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UV expose and development
As discussed before, the post-bake is important step during the photolithography. For the chemically amplified resists, post-exposure bake promotes the thermally activated diffusion of carboxylic acid formed during exposure from the photo active compound and catalytically completes the photo reaction initiated during exposure. AZ positive resist does not need post bake due to it is not belong to the chemically amplified resist. The effect of the post to the image reversal resist has been summarized before. The developer for the SU-8 and AZ resist are PM-Acetate and TMAH, respectively. To avoid the excess development, we can check the patterns under the microscopy and developing repeatedly until getting the good patterns.
Soft photolithography
Soft lithography represents a non-photolithographic strategy based on self-assembly and replica molding for carrying out micro- and nanofabrication. It provides a convenient, effective, and low-cost method for the formation and manufacturing of micro- and nanostructures [16, 17]. In soft lithography, an elastomeric stamp with patterned relief structures on its surface is used to generate patterns.
Mechanism and fabrication process
An elastomeric block with patterned relief structures on its surface is the key to soft lithography. The most common material used in soft lithography is polydimethylsiloxane (PDMS), which is inexpensive, nontoxic, and optically transparent. It is also chemically stable in a wide range of environments with desirable physical properties and the low surface energy (21.6×10-3 J m-2) allows easy release from templates and surfaces [18].
Fig. 2.7 introduces the regular soft lithography process. Briefly, the PDMS stamp is replicated from a patterned master obtained by photolithography, focused ion beam or electron beam lithography method. For preparing the PDMS stamp, a solution of A and B at different ratios is mixed evenly and then casted on the mold. After that, the system is transferred to a vacuum chamber to remove the bubbles.
Micro-contact printing (μCP)
Microcontact printing is considered one of the most useful techniques for large area (> cm2) patterning of functional organic surfaces. The process is very similar to using a common stamp to transfer ink from an ink pad to a piece of paper. The PDMS mold is « inked » with the material solution to be transferred to the substrate and contact is made between the substrate and the protruding features of the mold [28]. This allows the material to be transferred from the mold to the substrate for patterning the substrate as desired (Fig. 2.12). Depending on the type of the substrate, the inked molecule can be physically or chemically adsorbed on the substrate. In general, the resolution of microcontact printing is limited by the stamp feature size and the diffusion of the ink molecules around the contact areas [29]. In practice, the PDMS stamps provide a minimum feature size of ~200 nm but smaller features can be obtained by using stamps of a higher stiffness and/or less diffused molecules.
Flow rate and tip to collector distance
The flow rate of the solution influences the jet velocity and the material transfer rate. Normally, a lower feed rate is more desirable as the solvent evaporate relatively completed. It has been proved that with the increase of the polymer flow rate, the fiber diameter and the pore diameter increases and the morphological structure also can be slightly changed. Moreover, high flow rates result in beaded fibers due to unavailability of proper drying time prior to reaching the collector [44]. The distance between the tip and the collector has been examined as another approach to control the fiber diameters and morphology. A minimum distance is required to give the fibers sufficient time to dry before reaching the collector, otherwise with distances that are either too close or too far, beads have been observed. It has been reported that increasing the distance results in rounder fibers with the spinning of silk-like polymer, but with closer distances can produce flatter fibers [33, 43]. One important physical aspect of the electrospinning nanofibers is their dryness from the solvent used to dissolve the polymer. Thus, there should be optimum distance between the tip and collector which favors the evaporation of solvent from the nanofibers.
Alignment, collector-pattern and 3D structure
Electrospun nanofibers are normally collected with random orientation, whereas most native ECM found in tissues or organs, such as heart and blood vessel, have anisotropic architecture, which is crucial for tissue function [45, 46]. Therefore, a well-defined architecture is believed to be necessary in order to precisely imitate native ECM for guiding cell growth or tissue regeneration. To this end, electrospun fibrous scaffolds with alignment architecture have shown superior capacity in shaping cell morphology, guiding cell migration, and affecting cell differentiation [47].
A variety of strategies have been demonstrated to obtain alignment fibers. Overall, these methods can be classified into two major categories, i.e., collector configuration and controlling syringe moving. A number of collector have been tested to generate highly align fibers. Among them, the parallel rods and rotating cylinder are the most used collectors [47], as shown in Fig. 2.17a and b. Another approach is to control the moving of syringe, by which the deposition of fibers can be well controlled if the moving speed of syringe is fast enough. Moreover, nanofibers with various orientation integrated in one sample can be achieved (Fig. 2.17c and d) [48].
Table of contents :
OUTLINE
CHAPTER 1 GENERAL INTRODUCTION
1.1 Cell and cellular microenvironment
1.1.1 Cell and cell cytoskeleton
1.1.2 Cellular microenvironment
1.1.2.1 Extracellular matrix
1.1.2.2 Cell-ECM interaction
1.1.2.3 Cell-cell interaction
1.2 Cell-substrate interaction from mechanotransduction aspect .
1.2.1 Mechanotransduction
1.2.2 Substrate stiffness
1.2.3 Substrate topography
1.2.4 2D versus 3D culture
1.3 Technology of induced pluripotent stem cells
1.3.1 The induced pluripotent stem cells
1.3.2 Differentiation of induced pluripotent stem cells
1.3.3.1 Differentiation via EB formation
1.3.3.2 Substrate for stem cell differentiation
1.4 Scaffold engineering
1.4.1 Biomaterials
1.4.2 Fabrication techniques
1.4.3 State of the art and future directions
1.5 Research objectives of this work
References
CHAPTER 2 MICRO- AND NANO-FABRICATION TECHNIQUES
2.1 Introduction
2.2 UV photolithography
2.2.1 General principles
2.2.2 Mask design and exposure
2.2.3 Photoresist
2.2.4 UV expose and development
2.3 Soft photolithography
2.3.1 Mechanism and fabrication process
2.3.2 Micro-contact printing (μCP)
2.3.3 Replica molding
2.4 Electrospinning
2.4.1 Principles
2.4.2 Effects of various parameters on electrospinning
2.4.2.1 Applied voltage
2.4.2.2 Flow rate and tip to collector distance
2.4.2.3 Alignment, collector-pattern and 3D structure
2.5 Conclusion
Reference
CHAPTER 3 ELONGATION AND CELL MIGRATION ON DENSE ELASTOMER PILLARS WITH STIFFNESS GRADIENT
3.1 Introduction and motivation
3.2 Materials and methods
3.2.1 Materials
3.2.2 Fabrication of the substrate
3.2.3 Microfluidic device integration
3.2.4 Cell culture
3.2.5 Immunocytochemistry
3.2.6 Imaging and statistical analysis
3.3 Results and discussion
3.3.1 Fabrication of the micropillar substrate
3.3.2 Rigidity of the micropillar substrates
3.3.3 Cell motility and cell migration
3.3.4 Cell morphology and cell proliferation
3.3.5 Complex stiffness map
3.4 Conclusion
References
CHAPTER 4 NARROWLY SPACED MONOLAYER NANOFIBERS FOR THREE-DIMENSIONAL CELL HANDLING
4.1 Introduction and motivation
4.2 Materials and methods
4.2.1 Materials
4.2.2 Fabrication of tri-layer scaffold
4.2.2.1 PDMS mold fabrication
4.2.2.2 Aspiration-assisted PEGDA frame production and ring mounting
4.2.2.3 Electrospinning nanofibers
4.2.3 Cell culture
4.2.4 Immunocytochemistry
4.2.5 SEM observation
4.2.6 Live/dead assay
4.2.7 Cell proliferation
4.3 Results
4.3.1 Tri-layer scaffold formation
4.3.1.1 Frame pattern and size
4.3.1.2 Frame thickness
4.3.1.3 Materials for electrospinning
4.3.1.4 Nanofiber density
4.3.2 Cell attachment and spreading
4.3.3 Cell viability
4.3.4 Cell morphology and 3D cell culture
4.3.5 Cell migration
4.3.5.1 Top to bottom migration
4.3.5.2 Bottom to top migration
4.3.5.3 Migration on thick scaffold
4.3.6 Cell proliferation
4.4 Conclusion
References
CHAPTER 5 DIFFERENTIATION OF HUMAN INDUCED PLURIPOTENT STEM CELLS ON MICROPILLAR ARRAYS
5.1 Introduction
5.2 Materials and methods
5.2.1 Materials
5.2.2 Fabrication of elastomer micropillars
5.2.3 Fabrication of the PDMS stencil
5.2.4 hiPSCs culture
5.2.5 Scanning electron microscopy imaging
5.2.6 Immunocytochemistry
5.2.7 MTT assay
5.2.8 Intracellular Ca2+ transient assays
5.2.9 External electrical stimulation
5.3 Results
5.3.1 Sample characterization
5.3.1.1 SEM imaging
5.3.1.2 Effective stiffness of the elastomer pillars
5.3.3 Stencil assistant EBs formation
5.3.3.1 The effect of the substrate stiffness
5.3.3.2 The effect of cell seeding density
5.3.3.3 Pluripotency of hemisphere EBs
5.3.3.4 3D view of the EBs
5.3.4 Cardiac differentiation on micropillar substrates
5.3.4.1 Differentiation protocol
5.3.4.2 Immunostaining of differentiated cardiomyocytes
5.3.5 Beating behavior of differentiated cardiomyocytes
5.3.6 Impact of external electrical stimulation
5.4 Conclusion
References
CONCLUSION AND PERSPECTIVE
APPENDIX A A FACILE PHOTOLITHOGRAPHIC METHOD OF FABRICATING WAVY-LIKE PATTERNS
1 Introduction
2 Experiments
3 Results and discussion
3.1 Microwave and MLAs patterns
3.2 Rice leaf-like structure and the hydrophobic property
4 Conclusion
Reference
APPENDIX B FRENCH SUMMARY
