM lab. The intent of this part of the project was to assess the relative sterility in the cell culture lab, a critical component involved with the success or hindrance of cell and tissue cultures. Using microbiological validated methods, microbiological tests were conducted to examine the levels of microbial growth in and around the tissue engineering lab. Results were tracked over a two month period in the lab with several observations of aerobic microorganism growth on various counter and lab surfaces. Higher growth trends were found on surfaces outside the cell culture lab, in the general TE lab area. These findings were used to provide overall suggestions on tracking microbes for long-term durations in ongoing BVM setups to directly improve the overall sterility assurance of the studies. As the project reached its conclusion a look back at all the BVM setups and contamination assessments lead to a few suggestions for improving aseptic techniques within the TE lab, such as monitoring microbial growth in the culture processes, creating limit specifications, and creating a standardized way to regulate quality control within the lab environment. Furthermore, as the development BVM evolves, the findings from this report can be used with related research for improving the culture conditions of various BVM studies. 5v Acknowledgments I would like to thank several people who were constantly demonstrating the beauty of orchestrated madness in the form of science, engineering, and guidance to advance the minds of my generation into the new century. First, I want to extend big thanks and a hug to my thesis advisor Dr. Kristen O’Halloran Cardinal for giving me a chance to work with her TE team. I feel extremely lucky to have worked with such an intelligent, sharp, motivated, and fun professor over the past couple of years. The Tissue Engineering lab would not be what it is without Dr. Cardinal period. I also want to thank and appreciate my other committee members for their time and input into my thesis and all their great contributions to the department: Dr. Trevor Cardinal and Dr. David Clague. A special thanks to Dr. Clague for his mentorship as an undergrad and with helping me seek out the future of analytical methods and creative ideas that were related to my thesis as well as careers in the biotech world, and for just being a friend. Also my admirations go out to the rest of the biomedical engineering staff including Dr. Griffin and Dr. Walsh for bringing Cal Poly BMED to such high places. Of course, none of this today would be possible without my family and friends, and the big guy in the sky. I love you all. 6vi Table of Contents List of Figures __________________________________________ xiii List of Tables ___________________________________________ xvi CHAPTER 1: Introduction _____________________________________ 1 1.1 Overview _____________________________________________ 1 1.1.1 Blood Vessel Biology and Atherosclerosis _____________________________________ 3 1.1.2 Treatment Therapies and Justification of TEVGS ________________________________ 4 1.1.3 Tissue Engineering Impact _________________________________________________ 7 1.2 The Aims and Methodology of Tissue Engineered Vascular Grafts ____________________________________________________ 7 1.2.1 Introduction _____________________________________________________________ 7 1.2.2 Relevant Experimental Fields _______________________________________________ 7 1.2.3 BVM Research __________________________________________________________ 8 1.2.4 Graft Compliance and Biocompatibility ________________________________________ 9 1.2.5 Scaffold Development ____________________________________________________ 12 1.2.5.1 Scaffold Development with Well-Plate and Electrospun Scaffolds in Static Culture Conditions _____________________________________________________________________ 12 1.2.5.2 Dynamic Shear Flow Environments _________________________________________ 16 1.2.6 Vessel Conditioning _____________________________________________________ 18 1.3 Isolation and Cultivation Techniques of HUVECs __________ 20 1.3.1 Isolation Methods _______________________________________________________ 20 1.3.2 Characterizing Viable Culture and Growth Techniques __________________________ 23 vii7 1.3.2.1 Application of Different Growth Media Conditioning _____________________________ 24 1.3.2.2 HUVEC Cultures ________________________________________________________ 28 1.4 HUVEC Development in TEVGs _________________________ 29 1.4.1 In Vivo Studies _________________________________________________________ 29 1.4.2 HUVEC Development and Response in Static and Shear Environments ____________ 31 1.4.3 HUVEC Development on Stent Materials _____________________________________ 35 1.4.4 Effects of Scaffold Surface Roughness ______________________________________ 37 1.4.5 Growth Factor Influence in HUVEC Cultures __________________________________ 40 1.5 Methods for Cell Delivery onto Scaffolds and TEVGs _______ 41 1.5.1 Cell Sodding/Seeding Techniques __________________________________________ 42 1.5.2 HUVEC Sodding Trials in ePTFE Scaffolds ___________________________________ 44 1.6 Summary and Aims of the Thesis _______________________ 45 Chapter 2: Blood Vessel Mimic (BVM) Set-Ups Over One, Three, and Seven Days____________________________________________ 48 2.1 Introduction to Blood Vessel Mimics _____________________ 48 2.1.1 History of Blood Vessel Mimics _____________________________________________ 48 2.2 Materials and Methods ________________________________ 49 2.2.1 Materials and Methods for Study One _______________________________________ 49 2.2.2 Materials and Methods for Study Two _______________________________________ 55 2.2.3 Materials and Methods for Study Three ______________________________________ 56 2.3 Results and Observations _____________________________ 56 viii8 2.3.1 Results from Study One __________________________________________________ 56 2.3.1.1 BBI Imaging ____________________________________________________________ 58 2.3.1.2 Histology ______________________________________________________________ 61 2.3.2 Results from Study Two __________________________________________________ 65 2.3.2.1 BBI Imaging ____________________________________________________________ 66 2.3.2.2 Histology ______________________________________________________________ 69 2.3.3 Results from Study Three _________________________________________________ 74 2.3.3.1 BBI Imaging ____________________________________________________________ 75 2.3.3.2 Histology ______________________________________________________________ 81 2.4 Conclusion of BVM Studies ____________________________ 89 2.4.1 General Observations of BVM Studies _______________________________________ 89 2.4.2 Study One Discussion ____________________________________________________ 90 2.4.3 Study Two Discussion ____________________________________________________ 91 2.4.4 Study Three Discussion __________________________________________________ 93 2.4.5 Future Outlook/Next Steps ________________________________________________ 94 Chapter 3: Sterility Assurance of BVM Systems through Microbiological Practices ____________________________________ 96 3.1 Introduction
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