Elongated fascicle-inspired 3D tissues consisting of high-density, aligned, optogenetically ...

142 Pages·2014·12.49 MB·English

Elongated Fascicle-Inspired 3D Tissues Consisting of
High-Density, Aligned, Optogenetically Excitable
Muscle Tissue Using Sacrificial Outer Molding
by
Devin Michael Neal
S.M., Mechanical Engineering
'MSACHUSETTS INSTRrtTE,
Massachusetts Institute of Technology, 2009 M
OF TECFNIOL0OCUY
S.B., Mechanical Engineering AUG 1 5 2014
Massachusetts Institute of Technology, 2007
LIBRARIES
Submitted to the Department of Mechanical Engineering
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
June 2014
0 Massachusetts Institute of Technology, 2014. All Rights Reserved.
Signature redacted
Au thor..................................................................................... .... . .........
Department of Mechanical Engineering
23, 2014
______ z 4January
Certified by......................................................S a r acted.. .
V
Harry Asada
-
F *Wrofessor of Mechanical Engineering
Supervisor
Signature redacted
Accepted by......................................................
David Hardt
-man, Department Committee on Graduate Students
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Elongated Fascicle-Inspired 3D Tissues Consisting of High-Density, Aligned,
Optogenetically Excitable Muscle Tissue Using Sacrificial Outer Molding
by
Devin Michael Neal
Submitted to the Department of Mechanical Engineering
on January 23, 2014, in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy in Mechanical Engineering
ABSTRACT
The majority of muscles, nerves, and tendons are composed of fiber-like fascicle
morphology. Each fascicle has a) elongated cells highly aligned with the length of the construct,
b) a high volumetric cell density, and c) a high length-to-width ratio with a diameter small
enough to facilitate perfusion. Fiber-like fascicles are important building blocks for forming
those tissues of various sizes and cross-sectional shapes, yet no effective technology is currently
available for producing long and thin fascicle-like constructs with aligned, high-density cells.
Here we present a method for molding cell-laden hydrogels that generate cylindrical tissue
structures that are 100 pim in diameter with an extremely high length to diameter ratio (>100:1).
Using this method we have successfully created skeletal muscle tissue with a high volumetric
density (-50%) and perfect cell alignment along the axis. A new molding technique, Sacrificial
Outer Molding, allows us to i) create long and thin cavities of desired shape in a mold that is
solid at a low temperature, ii) release gelling agents from the sacrificial mold material after cell-
laden hydrogel is injected into fiber cavities, iii) generate a uniform axial tension between anchor
points at both ends that promotes cell alignment and maturation, and iv) perfuse the tissue
effectively by exposing it to media after melting the sacrificial outer mold at 37'C. Effects of key
parameters and conditions, including initial cavity diameter, axial tension, and concentrations of
hydrogel and gelling agent, upon tissue compaction, volumetric cell density, and cell alignment
are presented. Furthermore, the tissue is characterized in a custom designed mechanical
characterization system. Characterization has shown that an optimal diameter exists at which
muscle constructs exhibit the greatest contraction performance, and that optical and electrical
stimulation of optogenetic muscle cells result in similar performance if the tissue is developed
sufficiently.
Thesis Supervisor: H. Harry Asada
Title: Ford Professor of Mechanical Engineering
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To Mom
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ACKNOWLEDGMENTS
I want to start by thanking the members of my thesis committee: Professor Harry Asada,
Professor Roger Kamm, Professor Krystyn Van Vliet, and Professor Rashid Bashir. Your
comments, encouragement both in committee meetings and out have helped make this document
something I'm truly proud of. I'd like to thank my advisor in particular. He has shown me time
and again that his true product, his true pride is his students.
Thank you to the members of the d'Arbeloff lab. Every one of you was always willing to
help me with my research. I'd like to thank a few current members in particular for their
substantial contributions to the work presented in this thesis: Dr. Min-Cheol Kim, Dr. Sharon
Ong, and Dr. Vincent Chan. I would like to thank a former member in particular: Dr. M. Selman
Sakar. Selman was a mentor in every sense of the word. Thank you to members of the Kamm
Group. The Kammsters were always willing to help and share facilities and knowledge. With
them, I was privileged to be welcome in a lab away from lab. I'd like to thank two current
members for their contributions to the work presented here: Sebastien Uzel, and Jordan Whistler.
I want to thank Dr. Frances Hill for going through this process before me and being an
invaluable resource in managing the PhD process.
I want to thank the NSF and EBICS, and the NRF of Singapore and BioSyM. I am
grateful for these programs as funding sources as well as for all the people they allowed me to
work and collaborate with.
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TABLE OF CONTENTS
LIST OF FIGURES ........................................................................................................................ 9
Chapter 1: Introduction................................................................................................................. 11
Intro 1.1 Significance of engineered skeletal mu scle tissue...................................................... 11
Intro 1.2 Literature review of engineered skeletal m uscle tissue........................................... 13
Intro 1.3 Thesis Framing ....................................................................................................... 16
Chapter 2: Fascicle-Inspired 3D Tissues Using Sacrificial Outer Molding .............................. 18
2.1 Introduction ......................................................................................................................... 18
2.2 Results and Discussion..................................................................................................... 19
2.2.1 Sacrificial External M olding .................................................................................... 19
2.2.2 Fascicle-like tissue formation.................................................................................. 22
2.2.3 Importance of axial stress......................................................................................... 23
2.2.4 Construct diam eter................................................................................................... 23
2.2.5 Cell density................................................................................................................... 24
2.2.6 Fibrin com ponent variation ...................................................................................... 25
2.3 M aterials and M ethods..................................................................................................... 25
2.3.1 Fabrication of Perma nent M olds ............................................................................... 26
2.3.2 Culture of C2C12 M yoblasts.................................................................................... 26
2.3.3 Casting of Sacrificial Mold and Hydrogel Constructs ............................................. 26
2.3.4 Imm unostaim ng..................................................................................................... 27
2.3.5 Autom ated Im age Analysis ...................................................................................... 27
2.4 Conclusions ......................................................................................................................... 27
2.5 Acknowledgem ents.......................................................................................................... 28
2.6 Supporting Inform ation................................................................................................... 28
2.7 Figures and Captions........................................................................................................ 31
Chapter 3: Mechanical Characterization of Optically and Electrically Stimulated Fascicle-Like
Constructs ..................................................................................................................................... 39
3.1 Introduction ................................................... 39
3.2 M echanical characterization system design ................................................................... 41
3.2.1 Concept: lateral displacem ent with a cantilever..................................................... 41
3.2.2 Characterization System ............................................................................................ 42
3.2.3 Electrode design ....................................................................................................... 43
3.3 M aterials and m ethods ..................................................................................................... 44
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3.3.1 Tissue Constructs...................................................................................................... 44
3.3.2 Me chanical Characterization.................................................................................... 44
3.3.3 Im age and Vi deo A nalysis....................................................................................... 45
3.4 Results and discussion..................................................................................................... 46
3.4.1 Optical vs. Electrical Stim ulation............................................................................. 46
3.4.2 Di am eter variation..................................................................................................... 47
3.4.3 Probe stiffness variation ........................................................................................... 48
3.5 Conclusion........................................................................................................................... 49
3.6 Figures and Captions........................................................................................................ 52
Chapter 4: Design of Multi-Degree of Freedom Skeletal Muscle Powered Systems........ 61
4.1 Introduction ......................................................................................................................... 61
4.2 Bio-A ctuator Building Blocks......................................................................................... 63
4.3 Scaling from Tw o Unit to mu lti-Unit System s ................................................................... 64
4.3.1 M assively Parallel System s ...................................................................................... 65
4.3.2 Hi ghly Serial/Netw orked System ............................................................................. 65
4.4 Experim ental M aterials and M ethods ............................................................................ 66
4.5 Experim ental Results........................................................................................................ 67
4.5.1 Single Building-Block M uscle Ac tuator ................................................................... 67
4.5.1 Serial and Parallel Prototypes.................................................................................. 67
4.5.2 M ulti-U nit A ctuator System Prototypes....................................................................... 68
4.6 Conclusion........................................................................................................................... 68
4.7 Figures and Captions........................................................................................................ 69
CHAPTER 5: CONCLUSION AND FUTURE DIRECTIONS ............................................... 76
5.1 Conclusion........................................................................................................................... 76
5.2 Future directions.................................................................................................................. 76
5.3 A cknowl edgem ents .......................................................................................................... 77
5.4 Figures and captions........................................................................................................ 78
References..................................................................................................................................... 80
A ppendix A : Fascicle construct protocol.................................................................................. 89
Appendix B: Optogenetic Control of Live Skeletal Muscles: Non-Invasive, Wireless, and Precise
A ctivation of M uscle Tissues................................................................................................... 97
B .1 Introduciton ........................................................................................................................ 97
B.2 M yogenesis and Optogenetics......................................................................................... 99
B .3 Experim ental Evaluation .................................................................................................. 100
B Applications...................................................................................................................... 102
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B.4.1 M uscle-on-a-Chip Drug Screenings .......................................................................... 102
B.4.2 M any DO F Robotic Devices...................................................................................... 103
B.5 Conclusion........................................................................................................................ 103
B.6 Acknow ledgm ents ............................................................................................................ 104
B.7 Figures and Captions ........................................................................................................ 105
Appendex C: Patent Description of Patent Application PCT/US2012/027483.......................... 111
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LIST OF FIGURES
Fig. 2.11 Fascicle-like tissue construct production technique .................................................. 31
Fig. 2.2 1F ascicle-inspired tissue constructs............................................................................. 32
Fig. 2.3 1G eometric changes in fascicle construct structure over time. .................................. 33
Fig. 2.4 1V ariable parameters may be used to control the development of fascicle-like constructs.
....................................................................................................................................................... 34
Fig. 2.S1 I Demonstration of how the presented method may be expanded.................. 35
Fig. 2.S2 IL ow gelatin concentration..................................................................................... 35
Fig. 2.S3 IS uitable gelatin concentrations............................................................................... 36
Fig. 2.S4 IE ffects of gelatin viscosity ...................................................................................... 37
Fig. 2.S5 IN onuniformities from mixing thrombin and fibrinogen ........................................ 38
Fig. 3.1 1F orce probe diagram ................................................................................................... 52
Fig. 3.2 1M echanical characterization system.......................................................................... 53
Fig. 3.3 1E lectrode design.......................................................................................................... 54
Fig. 3.4 | Compliant electrodes functioning............................................................................. 55
Fig. 3.5 1S timulation protocol .................................................................................................. 55
Fig. 3.61 Twitch data analysis................................................................................................... 56
Fig. 3.7 1E lectric vs. optical performance results..................................................................... 57
Fig. 3.8 1S tress and force over various initial pin diameters..................................................... 58
Fig. 3.91 Uniform vs. nonuniform strip force.......................................................................... 59
Fig. 3.10 1T issue performance under varying load conditions................................................ 60
Fig. 4.11 Optogenetic control of skeletal muscles................................................................... 69
Fig. 4.2 1S keletal muscle structure ........................................................................................... 69
Fig. 4.3 1F ascicle-inspired building block muscle strip actuators........................................... 70
Fig. 4.41 The combination of parallel and serial connections of muscle strips........................ 71
Fig. 4.5 1N umerous parallel muscle strips on both sides of a floating node ............................ 72
Fig. 4.61 Increasing serial connections.................................................................................... 73
Fig. 4.7 | Individual building block actuator contracting with 4.3% measured strain .............. 74
Fig. 4.8 1P rototype muscle actuator systems consisting of fascicle-like muscle actuators.......... 74
Fig. 4.9 | Prototype muscle parallel and series actuator system................................................ 75
Fig. 5.1 1F uture Directions ....................................................................................................... 78
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Fig. 5.2 Initial PEG Fascicle-Like Strips ............................................................................... 79
Fig. A.1 I Schematic of fascicle tissue device with pins......................................................... 92
Fig. A.2 Schematic of fascicle tissue device with pins removed........................................... 93
Fig. B. 1 IO ptogenetic control of live cells and tissue. ............................................................... 105
Fig. B.2 Membrane-bound Channelrhodopsin 2 serves as an ion gate...................................... 105
Fig. B.3 I Plasmid containing ChR2............................................................................................ 105
Fig. B.4 Mouse skeletal cells (C2C12) transfected with ChR2. ................................................ 106
Fig. B.5 j Myogenic process........................................................................................................ 106
Fig. B.6 Experiment of optogenetic contraction control of skeletal muscles............................. 106
Fig. B.7 Experiment of spatial resolution of optogenetic control.............................................. 107
Fig. B.8 I Microfabricated tissue gauge for formation of 3D skeletal muscle microtissue......... 107
Fig. B.9 Cross-striations characteristic to sarcomere formation. Stained for a-actinin............ 107
Fig. B. 10 I Impulse response test of 3D skeletal muscle............................................................. 108
Fig. B.11I 2nd order ARMAX model siumulation with measured contraction data. ................ 108
Fig. B.12 Tetanus-like response from simulation..................................................................... 109
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Fig. B.13 Muscle-on-a-Chip Drug Screening........................................................................... 109
Fig. B.14 Highly Networked Mouth-Like Muscle System....................................................... 109
TABLE B.1 I Individual and group activations .......................................................................... 110
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