Preface ...................................................... xiii
PART 1 MATERIALS AND FABRICATION METHODS
CHAPTER 1 Microfluidic Cell Culture Platforms with
Embedded Nanoscale Features ..................................... 3
1.1 Introduction ............................................... 3
1.2 Engineering of nanoscale features .......................... 4
1.2.1 Fabrication of irregular nanoscale features ......... 5
1.2.2 Fabrication of regular nanoscale features ........... 6
1.3 Assembly of PDMS-based microfluidic platforms .............. 9
1.3.1 Reversible assembly ................................. 9
1.3.2 Irreversible assembly ............................... 9
1.3.3 Microtransfer assembly ............................. 10
1.4 Microfluidic platforms with embedded nanoscale features
for cell studies .......................................... 12
1.4.1 Cancer cell detection .............................. 12
1.4.2 Stem cell regulation ............................... 14
1.5 Summary ................................................... 19
Acknowledgment ............................................ 20
References ................................................ 20
CHAPTER 2 Microvascular Networks for Tissue Engineering ....... 27
2.1 Introduction .............................................. 27
2.2 Characteristics of branched vascular networks ............. 28
2.3 Fabrication of 2-D microvascular networks ................. 28
2.4 Fabrication of 3-D microvascular networks ................. 32
2.5 MicroChannel topologies ................................... 37
2.6 Engineering meets biology: toward tissue engineering
applications .............................................. 38
2.7 Outlook and future challenges ............................. 42
Acknowledgments ........................................... 47
References ................................................ 47
CHAPTER 3 Microfluidics for Engineering 3D Tissues
and Cellular Microenvironments ................................. 53
3.1 Introduction .............................................. 53
3.2 Fabricating 3D tissue scaffolds using microfluidics ....... 54
3.2.1 Microfluidic casting for engineering the
microstructure of natural ECM scaffolds ............ 54
3.2.2 Fabrication of composite photopolymerizable
hydrogel microstructures using microfluidics ....... 56
3.3 Dynamic 3D cell cultures within PDMS microfluidic
devices ................................................... 59
3.4 Hydrogel-based microfluidic culture devices and
tissue scaffolds .......................................... 61
3.4.1 Assembly of natural ECM multiphase tissues using
microfluidics ...................................... 62
3.4.2 Optimization of collagen-fiber interfacing for
directing capillary morphogenesis within
microfabricated tissue constructs .................. 65
3.5 Conclusion and future directions .......................... 75
References ................................................ 76
CHAPTER 4 Fabrication of Advanced Microcontainer Arrays
for Perfused 3D Cell Culture in Microfluidic Bioreactors ....... 81
4.1 Introduction .............................................. 81
4.2 Micromolding of cell container arrays ..................... 83
4.2.1 Micro injection molding ............................ 84
4.2.2 Hot embossing ...................................... 86
4.2.3 Microscale thermoforming ........................... 86
4.3 Introducing porosity ...................................... 89
4.3.1 Laser micromachining ............................... 90
4.3.2 Solvent vapor bonding of track-etched membranes .... 91
4.3.3 Combination of thermoforming and ion track
technology ......................................... 92
4.4 Functionalization of cell container arrays ................ 94
4.4.1 Protein coatings ................................... 94
4.4.2 SMART processes for overlaid patterned surface
and bulk modification of thermoformed cell
container arrays ................................... 94
4.5 Integration into microfluidic bioreactors ................. 99
4.6 Conclusion ............................................... 100
References ............................................... 100
CHAPTER 5 Mechanobiological Approaches for the Control
of Cell Motility .............................................. 105
5.1 Introduction ............................................. 105
5.2 Passive control of cell motility ......................... 107
5.2.1 Rome platform ..................................... 107
5.2.2 Materials and methods ............................. 110
5.2.3 Results and discussion ............................ 115
5.3 Active control of cell motility .......................... 123
5.3.1 Biological breadboard ............................. 123
5.3.2 Materials and methods ............................. 124
5.3.3 Results and discussion ............................ 127
References ............................................... 133
CHAPTER 6 Transport Models for Three-Dimensional Cell
Culture Systems ............................................... 111
6.1 Introduction ............................................. 137
6.1.1 Critical design criteria for in vitro models ....... 137
6.2 Fluid flow in cell culture systems ....................... 139
6.2.1 Equations of fluid motion ......................... 139
6.2.2 Boundary conditions ............................... 141
6.3 The theory of mass transport ............................. 142
6.3.1 Equation of mass transport ........................ 142
6.3.2 Boundary conditions ............................... 142
6.4 Binding kinetics ......................................... 143
6.4.1 Irreversible, first-order single-molecule
reactions ......................................... 143
6.4.2 Reversible, first-order single-molecule
reactions ......................................... 144
6.4.3 Reversible, first-order bimolecular reactions ..... 144
6.4.4 Enzyme kinetics (Michaelis-Menten kinetics) ....... 145
6.5 Nondimensionalization .................................... 146
6.6 Order of magnitude analysis .............................. 149
6.7 Bulk parameter models .................................... 151
6.8 Examples ................................................. 155
6.8.1 Ligand-receptor binding model ..................... 156
6.8.2 Mass transport in a membrane bilayer device ....... 158
6.9 Microfluidic approaches for flow and transport control ... 164
6.9.1 Membrane bilayer .................................. 164
6.9.2 Valves ............................................ 166
6.9.3 Grooves ........................................... 168
6.10 Conclusion ............................................... 168
Appendix A Navier - Stokes equations in other
coordinate systems ....................................... 168
A.l Navier - Stokes equations in radial coordinates ...... 168
A.2 Navier - Stokes equations in polar coordinates ....... 169
Appendix В Transport equation in other coordinate
systems .................................................. 170
References ............................................... 171
PART 2 TISSUE ENGINEERING STRATEGIES
CHAPTER 7 Microfluidic Systems for Controlling Stem Cells
Microenvironments ............................................. 175
7.1 Introduction ............................................. 175
7.2 Microfluidic elements for cell culture ................... 177
7.2.1 Cell sorting and filtering ........................ 177
7.2.2 Cell isolation and storage ........................ 179
7.2.3 Cell lysis ........................................ 182
7.2.4 Surface patterning ................................ 182
7.2.1 Microfluidic mixing, concentration gradients,
and combinatorial solutions ....................... 183
7.2.6 On-chip cell culture .............................. 185
7.2.7 Cell analysis on-chip ............................. 186
7.3 Controlling cellular microenvironments ................... 186
7.3.1 Soluble factors and chemical stimuli .............. 188
7.3.2 Mechanical stimuli ................................ 189
7.3.3 Electric stimuli .................................. 191
7.3.4 Cell-cell contact and coculture ................... 192
7.3.5 Development and mimicking of ECMs ................. 193
7.4 Challenges and outlook ................................... 194
Acknowledgments .......................................... 195
References ............................................... 195
CHAPTER 8 Vascularization of Microfluidic Hydrogels .......... 205
8.1 Introduction ............................................. 205
8.2 Design criteria for microfluidic scaffolds ............... 205
8.3 Forming and vascularizing microfluidic gels .............. 206
8.3.1 Forming microfluidic gels ......................... 206
8.3.2 Vascularizing gels ................................ 209
8.4 Design considerations .................................... 213
8.4.1 Perfusion ......................................... 213
8.4.2 Drainage .......................................... 215
8.5 Design algorithm ......................................... 217
8.6 Summary .................................................. 218
8.6.1 Future directions .................................. 219
Acknowledgments .......................................... 219
References ............................................... 220
CHAPTER 9 Microfluidic Vascular Networks for Engineered
Tissues ....................................................... 223
9.1 Introduction ............................................. 223
9.2 3D Microfluidics fabrication techniques .................. 224
9.2.1 Microfabrication of vasculature ................... 225
9.2.2 SFF techniques .................................... 227
9.2.3 Nontraditional techniques ......................... 230
9.2.4 3D fabrication issues ............................. 232
9.3 Materials for microfluidic vasculature ................... 232
9.3.1 Rigid materials ................................... 232
9.3.2 Elastomers ........................................ 234
9.3.3 Hydrogels ......................................... 236
9.3.4 Materials issues .................................. 240
9.4 Conclusion ............................................... 240
References ............................................... 241
CHAPTER 10 Microfluidic Approaches Toward Pulmonary
Tissue Constructs ............................................. 247
10.1 Introduction ............................................. 247
10.2 Lung design .............................................. 248
10.3 Engineering small airways ................................ 251
10.3.1 Overview .......................................... 251
10.3.2 Design parameters for small airways ............... 251
10.3.3 Microengineered airway models ..................... 258
10.3.4 Summary ........................................... 264
10.4 Engineering alveolar structures .......................... 265
10.4.1 Overview .......................................... 265
10.4.2 Design parameters for the air - blood barrier ..... 266
10.4.3 Conventional alveolar models ...................... 268
10.4.4 Microfluidic alveolar models ...................... 268
10.4.5 Summary ........................................... 270
10.5 Conclusions .............................................. 272
References ............................................... 272
CHAPTER 11 Microfabricated Kidney Tissue Models ............... 279
11.1 Introduction ............................................. 279
11.2 Significance of microfabricated kidney tissue models ..... 280
11.3 Kidney structure and function relationship ............... 280
11.4 Traditional kidney tissue models ......................... 283
11.5 Crucial signaling elements for kidney tissue models ...... 284
11.5.1 Signaling elements delivered via cell substrate ... 285
11.5.2 Signaling elements delivered via fluid flow ....... 287
11.6 Review of current microfabricated kidney tissue models ... 289
11.6.1 Microfabricated kidney models with controlled
FSS ............................................... 289
11.6.2 Microfabricated kidney models with porous
membranes and scaffolds ........................... 292
11.6.3 Microfabricated kidney models with user-defined
substrate topography .............................. 294
11.7 Summary and future direction ............................. 296
References ............................................... 297
CHAPTER 12 Microfluidic Cell Culture Techniques ............... 303
12.1 Fundamentals of microscale cell culture .................. 303
12.1.1 Cell culture ...................................... 303
12.1.2 Difference between macro- and microscale cell
culture ........................................... 303
12.1.3 Microscale cell culture considerations ............ 304
12.2 Microfluidic cell culture systems ........................ 304
12.2.1 Cell seeding ...................................... 305
12.2.2 Cell culture maintenance .......................... 306
12.2.3 Cell passaging .................................... 309
12.2.4 Cell concentration and dilution ................... 310
12.3 Microenvironmental stimuli ............................... 311
12.3.1 Biochemical stimuli ............................... 312
12.3.2 Physical stimuli .................................. 312
12.3.3 Compartmentalization .............................. 314
12.4 Microfluidic cell and tissue culture systems for drug
discovery and studies in physiology ...................... 315
12.5 Conclusions .............................................. 316
References ............................................... 316
PART 3 IN VITRO MODELS
CHAPTER 13 Functionalized Microfluidic Devices for
Separation of Cell Phenotypes ................................. 325
13.1 Introduction ............................................. 325
13.2 Negative selection for enrichment of target cells ........ 326
13.3 Positive selection of target cells for diagnostic
purposes ................................................. 328
13.4 Capture and release of target cells from positive
selection for tissue engineering purposes ................ 334
13.5 Effect of shear on changes of receptor expression in
cells .................................................... 335
13.6 Conclusions .............................................. 337
References ............................................... 337
CHAPTER 14 Microfluidic Hepatotoxicity Platform ............... 341
14.1 Introduction ............................................. 341
14.2 Liver tissue microenvironment ............................ 342
14.2.1 Liver acinus ...................................... 342
14.2.2 Sinusoid properties ............................... 343
14.2.3 Cell-cell interactions ............................ 343
14.2.4 Cell—matrix interactions .......................... 343
14.3 Microfluidic liver design ................................ 343
14.3.1 Microfabrication .................................. 344
14.3.2 Single unit layout ................................ 345
14.3.3 Perfusion barriers ................................ 346
14.3.4 Air channels ...................................... 347
14.3.5 Cell loading ...................................... 347
14.3.6 Gravity perfusion ................................. 348
14.3.7 Multiplexed plate ................................. 348
14.3.8 Automated system .................................. 348
14.3.9 Operation ......................................... 349
14.4 Long-term hepatocyte culture ............................. 350
14.4.1 Comparison with standard culture methods .......... 350
14.4.2 Morphology ........................................ 351
14.4.4 Viability and CYP450 activity ..................... 351
14.4.4 Gene expression ................................... 352
14.4.5 Albumin/urea production ........................... 353
14.4.6 Hepatocyte sources ................................ 353
14.5 Summary .................................................. 354
Acknowledgments .......................................... 354
References ............................................... 354
CHAPTER 15 Live Cell Analysis Under Shear Flow ................ 357
15.1 Introduction ............................................. 357
15.2 Flow control and well plate microfluidics ................ 358
15.2.1 Principles of well plate microfluidics ............ 358
15.2.2 WPM device fabrication ............................ 360
15.2.3 WPM device modeling and characterization .......... 360
15.3 Cell biology applications ................................ 362
15.3.1 In vitro cells under shear ........................ 362
15.3.2 Stem cell differentiation ......................... 362
15.3.3 Platelet biology: adhesion, aggregation, and
thrombus formation ................................ 364
15.3.4 Rolling and static cell adhesion .................. 372
15.4 Microbiology applications ................................ 373
15.4.1 Bacterial adhesion and biofilm development ........ 373
15.4.2 Biofilm compound screening ........................ 378
15.5 Summary .................................................. 380
References ............................................... 380
CHAPTER 16 Microfluidic Platforms for Evaluating
Angiogenesis and Vasculogenesis ............................... 385
16.1 Introduction ............................................. 385
16.2 Current methods in microfluidics ......................... 387
16.2.1 Mimicking angiogenesis in microfluidics ........... 388
16.2.2 Inducing angiogenesis ............................. 391
16.2.3 Coculture methods ................................. 393
16.2.4 Maturation/stabilization of blood vessels ......... 394
16.2.5 Quantification .................................... 395
16.2.6 Pros and cons of a microfluidic approach .......... 396
16.3 Conclusion and future directions ......................... 398
References ............................................... 398
CHAPTER 17 Cardiovascular Disease/Discovery Models ............ 405
17.1 Introduction ............................................. 405
17.2 Cell culture in cellix's Vena8 Endothelial +
microfluidic biochips .................................... 408
17.2.1 Validation of Vena8 Endothelial + biochip model) .. 408
17.3 Microfluidic cell culture biochip model for
atherosclerosis .......................................... 411
17.3.1 Monocyte (THP-1) adhesion to HUVECs in
Cellix's Vena8 Endothelial + microfluidic biochips ....... 411
17.4 Conclusion ............................................... 419
References ............................................... 419
Index ......................................................... 423
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