SCALABLE TOPOGRAPHY-INDUCED CELL ALIGNMENT FOR TISSUE ENGINERING ...

179 Pages·2014·9.76 MB·English

SCALABLE TOPOGRAPHY-INDUCED CELL ALIGNMENT FOR
TISSUE ENGINERING APPLICATIONS

ANENE-NZELU CHUKWUEMEKA ONYEKA GEORGE
(M.D) Cuba

A THESIS SUBMITTED
FOR THE DEGREE
DOCTOR OF PHILOSOPHY (PH.D)

DEPARTMENT OF BIOMEDICAL ENGINEERING
NATIONAL UNIVERSITY OF SINGAPORE
2014

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DECLARATION

I hereby declare that this thesis is my original work
and has been written by me in entirety. I have duly
acknowledged all sources of information which have
been used in the thesis

This thesis has not been submitted for any degree in
any university previously

Anene-Nzelu Chukwuemeka George Onyeka
th
4 August 2014

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ACKNOWLEDGEMENTS
I wish to thank my supervisors: Dr. Leo Hwa Liang, Prof Hanry Yu,
and Dr Gary Ng Sum Huan for their guidance, inspiration and mentorship
throughout my years of PhD study at NUS. They played very pivotal roles in
transforming me from an amateur student to a budding scientist. The journey
has not been easy but thanks to their support and insights I have made it this
far. I am hugely indebted to Dr. Yi-Chin Toh, her meticulous guidance and
excellent expertise went a long way in grooming me as a scientist. Thanks to
Dr. Toh for not giving up on me even though I seemed hopeless at the
beginning. My gratitude of course goes to NUS for the research scholarship
and the opportunity granted to me to study in this world-acclaimed university
I remain eternally grateful to the friends I met in the lab, namely: Dr.
Abhishek Ananthanarayanan, Dr. Tee Yee Han, Dr. Narmada Balakrishnan
and family, Dr. Lakshmi Venkatraman, Dr Justin and Family, Dr Ali Shazib
and Derek. Thanks for your support, you guys made living in Singapore
enjoyable. Thanks for all the laughter, outings and home visits, I really
appreciate everything. Million thanks to Dr Kuan Yee Han (Mr. President) for
all the travel adventures and for being a great friend. Thanks for opening my
eyes to the world of travelling; it has given me a better perspective of life. I
also want to extend my gratitude to other members of Cell and Tissue
engineering lab for all their support: Shu Ying has so much positive energy;
Phoebe is a giant with a gentle spirit, Pornteera, Yu Yu, Chan Way, Xiao Bei,
Xia Lei, Wei Yan, Inn Chuan and all other members of the lab. I’m really
grateful for the friendship over the years. Jason, Jia Loon, Ryan Lim Jean
Yves and Klement were awesome friends I met here in NUS. Thanks for all
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the times we shared together. Thanks as well to members of the Fluid
mechanics lab in Biomedical engineering: Sean, Foad, Munirah etc.
I wish to continue by thanking friends I made in church, worthy of
special mention are Keng Yee, Jes Mie, Pamela Koo and Ruth. You guys have
been a wonderful source of strength, inspiration and encouragement. I pray
God blesses you all richly. I’m also grateful to, Edward, Hansel, Jing Ting,
Jesse, and other members of the CG for the spiritual support I got from you
guys. Garuda house members were awesome, the list is endless, but just to
mention a few names: Ryan (Janet), Mikel, Melvin, Daniel, Claude, Abhi,
Minh Tu, Daryl, Tuck, Celine, Yityng, Charles, and all the members of
“collective calories” etc. Thanks for the daily dose of laughter and all the
crazy things you guys do. Dr. Tan was a great mentor, thanks for all your
words of wisdom. Members of the BME grad club deserve a special mention.
Ramesh, Soneela, Chaitanya, Poh Yong Cheng etc. It was great working with
you guys. I also had a great time working with the RA’s and RF’s in College
of Alice and Peter Tan.
I cannot forget to mention my “Amigos” for keeping me company
albeit from a distance. I always looked forward to whatsapp chat messages
from you guys. The crazy bunch includes: Ope girl, Chuki, Hajiya, Loco
wada, Queenie, Nike, Ijeoma, Tiny mama, Rafael, Afro. Thanks for all the
stories and jokes and words of encouragement. The friends in made in Cuba
were also very supportive and I love you guys immensely. Thanks for all the
calls, and words of encouragement I got from you guys. Your prayers
definitely helped level some of the mountains I met on the way. Sarita, Liz,
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Willyams, Milvia, Miguelito, Amos, Jimada, Okechukwu, Pastor Mayito and
family. I do hope to meet you guys some day in the future
Finally I want to thank my awesome family for the great support.
Words can’t describe how much I love you all, I’m sure the best years of our
lives are yet to come. Thanks to Dad, Mum, Amara, Ken, Uche, Onyi, Chinua,
Ebuka. I love you all immensely. Above all, all glory to Jesus for your great
love which is better than life. I am reminded that “though the mountains are
shaken and the hills are removed, your unfailing love for me would not be
shaken”.

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TABLE OF CONTENTS
ACKNOWLEDGEMENTS ............................................................................. iii
LIST OF PUBLICATIONS AND CONFERENCE PROCEEDINGS .......... xiii
LIST OF PATENTS ........................................................................................ xv
SUMMARY .................................................................................................... xvi
LIST OF FIGURES ..................................................................................... xviii
LIST OF TABLES ........................................................................................... xx
LIST OF SYMBOLS AND ABBREVIATIONS ........................................... xxi
CHAPTER 1: INTRODUCTION ...................................................................... 1
1.1 Cell alignment in vitro and in vivo ............................................................... 1
1.2 Cell alignment for differentiation and maturation ....................................... 2
1.2.1. Cell alignment for skeletal muscle differentiation and maturation...... 3
1.2.2 Cell alignment for neuronal differentiation .......................................... 5
1.3 Cell alignment incorporated into in vitro platforms for drug screening .... 11
1.3.1 In vitro drug testing ............................................................................. 11
1.3.2 Importance of drug induced cardiotoxicity and clinical manifestations
...................................................................................................................... 13
1.3.3. Mechanistic understanding of drug induced cardiotoxicity ............... 14
1.3.4. Importance of replication of tissue microenvironment in vitro.......... 16
1.4. Cell alignment in microfluidic chips as in vitro tools for biological
applications ...................................................................................................... 18
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1.4.1 Embedding micro and nanoscale cues in microfluidic chips .............. 19
1.5. Current methods used for creating cell alignment in vitro and the
drawbacks ........................................................................................................ 22
1.5.1 Mechanical stimulation ....................................................................... 23
1.5.2 ECM Patterning .................................................................................. 27
1.5.3 Electrical Stimulation .......................................................................... 28
1.5.4 Micro/Nanogrooves ............................................................................. 30
1.6 Commercially available optical media....................................................... 36
1.6.1 Mechanically Ruled Gratings ............................................................. 36
1.6.2 Holographically Ruled Gratings ......................................................... 37
1.6.3 Replication of the gratings .................................................................. 37
1.7 Use of optical media in biological research ............................................... 38
CHAPTER 2: OBJECTIVE AND SPECIFIC AIMS ...................................... 41
2.1 Specific Aim 1: Harnessing nanogrooved commercially available optical
media as platforms for aligning cardiac, skeletal and neuronal cells in culture
.......................................................................................................................... 42
2.1.1 Objective: ............................................................................................ 42
2.1.2 Experimental design ............................................................................ 42
2.2 Specific Aim 2: Engineering scalable 2D and 3D cellular constructs of
aligned skeletal myoblasts in PDMS microfluidic chips embedded with
nanogrooves ..................................................................................................... 42
2.2.1 Objective: ............................................................................................ 43
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2.2.2 Experimental design ............................................................................ 43
2.3 Specific Aim 3: Developing a high throughput in vitro platform for
physiological and pharmacological study of neonatal rat cardiomyocytes ..... 43
2.3.1 Objective: ............................................................................................ 43
2.3.2 Experimental design:........................................................................... 43
CHAPTER 3: HARNESSING NANOGROOVED COMMERCIALLY
AVAILABLE OPTICAL MEDIA AS PLATFORMS FOR ALIGNING
CARDIAC, SKELETAL AND NEURONAL CELLS IN CULTURE ........... 45
3.1. Introduction ............................................................................................... 45
3.2. Materials and methods .............................................................................. 48
3.2.1. Cell lines ............................................................................................. 48
3.2.2. Processing of CD-R/DVD-R/ and optical gratings for cell culture ... 48
3.2.3. Surface characterization by Atomic Force Microscopy (AFM) and
Scanning Electron Microscopy (SEM) ......................................................... 49
3.2.4. Nano indentation to determine Young’s modulus .............................. 49
3.2.5. Plasma treatment to render the surface hydrophilic .......................... 49
3.2.6. Water contact angle measurement of the surface .............................. 50
3.2.7. ECM coating of CD-R/DVD-R and optical gratings ......................... 50
3.2.8. Conjugation of RGD on PET film ...................................................... 50
3.2.9 Rat hepatocyte isolation and culture ................................................... 50
3.2.10. Picogreen assay................................................................................ 51
3.2.11. Alamar Blue assay ............................................................................ 51
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3.2.12. RNA isolation, cDNA synthesis and qPCR analysis ........................ 52
3.2.13. Differentiation of H9C2 cells ........................................................... 52
3.2.14. Differentiation of C2C12 cells ......................................................... 53
3.2.15. Differentiation of PC12 cells............................................................ 53
3.2.16. Immunofluorescent assessment of differentiation markers and F-
Actin ............................................................................................................. 54
3.2.17. Quantification of nucleus and cell orientation ................................. 54
3.2.18. Quantification of cell area ............................................................... 55
3.2.19. Statistical analysis ............................................................................ 55
3.3. Results and discussion .............................................................................. 56
3.3.1. Preparation and characterization of the optical media ..................... 56
3.3.2. Cell attachment and growth ............................................................... 60
3.3.3. Morphology and morphometric analysis ........................................... 62
3.3.4. H9C2 cell differentiation .................................................................... 66
3.3.5. C2C12 cell differentiation and maturation ........................................ 69
3.3.6. PC12 cell differentiation .................................................................... 72
3.3.7. Hepatocyte culture on RGD-conjugated optical media ..................... 74
3.4. Concluding remarks .................................................................................. 75
Chapter 4: ENGINEERING SCALABLE 2D AND 3D CELLULAR
CONSTRUCTS OF ALIGNED SKELETAL MYOBLASTS IN PDMS
MICROFLUIDIC CHIPS EMBEDDED WITH NANOGROOVES .............. 77
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4.1 Introduction ................................................................................................ 77
4.2 Material and Methods ................................................................................ 80
4.2.1. Pattern transfer from optical media to PDMS ................................... 80
4.2.2 Design, fabrication and assembly of microfluidic chip....................... 81
4.2.3 Surface characterization of PDMS micro-groove substrates by Atomic
Force Microscopy (AFM)............................................................................. 82
4.2.4 Cell culture .......................................................................................... 82
4.2.5 Total RNA isolation and gene expression analysis ............................. 83
4.2.6. Immunofluorescence staining ............................................................. 84
4.2.7. Quantification of nuclei and cell orientation ..................................... 84
4.2.8. 3D image rendering ........................................................................... 85
4.2.9. Shear stress modeling ........................................................................ 85
4.2.10. Statistical analysis ............................................................................ 85
4.3. Results and discussion .............................................................................. 86
4.3.1. Fabrication and incorporation of PDMS micro-grooved substrate into
microfluidic chips ......................................................................................... 86
4.3.2. Alignment of C2C12 myoblast in 2D and 3D cellular constructs ...... 91
4.3.3. Enhancement of differentiation in aligned 3D myoblast cellular
constructs ................................................................................................... 100
4.4. Concluding remarks ................................................................................ 103
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