Fabrication of hierarchic scaffolds by 3D printing and freeze–drying for drying for cell culture and neuron differentiationcell culture and neuron differentiation

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Key factors of scaffold affecting cell performance

The ability to predict and control the interactions of cells with scaffolds materials underlies the rational design of biocompatible implants and tissue engineered bio hybrid organs. T he following factors of scaffolds are of great significance to affect cell performance in tissue engineering and thus deser ve careful clarification

Surface wettability

Surface wettability of scaffolds plays an important role in cell adhesion after cell seeding. H ydrophobic surface can severely change the structure of ECM proteins deposited on it. T he denatured ECM proteins will lost their bioactivity and fail to provide enough structure motifs for integrin bonding during cell adhesion , resulting in few cell adhesion with minimal spreading [ 140]. E nhanced cell adhesion and spreading on hydrophilic su rface relatively to hydrophobic one has been proved by much research due to the enhanced deposition of bioactive ECM proteins on scaffold surface [ 141].
H ighly hydrophilic surface, however, will weaken cell adhesion because of low attachment of ECM protein s on the surface [ 142]. During implantation of scaffolds , the surface wettability also receives much attention due to its expos ure to numerous proteins present in blood, interstitial fluid, and damaged extracellular matrix, resulting in the formation of a complex layer of adsorbed proteins at the material surface.

Scaffolds for tissue engineering applications

During the past decades, scaffolds have shown superior advantages in tissue engineering field for improved cell culture and functional tissues reconstruction in vitro and implantation over traditional 2D method that lacks 3D structural support and growth guidance . Various kinds of functional tissues have been successfully generated with the help of functional scaffolds, including neurons, cardiac tissues, skeletal muscle, bone and cartilage, skin tissue, blood vessel, etc. Among all these applications, neuron and cardiac tissues generation has received intensively attention. A number of scaffold systems have been applied for functional tissue generations, in order to providing reliable results for alternative treatment for clinical neural and cardiovascular disease s that are difficult with conventional methods. Here we introduce the use of scaffolds in neural and cardiac tissue engineering.

Fabrication of hierarchic scaffolds

The PEGDA/porous gelatin scaffold is fabricated by combination of 3D printing technique and freeze drying o f protein. B riefly, a porous PEGDA frame was produced by 3D printing. T hen the gelatin solution was used to fill the free space of PEGDA frame. F inally freeze drying was used to make porous gelatin in PEGDA frame. T he detailed processes were described belo w:

3D printing of honeycomb lattice made of PEGDA

3D microstructures of PEGDA frame have been obtained using the 3D printer ProJet 1200 (3D SYSTEMS Company) and a green PEGDA solution from the same manufacturer. The printing principle of our printer is photopolymerization and the PEGDA we used is UV curable in pure liquid form with its density, tensile strength and le in pure liquid form with its density, tensile strength and tensile modulus being 1.04g/mL, 30MPa and 1.7GPa, respectively. Briefly, a scaffold tensile modulus being 1.04g/mL, 30MPa and 1.7GPa, respectively. Briefly, a scaffold model was designed by software SolidWorks. At first, we designed a simple honeycomb model was designed by software SolidWorks. At first, we designed a simple honeycomb frame with feature size, pframe with feature size, pitch size and thickness being 200, 1200 and 200 itch size and thickness being 200, 1200 and 200 μμm, m, respectively. We put great emphasis on the thickness of the scaffolds. So we started with respectively. We put great emphasis on the thickness of the scaffolds. So we started with small small thickness of 200 thickness of 200 μμm which is within m which is within severalseveral times of cellular size and times of cellular size and thus thus simplsimplififiesies the cell culture studthe cell culture study. The designed pattern was then loaded into the 3D y. The designed pattern was then loaded into the 3D printer. After parameter setting, the 3D printprinter. After parameter setting, the 3D printerer can calculate the printing time according can calculate the printing time according to the height of targeted scaffold. The fabricated 3D PEGDA frame together with to the height of targeted scaffold. The fabricated 3D PEGDA frame together with printing platform was washed in iprinting platform was washed in isopropanol for 5 min, dried carefully with air flow sopropanol for 5 min, dried carefully with air flow and postand post–baked with UV light for 10 min. Then the 3D printing of PEGDA frame was baked with UV light for 10 min. Then the 3D printing of PEGDA frame was finished and the finished and the PEGDA PEGDA frame was carefully detached from the platform. frame was carefully detached from the platform. Fig. Fig. 3.3.11 shows the 3D printed PEGDA honeycomb frame.shows the 3D printed PEGDA honeycomb frame.

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Freeze-drying of porous gelatin in PEGDA lattices

The printed 3D PEGDA frame is used as backbone to support the formation of gelatin porous structures inside. Freeze drying method is a convenient way to make highly porous 3D scaffold meanwhile can both keep activity of biomaterials and avoid deformation of 3D scaffold to great extent , which is widely used in tissue engineering [19, 20]. Without freeze drying, air drying, for example, will cause shrinkage, deformation and even denaturation of gelatin scaffold with much lower porosity. Briefly, 0.2 g of gelatin powder (from porcine skin , Sigma) was dissolved in 20 mL of deionized water containing 0.02g of citric acid under vigorous stirring at room temperature. The 3D PEGDA frame was treated with oxygen plasma to promote its hydrophilicity and then was immersed in the gelatin solution wh ich was frozen at 20 overnight and dried in vacuum for 24h at room temperature. Afterwards the porous gelatin together with PEGDA frame was heated at 140 for 4h, in which porous gelatin can be cross linked in the presence of citric acid. Finally, excess ive gelatin was removed with a sharp knife, remaining a porous gelatin layer inside the hexagonal lattices with similar thickness of PEGDA frame, as is shown in Fig. 3. 3

Table of contents :

Outline
Chapter 1 Introduction
1.1 Cell and cellular environment
1.1.1 Cell
1.1.2 Cell microenvironment
1.1.3 Cell culture
1.2 Stem cells and induced luripotent stem cells
1.2.1 Stem cells
1.2.2 Neural progenitor cells
1.2.3 Induced pluripotent stem cells
1.3 Bioscaffolds for tissue engineering
1.3.1 Biomaterials
1.3.2 Scaf folds requirements and fabrication methods
1.3.3 Key factors of scaffold affecting cell performance
1.3.4 Scaffolds for tissue engineering applications
1.4 Research objectives
Reference
Chapter 2 Fabrication methods
2.1 UV lithography
2.1.1 Mask preparation
2.1.2 Substrate cleaning
2.1.3 Photoresist processing
2.2 Soft lithography
2.3 Electrospinning
2.3.1 Principle and parameters
2.3.2 Materials
2.3.3 Applications
2.3.4 Limitations
2.4 3D pri nting
2.4.1 Advantages
2.4.2 Printing technologies
2.4.3 Materials
2.4.4 Applications
2.4.5 Limitations
2.5 Freeze drying
2.5.1 Principle
2.5.2 Applic
2.5.2 Applicationsations
2.6 SelfSelf–organization of polymer scaffoldsorganization of polymer scaffolds
2.7 Parylene C depositionParylene C deposition
2.7.1 Introduction of parylene C
2.7.2 Principle
2.7.3 Working process
2.7.4 Substrates for Parylene C coating
2.7.5 Applications
Reference
Chapter 3 Fabrication of hierarchic scaffolds by 3D printing and freeze–drying for drying for cell culture and neuron differentiationcell culture and neuron differentiation
3.1 Introduction
3.2 FabricatFabrication of hierarchic scaffoldsion of hierarchic scaffolds
3.2.1 3D printing of honeycomb lattice made of PEGDA3D printing of honeycomb lattice made of PEGDA
3.2.2 Freeze–drying of porous gelatin in PEGDA latticesdrying of porous gelatin in PEGDA lattices
3.3 Cell culture studiesCell culture studies
3.4 3D printed PEGDA/porous gelatin scaffold for neuronal 3D printed PEGDA/porous gelatin scaffold for neuronal differentiationdifferentiation
3.4.1 Fabrication of 3D printed PEGDA/porous gelatin scaffold
3.4.2 Culture and differentiation of neural progenitor cells
3.5 ConclusionConclusion
Reference
Chapter 4 Fabrication of self–organized porous PCL membrane for improved cell organized porous PCL membrane for improved cell culture and hiPSCs differentiationculture and hiPSCs differ
4.1 Introduction
4.2 Fabrication of the culture patchation of the culture patch
4.2.1 Fabrication of PEGDA honeycomb frame
4.2.2 Self–organization of porous PCL membraneorganization of porous PCL membrane
4.3 Cell based assays
4.4.1 Cell proliferation on 2D porous PCL patch
4.4.2 Cellular uptake
4.4.3 Gene transfectiontransfection
4.5 Cardiac differentiation of hiPSCs on 2D porous PCL
4.5.1 HiPSCs culture on 2D porous PCL
4.5.2 Pluripotency of hiPSCsency of hiPSCs
4.5.3 Proliferation of hiPSCs
4.6 Biodegradation
4.7 Conclusionlusion
Reference
Chapter 5 Monolayer gelatin nanofibers on PDMS frame for cardiac differentiationdifferentiation
5.1 Introductionon
5.2 Fabrication of PDMS/gelatin nanofibers patch
5.2.1 Fabrication of PDMS honeycomb frame
5.2.2 Electrospinning of gelatin nanofibersgelatin nanofibers
5.3 Cell based assays
5.3.1 Culture and seeding of hiPSCs
5.3.2 Cell viability assay
5.3.2 Cell viability assay
5.3.3 Pluripotency of hiPSCs
5.3.4 Cardiac differentiation
5.3.5 Immunostaining
5.3.6 Calcium imaging
5.3.7 Electric stimulationElectric stimulation
5.3.8 Drug test
5.3.9 Deformation of PDMS frame
5.4 Conclusion
Reference
Chapter 6 Conclusion and perspective
Reference
Appendix A Patterned parylene C for cell adhesion, spreading and alignment ned parylene C for cell adhesion, spreading and alignment studiesstudies
A.1 Introduction
A.2 Parylene C pattern fabrication
A.2.1 Fabrication process2.1 Fabrication process
A.2.2 SEM observation
A.3 Cell culture studiesll culture studies
A.3.1 Gelatin coating on parylene C film
A.3.2 Cell adhesion and spreading
A.3.3 Cell alignment
A.4 Conclusion
Reference
Appendix B Porous gelatin patch for VEGF loading and controlled release 
B.1 Introduction
B.2 Fabrication of VEGF loaded porous gelatin patch
B.2.1 Fabrication of porous gelatin patch
B.2.2 VEGF loading on porous gelatin patch
B.3 Results
B.3.1 Porous gelatin patch
B.3.2 VEGF loading and release
B.4 Conclusion
Reference
Appendix C French summary

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