Assessing differentiation potential of C2C12 myoblastic cells on hydrogels, and development of stimulation device to induce contraction on regular and micropatterned C2C12 cells Submitted By: Adriana Martinez-Betancourt ____________________________________________ Jeffrey Lessard ____________________________________________ Kien Dao ____________________________________________ Yow-Chyuan Yeh ____________________________________________ Submitted to: Professor Sakthikumar Ambady __________________________________________ Profressor Raymond Page __________________________________________ April 26, 201 Page | 1 Abstract We examined the differentiation characteristics of C2C12 mouse myoblasts on polyacrylamide hydrogels of varying stiffness’ in comparison to widely used polystyrene tissue culture plates. The benchmarks we used included formation of myotube fibers, BrdU incorporation, myosin heavy chain expression, and fiber contraction. As a method of observing fiber contraction, we designed, built, and used an electrical stimulation device which allowed us to run a current through the cells after they had differentiated while viewing and recording the cells in real time. The data suggest that cells grown on hydrogels proliferate less but differentiate more similar to those in vivo. Page | 2 Table of Contents Abstract ......................................................................................................................................................... 2 Acknowledgements ....................................................................................................................................... 5 Authorship Page ............................................................................................................................................ 6 Table of Figures ............................................................................................................................................ 7 Table of Tables ............................................................................................................................................. 8 Table of Equations .................................................................................................................................... 9 Chapter 1: Introduction ............................................................................................................................... 10 Chapter 2: Literature Review ...................................................................................................................... 11 2.1 Tissue Engineering ............................................................................................................................ 11 2.2 Hydrogels .......................................................................................................................................... 14 2.3 Electrical Stimulation ........................................................................................................................ 18 2.4 Micropatterning ................................................................................................................................. 21 Chapter 3: Project Strategy ......................................................................................................................... 25 3.1 Design ............................................................................................................................................... 25 3.2 Initial Client Statement ..................................................................................................................... 26 3.3 Objectives, Constraints, and Functions ............................................................................................. 27 3.3.1 Feasible Objectives .................................................................................................................... 29 3.3.2 Quantitative Analysis of Objectives........................................................................................... 32 3.4 Revised Client Statement .................................................................................................................. 39 3.5 Project Approach .............................................................................................................................. 39 Chapter 4: Alternative Designs ................................................................................................................... 40 4.1 Needs Analysis .................................................................................................................................. 40 4.2 Previous Designs ............................................................................................................................... 40 4.2.1 Mechanical stretching device ..................................................................................................... 40 4.2.2 Electrical stimulation: initial design........................................................................................... 41 4.3 Feasibility Analysis ........................................................................................................................... 42 4.3.1 Mechanical Stimulation ............................................................................................................. 42 4.3.2 Electrical Stimulation: initial design .......................................................................................... 42 4.4 Final Design Choice .......................................................................................................................... 43 4.5 Materials for Electrical Stimulator .................................................................................................... 44 4.6 Device Specifications ........................................................................................................................ 44 Page | 3 4.6.1. Stimulation procedure .................................................................. Error! Bookmark not defined. 4.7 Design Validation ............................................................................................................................. 46 Chapter 5: Design Verification ................................................................................................................... 46 5.1 Determination of Myoblast Differentiation ...................................................................................... 46 5.2 Differentiation of C2C12 cells on Polystyrene culture plates ........................................................... 48 5.3 Softwell™(Polyacrylamide hydrogels) .............................................................................................. 51 5.4 Hydrogel Stiffness Testing................................................................................................................ 53 5.5 Micro-Patterning ............................................................................................................................... 55 Chapter 6: Discussion ................................................................................................................................. 56 Chapter 7: Final Design and Validation ...................................................................................................... 58 7.1 C2C12 Mouse Myoblast cells ........................................................................................................... 58 7.2 C2C12 culture and differentiation. .................................................................................................... 59 7.2.1. Proliferation: ............................................................................................................................. 59 7.2.2 Differentiation: ........................................................................................................................... 59 7.3 Freezing C2C12 Cells ....................................................................................................................... 59 7.4 Immunocytochemistry ................................................................................................................ 60 7.4.1 BrdU proliferation assay ............................................................................................................ 60 7.4.2 Myosin Heavy Chain Expression analysis ................................................................................. 60 7.5 Micro-Patterning ............................................................................................................................... 61 7.5.1. Coverslip method ...................................................................................................................... 61 7.5.2. Cell Printing Method ................................................................................................................. 62 7.6 Testing Hydrogel Stiffness................................................................................................................ 63 7.7 Electrical Stimulation of C2C12 Multinucleated Fibers ................................................................... 64 Chapter 8: Future Work and Recommendations ......................................................................................... 64 Chapter 9: Conclusions ............................................................................................................................... 68 References ..................................................................................................................................................