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Chapter 1 : Background and Introduction DEVELOPMENT OF IN VITRO SYSTEMS TO STUDY ENDOCRINE DISRUPTORS A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Hui Xu May 2009 © 2009 Hui Xu DEVELOPMENT OF IN VITRO SYSTEMS TO STUDY ENDOCRINE DISRUPTORS Hui Xu, Ph. D. Cornell University 2009 Environmental estrogenic endocrine disruptors (EDs) are a health concern as the general population is exposed to a variety of EDs at low doses. Rapid screening and in depth understanding of how the body responds to EDs and their mixtures are crucial for toxicological evaluation and health protection. In this work, in vitro systems, including stable cells lines with estrogen induced green fluorescence protein (GFP) expression and micro cell culture analog (microCCA) devices, were developed to study EDs. A dual cell-line GFP expression system was constructed to study EDs with activities as estrogen receptor agonists or antagonists. The two stable cell lines, Ishikawa-GFP (uterine origin) and MCF7-GFP (breast origin), displayed increased intracellular GFP intensity with estrogens. This system also responded in a tissue specific manner to selective estrogen receptor modulators, raloxifene and tamoxifen. Our results also suggested no low dose synergistic effects between the tested estrogenic compounds. Physiologically realistic microCCA devices were developed to help predict in vivo responses to EDs. The silicon microCCA device contained a co-culture of three cell lines: MCF7-GFP, Ishikawa-GFP and HepG2/C3A (liver origin). Recirculation of medium mimicked the time dependent changes in drug concentrations and the microfluidic shear conditions inside a body. An in-line bubble trap was incorporated into the system to overcome air bubble related device failure. Interactions between hydrophobic surfaces and the fluid were investigated. Significant surface adsorption of estrogen to polypropylene based tubing (BPT) was observed. Contaminants leaching out of the BPT tubing were also found to be estrogen receptor agonist. A pneumatic micropump with a pumping rate ~3 ul per min was build with PTFE tubing replacing BPT tubing. Detection of estrogens was demonstrated on a microCCA device integrated with the micropump. Further, a photopolymerizable hydrogel based method was developed for direct patterning of multiple types of cells into their individual cell culture chambers in a PDMS microCCA. The use of this device as a biosensor was demonstrated by monitoring PEG encapsulated cells in microCCA exposed to a model toxicant, Triton X-100. The results indicate the potential use of PDMS microCCA devices to study EDs. BIOGRAPHICAL SKETCH Hui Xu was born to Xiulan Bai and Qinghua Xu on a bright sunny winter day. Her home town, Pingyi in the Shandong Province, China, was full of lovely friends and wonderful memories. She went to Tsinghua University in Beijing at seventeen years old and earned her bachelor’s degree in Applied Physics. Three years later she received her Master of Science degree studying the development of mouse embryos with multiphoton microscopy. This combination of engineering, physics and biology led her into the world of Biomedical Engineering. She also first met Xiaodong Chen, her future husband, when she served as the vice president of the Student Technology Innovation Center. In 2003, Hui attended the Biomedical Engineering program at Cornell University with Dr. Michael L. Shuler as her advisor. She worked on the “Body-on-a-Chip” project and enjoyed her time working in the clean room. In the summer of 2008 she gave birth to a lovely baby girl named Yilan (Sophia). Hui and her family are moving to Northern California, where she will be sure to miss the white snow and gorgeous waterfalls in Ithaca. iii To My Parents, Xiulan Bai & Qinghua Xu 致我的父母, 白秀兰和徐清华 To Xiaodong, Zhen and Yilan 致晓东, 真真和沂岚 For Their Unconditional Love 感谢他们无尽的关爱 iv ACKNOWLEDGMENTS First I would like to acknowledge my thesis advisor, Dr. Michael Shuler. His knowledge, foresight and enthusiasm have guided me through my Ph.D. study. Working with him has improved me as an independent thinker and scientific researcher. I am grateful to my committee member Dr. W. Lee Kraus for his kind instructions on molecular biology experiments and his generosity for allowing me full access to his laboratory. I also thank Dr. Harold Craighead for all the helpful advice in my study of biophysics. This project is finished with the support from many people. I am grateful to the whole Shuler research group for their support and friendship. I thank Paula Miller for cell culture and facility trainings and for always being there to help. Within the microCCA team, Dan Tatosian, Gretchen McAuliffe, Mandy Esch and Jay Jung Hwan Song have been the best co-workers one may ask for. I also have very much enjoyed all the nice talks with BJ Kim, Tricia Echtenkamp, Jordan Altas, Jinpian Diao and many others working on the second floor, Olin Hall. Wangyu Tong and Wei Li, two visiting scientists in out lab, gave me many valuable advices on my project. Jun Wu has been the best collaborator and Dr. C.C Chu has given me many useful suggestions on photopolymerizable polymers. I thank Gary Isaacs, Edwin Cheung and Tong Zhang from Dr. Kraus’s lab for their assistance with plasmid construction and cell infection. Special thanks to Shivaun Archer for allowing me to use the BME lab; Glenn Swan for fabricating the chip housings; Dr. Leonard W. Lion and Dr. David Putnam for helpful discussions and technical inputs. I thank Dr. Volker Vogt for his kind gifts of pQCXIP-GFP plasmid and 293T cell line. I also acknowledge Dr. Myles Brown at the Dana-Farber Cancer Institute (Boston, MA) for supplying Ishikawa cell line; and Dr. v Ana Soto and Dr. Carlos Sonnenschein at Tufts University of Medicine (Boston, MA) for providing MCF7-BOS cells. Financial support for this work was provided in part by the Cornell NanoBiotechnology Center (NBTC), an STC Program of the National Science Foundation (ECS-9876771); A New York State Office of Science, Technology and a Academic Research (NYSTAR) Distinguished Professorship Award (M. L. Shuler); National Science Foundation (BES0342985); and NIH/NIDDK (DK058110) to W. L. Kraus. Lastly I wish to thank my family especially my parents. I want to thank my parents for giving me a free spirit, for their unconditional love, for all of the sacrifices they have made for me, and for everything. I thank my husband for his support, his encouragement, for his smile and for making me smile. I appreciate my sister for the memorable childhood and for all the beautiful time we have spent together. Last I thank my daughter for making me a mother and for bringing new meaning to my life. vi TABLE OF CONTENTS Biographical sketch ................................................................................................... iii Dedication ................................................................................................................. iv Acknowledgement...................................................................................................... v List of figures ........................................................................................................... x List of tables ........................................................................................................... xii List of abbreviations ................................................................................................ xiii Chapter 1 : Background and Introduction ................................................................... 1 1.1 In vitro cell co-culture systems ............................................................................. 1 1.2 Micro and nano fabrication technique for micro cell culture devices ..................... 6 1.3 Materials for miniature cell culture devices ....................................................... 13 1.4 Biological problem of interest: estrogenic endocrine disruptors ........................ 15 References................................................................................................................ 19 Chapter 2 : Development of a Stable Dual Cell-Line GFP Expression System to Study Estrogenic Endocrine Disruptors ............................................................................ 27 2.1 Abstract .............................................................................................................. 27 2.2 Background and introduction .............................................................................. 28 2.3 Materials and methods ........................................................................................ 32 2.3.1 Chemicals and reagents ............................................................................ 32 2.3.2 Cell culture ............................................................................................... 32 2.3.3 Plasmid construction ................................................................................ 33 2.3.4 Retrovirus infection of MCF7-BOS and Ishikawa cells ............................. 34 2.3.5 GFP assay with stably transfected MCF-7 and Ishikawa cells ................... 35 2.3.6 Optical imaging and data analysis ............................................................. 35 2.4 Results and discussions....................................................................................... 36 2.4.1 Establishment of an ERE-regulated dual-cell line GFP expression system 36 2.4.2
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