Microsystems to Study the Mechanobiology of Cell Adhesion

297 Pages·2015·12.08 MB·English

MICROSYSTEMS TO STUDY THE MECHANOBIOLOGY
OF CELL ADHESION

A DISSERTATION
SUBMITTED TO THE DEPARTMENT
OF MECHANICAL ENGINEERING
AND THE COMMITTEE ON GRADUATE STUDIES
OF STANFORD UNIVERSITY
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

Joo Yong Sim
January 2015
ABSTRACT
Adherent cell functions can be altered by mechanical stimuli through
cytoskeleton remodeling and cell-cell junction disruption. Thus, a better
understanding of the mechanical response of adherent cells is crucial to
the design of pharmacological therapies for cancers and skin blistering
diseases. However, a lack of reliable tools to apply mechanical stimuli
and probe the cellular response has limited research on the effects of
varying strains on adherent cells. Therefore, I develop systems to probe
cellular mechanics using microfabrication technology with soft materials
specifically designed to exert controlled strain on adherent cells and probe
their mechanical response.

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ACKNOWLEDGEMENTS
Fellowships and Funding
Ilju Study Abroad Doctoral Fellowship, Stanford Bio-X Fellowship.
National Science Foundation’s EFRI CBE0735551, EFRI MIKS-1136790 and Bio-X seed
grant.
Personal Thanks
This research and, in fact, my PhD career would not have been possible without so many
people whom I admire, care, and love. I am deeply grateful to the list of people who have
supported me and provided the guidance and inspiration throughout my PhD for my thesis
work.
Most important of all, I would like to thank my advisor, Dr. Beth Pruitt for guiding me along
this academic journey and for being patient with me. Throughout the last six years, she has
provided an immense amount of technical, intellectual and personal support. I must express
my sincere appreciation to her. Thank you, Beth for your invaluable advice and mentorship,
guiding me this wonderful journey to become a doctor and teaching me a solid confidence that
I can learn anything I want.
I'd also like to thank my committee Dr. W. James Nelson for his long lasting support and
friendly advice. Working with him and his lab has been a great privilege and keeping me
inspired on this thesis work. I had been benefited tremendously by learning from his
publications and lecture. I must also thank my committee Dr. Alex Dunn whom I owe much of
my progress in the molecular and biological aspects of my thesis for their insightful questions
and critical reviews and discussions of my work. I'd also like to say a big thank you to Prof.
Ovijit Chaudhuri and Prof. Roger T. Howe for being my PhD defense committee member.
I am grateful to have been the group of Pruitt lab. I have learned something from each of you.
I owe a special acknowledgement to Keekyoung Kim and Sun Jin Park for their mentorship
when I joined the group. Thank you for all of our conversations and supports for the last six
years. I'd also like to thank Chelsey Simmons, Jens Moeller, Kevin C. Hart, Diego Ramallo
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and Ehsan Sadeghipour for their kind friendships, positive energies and wise words not only in
research as well as in my graduate life at Stanford and ETH Zurich.
I am indebted to friends in KME (Korea Mechanical Engineering) and KBAS (Korea
Basketball Association at Stanford) for friendship and joy. Especially, Hyungchai Park who
gave me invaluable friendship and who I admire his humaneness, leadership and integrity.
Last but not least, I would like to thank my family. My parents and sister provided me endless
support and resources so I can be where I am today. They have offered me so much energy
that allowed me to keep strong regardless of the circumstances, and I look forward to the
future ahead of us.

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TABLE OF CONTENTS
Abstract ························································································· iv
Acknowledgements ············································································ v
List of Figures ················································································· xii
List of Tables ··················································································xvi
1. Introduction and Background: Microengineered Platforms to Study Cell
Mechanobiology ················································································ 1
Abstract ···················································································· 1
1.1 Mechanical Aspect of Biological Cells and Tissues ···························· 1
1.2. Microengineered Platforms to Study Mechanobiology of Cells ············· 5
1.2.1 Techniques to Study Cell Mechanics ···································· 6
1.2.2 Mechanically Dynamic Cell Culture Systems ························· 8
1.3 Cell Mechanics Models ··························································· 13
1.4 Techniques to Measure Cell-Generated Forces ································ 15
1.5 Recent Developments on Techniques To Study Cell-Cell Adhesion
Mechanobiology ································································· 18
1.6 Outline of Dissertation ···························································· 21
References of Chapter 1 ······························································· 24

2. Integrated Strain Array for Dynamic Cell Culture ······························· 35
Abstract ················································································ 35
2.1 Design Space of Integrated Strain Array ···································· 35
2.2 Concept of the Integrated Strain Array ······································ 38
2.3 Design Specs and Design Decisions ········································· 40
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2.3.1 Base Plate Design ························································ 40
2.3.2 Cell Culture Layer Design ·············································· 43
2.3.3 Pressure Controller Design ·············································· 45
2.4 Fabrication of the Integrated Strain Array··································· 47
2.4.1 Pneumatic Chamber Fabrication ································· 47
2.4.2 Cell Culture Chamber Fabrication ······························ 47
2.4.3 Device Assembly and Lubrication ······························· 49
2.4.4 Integration with Pneumatic Control System ···················· 50
2.5 Methods for Finite Element Model and Calibration ······················· 52
2.5.1 Finite Element Model (FEM) ····································· 52
2.5.2 Calibration ··························································· 53
2.6 Results and Discussion ························································· 54
2.6.1 Finite Element Model ·············································· 54
2.6.2 Calibration ··························································· 56
2.6.3 Cell Studies on Integrated Strain Array ························ 58
2.7 Conclusion ······································································· 61
References of Chapter 2 ····························································· 62

3. Micropost Array Calibration and Cross-Correlation Image Processing ····· 65
Abstract ··············································································· 65
3.1 Introduction ······································································ 66
3.2 Image Processing algorithm to Track the Micropost Arrays with Sub Pixel
Resolution ············································································ 68
3.2.1 Previous Studies to Find Micropost Array Displacement ···· 68
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3.2.2 Experimental Set-up for Imaging Neonatal Cardiomyocytes on
Micro Post Arrays ························································· 68
3.2.3 Imaging Processing Using Hough Transform to Find Region of
Interest ······································································ 69
3.2.4 Imaging Processing Using Cross-Correlation Algorithm ····· 71
3.3 Calibration and Characterization of Micropost Stiffness ················· 73
3.3.1 Calibration of Micropost Array Using a MEMS force Sensor 74
3.3.2 Measurement of the Mechanical Properties of Stiff Skin using
Surface Film Buckling ···················································· 77
3.2.3 Theoretical Modeling of Micropost with Stiff Skin ··········· 79
3.3.4 Characterization of Micropost Array Stiffening due to Plasma
Oxidation ··································································· 83
3.3.5 Implication of Calibrated Micropost with Considering Oxidation
Stiffening ··································································· 86
3.4 Conclusion ······································································· 88
References of Chapter 3 ····························································· 90

4. Microfluidic Cell Stretcher for High Resolution Live Cell Imaging ··········· 93
Abstract ··············································································· 93
4.1 Introduction ······································································ 94
4.2 Principle of Microfluidic Cell Stretcher ····································· 96
4.3 Fabrication of the Uniaxial Cell Stretcher ··································· 97
4.4 Surface Treatment for Extracellular Matrix Coating ······················ 98
4.5 Cell Culture and Transfection ················································· 99
4.6 Straining cells by Programmable Modulation ····························· 100
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4.7 Cell Straining Experiment with FRET Probes ····························· 102
4.8 Lessons Learned and Fabrication Trouble Shooting ······················ 104
4.9 Alternative Approaches for Live Cell Stretching ························· 106
4.9.1 Scaling Down Integrated Strain Array ······························· 106
4.9.2 Scaling Up Microfluidic Cell Stretcher ······························ 110
4.10 Integration of Micropost arrays with Uniaxial Loading ················ 112
4.6 Conclusion ······································································ 114
References of Chapter 4 ···························································· 115

5. Molecular Force of E-cadherin in Various Micromechanical Environments 117
Abstract ················································································· 117
5.1 Introduction ········································································ 117
5.2 Intensity-based Measurement of Ratiometric FRET ························ 118
5.2.1 Spectral Bleed-Through Correction for the Intensity-based
Measurement of Ratiometric FRET ········································· 119
5.2.2 High FRET and Low FRET Controls ································ 121
5.2.3 Kinetics of EcadTSMod Sensor Agrees with E-cadherin DsRed 122
5.3. Substrate Stiffness Affects Molecular Tension on E-cadherin ············ 125
5.3.1 PDMS Substrate Stiffness Characterization ························· 125
5.3.2 PDMS Spin Speed and Film Thickness Characterization ········· 126
5.3.3 Molecular Tension on E-cadherin on Varying Stiffness of PDMS
Substrate ········································································· 127
5.4 E-cadherin Tension Decreases as the Number of Neighboring Cells Increases
···················································································· 128
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5.5 E-cadherin Tension Decreases as the Density of Cells Increases ·········· 130
5.6 Conclusion ········································································· 131
References of Chapter 5 ···························································· 133

6. Spatial Distribution of Cell-Cell and Cell-ECM Adhesions Regulates Force
Balance and E-cadherin Tension in Cell Pairs ········································ 135
Abstract ················································································· 135
6.1 Introduction ········································································ 136
6.2 Techniques to Modulate Mechanical Environments and Probe the Cellular
and Molecular Forces ······························································· 140
6.2.1 Micropatterning Technique Using Photoresist-Assisted Lift-Off
Technique ······································································· 140
6.2.2 Traction Force Microscopy on Micropatterns ······················· 141
6.2.3 Fluorescence Lifetime Imaging Microscopy (FLIM) ·············· 143
6.3 Spatial Distribution of Cell-Cell and Cell-ECM Adhesions Regulates Force
Balance ·········································································· 144
6.3.1 Constraining the Shape of Cell Pairs and Measuring F and F · 144
∥ ⊥
6.3.2 Forces at Cell-ECM and Cell-Cell Contacts Increase with Cell
Spread Area on Squares ······················································· 145
6.3.3 F and F Increase on Elongated Rectangles ······················· 146
∥ ⊥
6.3.4 F Maintains a Constant Level with Isotropic Increase in the I-Shape
⊥
Area ·············································································· 147
6.3.5 F⊥ and F∥ Stays Constant on Elongated I-Shapes ················ 148
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6.3.6 Cell-Generated Forces and Strain Energies in Cell Pairs Are
Regulated by Cell Spread Area ··············································· 149
6.3.7 Cell Pairs Generate Larger Per-Cell Forces than Single Cells Even
With Smaller Per-Cell Spread Area ········································· 150
6.4 E-cadherin Tension Independent of F ······································· 152
⊥
6.5 The Actin Cytoskeleton and E-cadherin Are under Tension at the Edges of
Cell-Cell Contacts in Cell Pairs on I-Shapes ···································· 154
6.6 E-cadherin Localized at the Edges of Cell-Cell Contact but Molecular
Tension of E-cadheirn Is Evenly Distributed Along the Cell-Cell Contact · 157
6.7 Discussion ·········································································· 158
6.8 Conclusion ········································································· 161
References of Chapter 6 ···························································· 163

7. Conclusion and future work ··························································· 167
7.1 Concluding remarks: for the next generation of tools for mechanobiology
studies ················································································ 167
7.2 Future work and lessons learned ················································ 168
7.2.1 Improvements on Integrated Strain Array ··························· 168
7.2.2 Vinculin Localization at the Cell-Cell Contacts of MDCK Cells
Undergoing Strain ······························································ 169
7.2.3 Mechanical Characterization of Oxidized PDMS Surfaces ······· 173
7.2.4 Improvements on the Microfluidic Live Cell Stretching Device · 174
7.2.5 Alternative Molecules Contributing to the Force Bearing ········· 174
References of Chapter 7 ······························································ 176

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