Clemson University TigerPrints All Theses Theses 12-2018 System for the Application of Hydrostatic Pressure and Mechanical Strain to Cell Cultures Justin Bacaoat Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_theses Recommended Citation Bacaoat, Justin, "System for the Application of Hydrostatic Pressure and Mechanical Strain to Cell Cultures" (2018). All Theses. 2966. https://tigerprints.clemson.edu/all_theses/2966 This Thesis is brought to you for free and open access by the Theses at TigerPrints. It has been accepted for inclusion in All Theses by an authorized administrator of TigerPrints. For more information, please contact [email protected]. SYSTEM FOR THE APPLICATION OF HYDROSTATIC PRESSURE AND MECHANICAL STRAIN TO CELL CULTURES A Thesis Presented to the Graduate School of Clemson University In Partial Fulfillment of the Requirements for the Degree Master of Science Bioengineering by Justin Bacaoat December 2018 Accepted by: Jiro Nagatomi, PhD, Committee Chair William Richardson, PhD Ken Webb, PhD ABSTRACT Pressure and stretch are two of the primary forces that result in mechanotransductory events which regulate certain aspects of human health and disease. Laboratory systems such as the commercially available Flexercell® system and a variety of custom-made setups are currently used in research to systematically apply stretch and hydrostatic pressure independently, or in conjunction to cell and tissue cultures. However, these systems do not allow for the decoupling of pressure and stretch under the same culture conditions. The present study aims to design, fabricate, and calibrate a device that can apply pressure and stretch simultaneously, as well as independently to cells in culture. Moreover, in order to characterize the mechanical behavior of the cell substrate, equibiaxial mechanical testing was conducted on the substrate and the resulting data were used to generate a finite element simulation of the device. Moreover, an analytical approach was used in an attempt to validate the simulation. To our knowledge, this is the first system that can definitively distinguish between the mechanotransductive events activated in response to pressure and stretch. Using this novel device, MYP3 cells cultured on fibronectin-coated substrates were exposed to pressure and stretch, together or independently. The cells exhibited the morphology characteristic of healthy urothelial cells. The extracellular ATP data indicated an increase in ATP release in response to mechanical stimuli. Caspase-1 activity decreased in response to mechanical stimuli. The present study was successful in creating a unique device capable of applying pressure and stretch, together and independently, to cells in culture to allow examination of the relative contributions of these stimuli in various mechanobiological events. ii ACKNOWLEDGMENTS Firstly, I would like to thank my advisor Dr. Nagatomi for all of his patience and support. His guidance and persistence have helped me grow as a bioengineer and as a person. I would also like to thank the members of the Cell Mechanics and Mechanobiology Laboratory, especially Cody Dunton for always being available for assistance and for all of the contributions he made to this project. I would like to thank my committee members for dedicating their time and effort to this project. I want to thank Dr. Richardson for allowing the use of his lab’s BioTester. I also would like to thank Dr. Vladimir Reukov for allowing the use of his lab’s 3D printer. Special thanks go to Chad McMahan who has provided assistance many times throughout this project and also Maria Torres who has always been helpful in keeping me on track to graduate. Thanks to the IBIOE department for allowing the use of their laser cutter, and a huge thanks to the Clemson MTS department. Lastly, I would like to thank my family and close friends whose support has carried me through my entire graduate experience. iii TABLE OF CONTENTS Page TITLE PAGE .................................................................................................................... i ABSTRACT ..................................................................................................................... ii ACKOWLEDGMENTS ................................................................................................. iii LIST OF TABLES .......................................................................................................... vi LIST OF FIGURES ....................................................................................................... vii CHAPTER 1. INTRODUCTION AND BACKGROUND ........................................................ 1 1.1 Introduction ........................................................................................ 2 1.2 Mechanobiology ................................................................................ 2 1.3 Bioreactors ......................................................................................... 7 2. RATIONALE AND SPECIFIC AIMS .............................................................. 11 3. MATERIALS AND METHODS ....................................................................... 14 3.1 Description of Pressure-Stretch Bioreactor ..................................... 14 3.2 Calibration of Pressure-Stretch Bioreactor ...................................... 15 3.3 Mechanical Characterization of Culture Substrate .......................... 18 3.4 Exposure of Urothelial Cells to Pressure-Stretch ............................ 23 4. RESULTS .......................................................................................................... 26 4.1 Description of Pressure-Stretch Bioreactor ..................................... 26 4.2 Mechanical Characterization of Culture Substrate .......................... 27 4.3 Calibration of Pressure-Stretch Bioreactor ...................................... 37 4.4 Exposure of Urothelial Cells to Pressure-Stretch ............................ 38 5. DISCUSSION .................................................................................................... 41 5.1 Mechanical Characterization of Culture Substrate .......................... 41 5.2 Calibration of Pressure-Stretch Bioreactor ...................................... 45 5.3 Exposure of Urothelial Cells to Pressure-Stretch ............................ 46 iv Table of Contents (Continued) Page 6. CONCLUSIONS AND RECOMMENDATIONS ............................................ 49 APPENDIX .................................................................................................................... 51 A: COMSOL Outputs ............................................................................................. 51 REFERENCES .............................................................................................................. 53 v LIST OF TABLES Table Page 4.1 Pressure-loss results from calibration test.................................................... 37 A.1 2-parameter Mooney Rivlin model fitting data as seen in Figure 4.7 ......... 51 A.2 5-parameter Mooney Rivlin model fitting data as seen in Figure 4.8 ......... 51 A.3 Displacement field data for the COMSOL simulation of 122.3 cmH2O as seen in Figures 4.11 and 4.12. ........................................................................ 52 A.4 Displacement field data for the COMSOL simulation of 180.5 cmH2O as seen in Figures 4.11 and 4.12 ......................................................................... 52 A.5 Displacement field data for the COMSOL simulation of 225 cmH2O as seen in Figures 4.11 and 4.12 ............................................................................. 52 vi LIST OF FIGURES Figure Page 3.1 Schematic diagrams of the pressure device assembly ................................. 17 3.2 Custom stamp used for imprinting coordinate system onto silicone substrate samples .............................................................. 21 3.3 Side-view image of a wax replica of the deformed substrate ...................... 21 3.4 Diagram of strain quantification values used in image analysis of wax replicas ........................................................................ 22 4.1 Assembly of the upper and lower chambers for the Pressure-Stretch Bioreactor ................................................................... 26 4.2 Silicone cell culture substrate assembly ...................................................... 27 4.3 Stress-strain curve data from first round of equi-biaxial load testing ............................................................................................. 28 4.4 Stress-strain curve data for the first and fifth cycles of the second round of equi-biaxial load testing .............................................. 29 4.5 Stress-strain curve data for the fourth cycle of the third round of equi-biaxial load testing. Data are mean +/- the standard error of mean (n=3). .......................................................... 29 4.6 Average stress for each axis at the maximum strain for round 3, cycle 4. Data are mean +/- the standard error of mean ........................ 30 4.7 2-parameter Mooney-Rivlin model fitting data using the stress-strain data obtained from round 2, cycle 5, of planar biaxial testing. ......................................................................... 31 4.8 5-parameter Mooney-Rivlin model fitting data using the stress-strain data obtained from round 2, cycle 5, of planar