Engineering Contractile 2D and 3D Human Skeletal and Cardiac Muscle Microtissues Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical engineering Rebecca Marie Duffy B.S., Biomedical Engineering, Virginia Commonwealth University Carnegie Mellon University Pittsburgh, PA August, 2016 Dedication In memory of Sue, Phil, and Bill. You are always in my heart Thank you to my parents, Jerry and Lisa, for always encouraging me to do my best. I wouldn't be the person I am today without all of the valuable lessons you've taught me - following through on things you start, understanding how to juggle priorities, the importance of family, how to stay true to yourself, and how to stand up for yourself. You've always told me you'd be proud of me as long as I did my best...and it looks like you've served me pretty well with that attitude. I'm so grateful for all of the sacrifices the two of you have made (that I'm probably unaware of) to make my life better and give me all of the best opportunities you could over the years. Thank you to my grandparents, Shelby and Jimmy - you two are such an important part of my life, and I want to thank you for busting your butts to raise two amazing kids and for reminding me to have perspective on how lucky I have been to be blessed with the opportunities life has given me. My grandparents Big Ed, Margaret, and BK. You all were also testaments to working hard to achieve your goals and provide for your families, and I just hope I continue to make you proud. To Kevin, thank you for keeping me balanced through this whole experience. The distance hasn't made things easy the last 3 years, but it's been so worth it. Thank you for believing in me even when I don't believe in myself, for bringing me back to reality when I start to take myself too seriously, and for always being able to make me laugh when I need it most. Here's to starting our next adventure (together)! To Lacy - my sister from another mister. You've been my partner in crime for almost 20 years now! I have always been able to lean on you through good times and bad, and I'm so excited that we've been able to encourage each other to follow our dreams (and actually see it happen in real time), from running around like hooligans on the playground, to the hilariously nerdy phases of middle and high school, to the current chapter where you're totally killing it as an editor, and I'm going to try and be the crazy mad scientist. ii To Kristy - I can't believe how lucky I was to start of my Pittsburgh adventure with you as my roommate. So many things have changed since I moved in with you 5 years ago, and I'm excited to see where the next 5 years take us. Thank you for everything you brilliant, glittering, witty, incandescent lioness! To Savannah, Zak, and Gabe. I love you guys like my own siblings and can't imagine getting to this step without all of your love and support. I decided to head off on this journey right after we all went through one of the most devastating losses I've ever experienced. But you all helped keep me strong and have always encouraged my nerd-ness (even if you show it by relentless teasing). Just don't let me turn into Dr. Steve.... And to all of my extended Duffy, Loehr, Durham and Aycock families. I've been so blessed to have such large families on all sides of my family. You all make life way more fun, and I've been able to count on all of you throughout the years. Acknowledgements The biggest of thank yous to all of the friends, mentors, collaborators, and funding sources that made all of this possible. To Professor Feinberg, thank you for taking me under your wing in a new lab and always encouraging me to do more than I thought I could. It's been a rollercoaster at times, but I've learned more than I even thought possible when I first started. You've enabled me to continue to work on projects that I still get very excited about (even if it's not as obvious to others that I am, in fact, very excited), and your encouragement helped me land a job that I didn't even think I'd be able to get. I'm excited to see where the next few years lead. My committee members - Professors Phil Campbell, Chris Bettinger, and Fabrisia Ambrosio. Thank you for your words of encouragement, insightful discussions, and input through the years. iii Thank you also to all of the faculty and staff that have made my experience here at CMU a really enjoyable one! Thank you to Dr. Wang, I remember our initial conversations when I was visiting before I chose grad school, and your commitment to having happy and healthy students was a significant part of why I chose to come here. To all of the staff (Kristin, Vanessa, Kristyn, Ryan, Keri, Julie and Crystal and Maryia) who have made my years as a grad student fun and have helped deal with all of the fun administrative stuff. For specific contributions to this dissertation: Yiyi Yu from Ge Yang's lab in Biomedical Engineering at CMU assisted with initial SDS-PAGE gels for analysis of laminin weight in Chapter 4. Logan Plath from Mark Bier's lab in Chemistry at CMU for performed the MALDI-TOF mass spectroscopy analysis of laminin in Chapter4. Breanna Duffy from Lauren Black's lab at Tufts performed LCMS analysis of laminin compositions in Chapter 4. Tong Lu from Carmel Majidi's lab in Mechanical Engineering at CMU - thank you for providing countless laser cut inserts for the 3D cardiac muscle tissues in Chapter 6. Wei Chen and Recep Onler from the Burak Ozdoganlar lab in Mechanical Engineering for 3D prints of molds used in Chapter 6 and for use of the profilometer to obtain insert thicknesses. Mathilde Vermeer and Dr. Peter Van Der Meer for getting our human embryonic stem cell culture off the ground. Seth Thompson from Professor Huard's lab at University of Pittsburgh for providing the first vial of human SkMDC cells and insights into the fickle nature of human derived cells. To all of my lab mates, thanks for making the adventure interesting. John Szymanski for being the brave one as the first grad student in our lab (and for training me in the nanofab facility for the SU-8 stamps used in Chapter 5). Quentin Jallerat and Ivan Batalov, thank you for all of your stem cell culture expertise and the countless laughs and pain that went along with differentiating cardiomyocytes. Thank you to TJ Hinton for initial 3D printed molds, designing the physical temperature controlled stage used for 2D and 3D experiments, and laser cutter training. Thank you to Yan Sun, visiting scholar from Beijing University, for training me and iv passing the torch on the initial muscular thin film project. Thanks to Alkiviadis Tsamis for generating bending models of inserts and lookup tables for bending inserts for Chapter 6. Rachelle Palchesko Simko, I can't thank you enough for all of the mentoring, sound boarding, lunches, and generally keeping me sane and encouraging me to keep going. You've been an amazing role model and I'm going to feel like I'm missing my right hand when I move to my new job without you around. Molly Bank, I’m so happy that grad school brought us together. We’ve had countless adventures through the years, and I’m so excited to see what your career evolves into. I can’t thank you enough for always being there – you let me be a temporary live in roommate during my proposal exam when my leases didn’t overlap, you let me vent to you on an almost daily basis while you were wrapping up your own PhD and made it look easy, and you’ve always been a shoulder to lean on, an inspiration, and one of the most generous people I’ve had the honor of knowing. Stephanie Wong, Elaine Soohoo, Stephanie Chang, Natasha Loghmanpour - you ladies have also been such an important part of this whole experience, general commiserating, bouncing ideas around, developing cat lady habits, and making a lot of wonderful memories inside and outside of the PTC and Scott Hall. All of my undergrads I've had the pleasure of working with: Briana Larkin, Lucas Friedman, Sabrina Liu, Stacy Chang, Eileen Ge, and Ally Vendetti. I wish you the best in your future endeavors! Also, to Jaci who is undertaking the behemoth that is the 3D cardiac MIFIs, I'm so happy to be leaving this in capable hands! I look forward to us working together, even if it's from afar, to wrap up some of my initial work. Best of luck during your grad career. Finally, I was fortunate to receive the John and Clair Bertucci Fellowship as part of my funding during my studies here. My work (and stipend...and tuition) has also been made possible by Professor Feinberg's funding sources from the NIH and HFSP. I also want to thank the Biomedical Engineering Department as a whole for accepting me in the program and providing so many wonderful resources throughout the completion of my degree.