INDEX
INTRODUCTION ...3
REFERENCES...6
1 MICROFLUIDIC GRADIENT GENERATION ...7
1.1 INTRODUCTION TO MICROFLUIDIC SYSTEMS... 8
1.2 FLUID PHYSICS AT THE NANOLITER SCALE ... 9
1.3 STATE OF THE ART ON GRADIENT GENERATING SYSTEMS ... 12
1.3.1 The traditional in vitro methods ... 12
1.3.2 The microfluidic methods ... 18
1.4 THE MICROFLUIDIC MULTI-INJECTOR (MMI) ... 23
REFERENCES... 26
2 DYNAMIC SPATIO-TEMPORAL GRADIENT GENERATOR ...30
2.1 OBJECTIVES ... 30
2.2 GRADIENT GENERATOR DESIGN... 32
2.3 SUBSTRATE PATTERN DESIGN ... 36
REFERENCES... 39
3 MATERIALS AND METHODS ...40
3.1 MICROCHIP FABRICATION: MATERIALS AND METHODS ... 41
3.1.1 Multilayer Soft Lithography: basic concept... 41
3.1.2 Monolithic active valve and its control ... 45
3.1.3 Micro Contact Printing for patterning of proteins... 46
3.2 MATERIALS... 47
3.2.1 Soft lithography material... 47
1
2
3.2.2 Substrate material... 51
3.2.3 Microcontact printing materials... 52
3.3 CELL CULTURE STANDARD PROTOCOLS... 53
3.3.1 Cell culture ... 55
3.3.2 Cell seeding and duplication... 57
3.3.3 Manual cell counting... 59
3.3.4 Cryopreservation... 60
3.3.5 Thawing frozen cells... 61
REFERENCES... 62
4 MICROFLUIDIC CHIP: EXPERIMENTAL RESULTS...65
4.1 MICROFLUIDIC CHIP FABRICATION ... 65
4.2 MICROCHIP TESTING... 72
4.2.1 Valve characterization ... 76
4.2.2 Concentration gradient characterization... 78
4.3 CELL VIABILITY TEST ... 89
4.4 MICROPATTERNED SUBSTRATE ... 91
4.5 CELL ADHESION ON FIBRONECTIN ... 94
5 CONCLUSIONS AND FUTURE WORK ...98
5.1 CONCLUSIONS... 98
5.2 FUTURE WORK... 99
REFERENCES...101
