Porous Nanocrystalline Silicon Membranes as Highly Permeable and Molecularly Thin Substrates for Cell Culture by Anant Akash Agrawal Submitted in Partial Fulfillment of the Requirements for the Degree Master of Science Supervised by Professor James L. McGrath Department of Biomedical Engineering Arts, Sciences and Engineering Edmund A. Hajim School of Engineering and Applied Sciences University of Rochester Rochester, New York 2009 CURRICULUM VITAE The author was born in Raipur, India on January 18, 1984. He attended M. S. Ramaiah Institute of Technology, Bangalore, India under the Visveswaraiah Technological University from 2003 to 2007, and graduated with a Bachelor of Engineering (Biotechnology) degree in 2007. Anant began his graduate studies in Biomedical Engineering at the University of Rochester in Fall of 2007. He pursued his research in using recently discovered nanomembranes for cell culture under the direction of Professor James L. McGrath and received the Master of Science degree from the University of Rochester in 2009. i ACKNOWLEDGEMENTS I thank my advisor Professor James McGrath for giving me the opportunity to work in a wonderful lab environment. His supportive, dedicated and compassionate guidance helped me grow my scientific insight during this research work and I wish to carry the lessons he has taught me through my life. I also thank my defense committee, Professors Philippe Fauchet and Harold Smith. I really appreciate the time and effort they have put in this process. There are many people from the Nanomembrane Research Group that have helped me with their valuable inputs. I would like to start with thanking Karl Reisig, SiMPore Inc. co-op student and a friend, for helping significantly with the cell culture experiments. I thank Barrett Nehilla, a post-doctoral fellow in McGrath lab, for his immense support and guidance in lab as well as in preparing this thesis. I am grateful to Henry Chung and Jess Snyder for their contributions in diffusion experiments as well as their friendship and encouragement. I thank other past and present members of the McGrath lab, particularly Mort Ehrenberg, Tom Gaborski, Mike Hoffman, Rachel Twardowski, Derek Crowe for thoughtful discussions and general camaraderie. Importantly, I would like to thank Dave Fang, Chris Striemer and other employees of SiMPore Inc. for their role in nanomembrane fabrication and testing. I thank Loel Turpin at University of Rochester Medical Centre for providing human vascular cells used in this study. I appreciate all the help from the BME office staff, especially Donna Porcelli for her efforts in answering my queries. Last but not the least; I thank my parents (Alka and Suresh) for their unconditional love, support and belief. I would like to thank my sister and brother-in-law (Avanti and Amit) for their affection and good wishes. I thank my circle of friends, from both here and back in India (Rajesh, Nakul, Shilpa, Dhiren, Ryan, Carlos, Dan, Manoj, Justin, Nate, Shikhir, Shail, Rahul, Vignesh, Chirag, Ritesh, Sunaina and more) for making me feel lucky. I particularly thank Vijaya, my best friend, for her undying encouragement and cooperation, not forgetting all the meals she cooked for me during this period. ii ABSTRACT Porous nanocrystalline silicon (pnc-Si) is a novel silicon based material with potential uses in lab-on-a-chip devices, drug delivery, cell culture, tissue engineering and biosensing. The pnc-Si material is an ultrathin (15 nm), free-standing, nanoporous membrane made with highly scalable semiconductor technology. Because pnc-Si membranes are approximately 1000 times thinner than any polymeric membrane, the permeability of pnc-Si to small solutes is orders-of- magnitude greater than conventional materials. As cell culture substrates, pnc-Si membranes could overcome the inadequacy of membrane materials used in conventional transwells and enable new types of culture devices for the precise control and manipulation of cellular microenvironments. The objective of this thesis is to investigate the feasibility of pnc-Si as a cell culture substrate by comparing cell adhesion, spreading, growth kinetics and viability on pnc-Si to conventional tissue culture substrates. By each of these metrics, the behaviors of both immortalized cells (3T3-L1 fibroblast cell line) and primary vascular endothelial cells (HUVEC) were found to be highly similar on pnc-Si, tissue culture grade polystyrene and glass. Significantly, pnc-Si was found to degrade in cell culture media over several days without cytotoxic effects. Membrane stability could be tuned by modifying the density of a superficial native oxide layer through post production rapid thermal processing and additional surface treatment like amino-silanization. Pnc-Si was assembled in a custom designed tube to form a pnc-Si transwell device that could replace existing 24-well plate inserts. The adhesion of HUVEC to treated pnc-Si transwells was found to be comparable to commercial transwells. Collectively, the results establish pnc-Si as a viable new substrate for cell culture and a new type of degradable biomaterial. Pnc-Si membranes should find many uses in bioscience including the study of molecular transport through cell monolayers, as molecularly thin dividers for the study of cell-cell communication in co-cultures, and as biodegradable scaffolds for three-dimensional tissue constructs. iii TABLE OF CONTENTS Chapter 1: Introduction 1.1 Nanotechnology…………………………………………………………………………………..…2 1.2 Nanoporous Membrane Technology……………………………………………………..……….4 1.2.1 Overview of Membrane Technology……………………………………………..…………4 1.2.2 Polymeric Membranes……………………………………………………………..…………5 1.2.3 Inorganic Membranes………………………………………………………….……………11 1.2.4 Advanced Membrane Technology: Microsieves and Nanosieves…...……...…………15 1.2.5 Membrane Processes and Applications………………………………………..…………25 1.3 Porous Silicon (P-Si): A Biomaterial…………..…………………………………..……………..30 1.3.1 Formation of P-Si………….……………………………………………………...…………30 1.3.2 Biocompatibility of P-Si……………..………………………………………………………31 1.3.3 Biodegradation of P-Si…………………...…………………………………………………32 1.3.4 Enhancing Stability in Physiological Fluids…………….…………………………………33 1.4 Porous Nanocrystalline Silicon (Pnc-Si): A Novel Material……………………………………35 1.4.1 Characteristics of Pnc-Si Membranes……………………………………….……………36 1.4.2 Thermodynamically Driven Fabrication of Pnc-Si…………………………..……………37 1.4.3 Molecular Separations using Pnc-Si………………………………………………………38 1.5 Membrane Based Cell Culture for Cell Biology Research………………………….…………40 1.5.1 Three Dimensional Transwell Cell Culture as In Vitro Models …………………………40 1.5.2 In Vitro Cell Culture Based Assays…………………………………………………..……42 1.5.3 Transwell Membrane Properties for Better Assay Sensitivity……………………..……44 1.5.4 Potential Advantages of Pnc-Si Membranes over Traditional Membranes…………...45 1.6 Thesis Outline and Research Objectives……………………………………………………..…46 iv TABLE OF CONTENTS (continued) 1.6.1 Investigation and Control of Pnc-Si Membrane Biodegradation in Physiological Media………….…………………………………………………………...…46 1.6.2 Demonstration of Pnc-Si Membranes as a Biocompatible Support for Cell Culture Growth……………...………………………………………………………….46 1.6.3 Demonstration of Pnc-Si Membrane Performance in Three Dimensional Transwell Format…………………………………………….………………47 1.7 References…………………………………………………………………………….……………48 Chapter 2: Stability of Porous Nanocrystalline Silicon (Pnc-Si) in Cell Culture Media 2.1 Introduction…………………………………………………………………………………………62 2.2 Material and Methods……………………………………………………………………………...68 2.2.1 Materials..…………………………………………………………………………….………68 2.2.2 Cell Culture…………………………………………………………………………………..69 2.2.3 Pnc-Si Fabrication ………………………………………………………………………..…69 2.2.4 Post Production Rapid Thermal Processing……………………………………...………70 2.2.5 Pore Processing………………………………………………………………..……………70 2.2.6 Rhodamine Diffusion ………………………………………………………………….……71 2.2.7 Chemical Stability and Biodegradation ………………………………………………...…72 2.3 Results and Discussion…………………………………………………………….....................74 2.3.1 Correlation between Pnc-Si Degradation and Discoloration ……………...……………74 2.3.2 Other Influences on Pnc-Si and Its Dissolution…………………………………..………82 2.3.3 Enhancing Stability of Pnc-Si..………………………………………………………..……87 2.4 Conclusion………………………………………………………………………………………….89 v TABLE OF CONTENTS (continued) 2.5 References……………………………………………………………………………….…………90 Chapter 3: Investigating Pnc-Si Biocompatibility: Cell Culture in Two Dimensional Format 3.1 Introduction…………………………………………………………………….………...…………94 3.2 Material and Methods……………………………………………………………………………...96 3.2.1 Materials..…………………………………………………………………………….………96 3.2.2 Cell Culture…………………………………………………………………………………..97 3.2.3 Cell Adhesion……………………………………………………………...…………………97 3.2.4 Cell Spreading...…………………………………………………………………..…………98 3.2.5 Cell Proliferation…………………………………………………………………………..…98 3.2.6 Cell Viability………………………………………………………………………..…………99 3.2.7 Cell Counting………………….………………………………………………………..……99 3.2.8 Data Analysis……………………………………………………………………………….100 3.3 Results and Discussion………………………………………………………………………….100 3.3.1 Cell Adhesion………….……………………………………………………….…………..100 3.3.2 Cell Spreading……………..………………………………………………………….……103 3.3.3 Cell Proliferation……………………………………………………………………………104 3.3.4 Effects of Pnc-Si Degradation on Cell Proliferation ……………………………………106 3.3.5 Cell Viability…………………………………………………………………………………107 3.4 Conclusion………………………………………………………………………………………...110 3.5 References……………………………………………..…………………………………………111 vi TABLE OF CONTENTS (continued) Chapter 4: Pnc-Si