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Title Page
Abstract
Contents
List of abbreviations 11
1. General introduction 14
1.1. Definition of the terminology of "cell aggregate". 14
1.2. Importance to consider cell-to-cell interaction in the field of tissue engineering. 14
1.3. Principles and methodologies to generate cell aggregates. 15
1.4. Research objectives 16
2. Development of the bioprinting method of cells with in-situ aggregation in high precision. 17
2.1. Introduction 17
2.1.1. Necessity of patterning cell aggregates for paracrine interaction in tissue models 17
2.1.2. Technical limitations of patterning of cell aggregates 17
2.1.3. Highly precise dot printing technique using multiple types of cells. 18
2.2. Materials and method 19
2.2.1. Customized extrusion-based 3D bioprinting system 19
2.2.2. Preparation of bioinks and analysis of rheological properties 19
2.2.3. Isolation of primary hepatocyte 21
2.2.4. Preparation of cells-laden bio-inks 21
2.2.5. Dot printing process 23
2.2.6. Preparation of microwell cell aggregates 24
2.2.7. Scanning electron microscopy (SEM) 24
2.2.8. Characterization of dot-printed cell aggregates 24
2.2.9. DNA quantification 25
2.2.10. Cytocompatibility test 25
2.2.11. Albumin and urea secretion measurements 25
2.2.12. Sectional analysis with tissue staining. 26
2.2.13. Immunocytochemistry assay 26
2.2.14. Measurement of CYP1A2 activity 26
2.2.15. Statistical analysis 26
2.3. Results and discussion 27
2.3.1. Characterization of bio-inks and the printing procedure. 27
2.3.2. Diameter controllability of cell aggregate using dot printing 32
2.3.3. Controllability of the position with cell aggregates using dot printing. 35
2.3.4. Verification of effects on cytocompatibility and functionality upon dot printing process. 38
2.3.5. Effect of position controllability of PMH aggregates with EC on hepatic function. 40
2.3.6. Expending application (1): cancer invasion model 43
2.3.7. Expending application (2): Incorporation of polydopamine-coated fragmented fibers with mesenchymal stem cells (MSCs) 45
2.4. Conclusion 47
3. Development of a high-efficient islet macroencapsulation sheet for subcutaneous transplantation 48
3.1. Introduction 48
3.1.1. Xenotransplantation as an alternative to autologous requirement 48
3.1.2. Islet transplantation for type 1 diabetes 48
3.1.3. Islet encapsulation for immune protection and its limitations 49
3.1.4. Potentials and hurdles of current subcutaneous transplantation 50
3.1.5. 3D printing islets for high-efficient macroencapsulation system 50
3.2. Materials and method 51
3.2.4. Islet isolation 51
3.2.1. Preparation of bio-inks 52
3.2.4. Bioprinting of the encapsulation sheet 52
3.2.2. Permeability test to verify immune-protection of the bio-ink. 53
3.2.5. Mechanical test 53
3.2.6. COMSOL finite element analysis 53
3.2.7. In vitro islet viability test 54
3.2.8. Glucose stimulated insulin secretion (GSIS) test. 54
3.2.9. Subcutaneous transplantation 54
3.2.10. Histological analysis 54
3.2.11. Evaluation of glycemic control in-vivo 55
3.2.12. Intraperitoneal glucose tolerance test (IPGTT) 55
3.2.13. Quantification of macrophages 55
3.2.14. Statistical analysis 56
3.3. Results and discussion 56
3.3.1. Characterization of the bio-ink 56
3.3.2. Characterization of the printed sheet 57
3.3.3. The effect of shell-positioned islets on islet viability 61
3.3.4. Adjusting islet packing density and its effect on islet viability and functionality 62
3.3.5. Long-term glycemic control compared to injection of free islets 64
3.3.6. Potential of xeno-islet transplantation using the sheet co-delivery of immunomodulatory drug 65
3.3.7. Further design consideration to inhibit fibrosis: Effect of control of line width in lattice structure. 67
3.4. Conclusion 70
4. Suggestion for future works 71
4.1. Extension of applications using dot printing. 71
4.2. Scenario of clinical translation of islet transplantation from our developed macroencapsulation system. 72
5. References 74
Figure 1. Schematic illustration of typical generation methods of cell aggregates. (a) Cell aggregation in non-adherent concave well. (b) Cell aggregation using droplet microfluidic technique (c) Hanging... 16
Figure 2. A schematic illustration of different printing techniques of cell aggregates. 18
Figure 3. Schematic representation and photographic depiction of the customized bioprinter and its components. 19
Figure 4. Schematic illustration (a) and photos (b) of dot printing process. 28
Figure 5. Dot-printing results based on the viscosity of bio-inks. (a) Measured viscosity of the bio-inks. (b) Shear stress and shear rate plotted against increasing shear rate. (c) Confocal images of constructs... 29
Figure 6. Cell aggregate formation using the dot printing process. (a) Microscope images of HepG2 cultured for 3 days after dot printing (SBs, 200 μm). (b) Confocal images of fluorescent-stained HepG2... 31
Figure 7. Diameter controllability of cell aggregates with dot printing process. (a) Microscopic images of dot-printed HepG2 cells at right after printing (day 0) and formed cell aggregates (day 3) in variation... 34
Figure 8. Position controllability of cell aggregate using dot printing process. (a) Generated printing path of dots in motion program and microscope images showing printed results in variations of interval... 36
Figure 9. 3D patterning and fabrication application using dot printing technique. (a) Photograph (top) and microscopy (bottom) images of PCL sheet with dot-printed HepG2 aggregates (SB for the top image,... 37
Figure 10. Verification of effects on cytocompatibility and functionality upon dot printing process. (a) Confocal z-stack images of live and dead staining of PMH aggregates on day 3 and day 11. (b)... 39
Figure 11. Schematic illustrations and fabrication results of hepatic models with controlled positioning of EC and PMH aggregates. (a) Schematic illustration depicting each experimental group. (b) Confocal... 41
Figure 12. Effect of distance between hepatocyte aggregates and ECs on hepatic functionality. Microscope images (a), live and dead stained images (b), and quantified viability (c) of Proximal and... 42
Figure 13. Expanded application: development of a cancer invasion model (a) Microscopy and fluorescence images to visualize the progress of invasion assay of cell aggregates using dot-printed... 44
Figure 14. Cancer aggregates were patterned to form the word 'CANCER' within a matrix that included CAFs. 45
Figure 15. Expending application of incorporation of polydopamine-coated fragmented fibers with MSCs. (a) Schematic of the concept and experiment. High magnification image (b) and low... 46
Figure 16. Illustrations of micro- and macro-encapsulation and their examples. 49
Figure 17. Schematic conceptual description of our developed high-efficient islet macroencapsulation sheet. 51
Figure 18. Freshly isolated islets from a rat pancreas 52
Figure 19. Permeability test for immune protection. Confocal images (a) and the profile of intensity (n＝3) after 10 minuate (b) and 20 hours (c) of fluorescence-labeled IgG comparing pure alginates... 56
Figure 20. Printing results of the developed sheet. (a) Top view photograph of the sheet, showcasing the uniform distribution of islets (SB in the right image, 300 µm). (b) Microscope image displaying the... 57
Figure 21. Control of shell layer thickness with printing speed. (a) Fluorescence image of the printed shell layer at different printing speeds. (b) Quantification of the thickness of the shell layer... 58
Figure 22. Characterization of mechanical property of our developed sheet. (a) Load-displacement profile in variation of thickness of the sheets. Photographs (b) and folding frequency (c) in variation of... 59
Figure 23. Result of pre-and post-crosslinking using barium chloride solution directly to the alginate-based bio-ink. 60
Figure 24. Islet viability and functionality over crosslinking time with using barium chloride solution to the alginate-based bio-ink. (a) Dead staining results of islets in variation of crosslinking time. (b)... 60
Figure 25. Impact of islet positioning in the shell layer on oxygen diffusion and in vitro cell viability. (a) Schematic representation of experimental groups and simulated oxygen concentration color map. (b)... 62
Figure 26. Effect of controlling islet packing density on in-vitro viability and functionality (a) Live and dead staining results on day 8 in variation of islet packing density (SBs, 100 μm, arrow, the spot of... 63
Figure 27. Effect of controlling packing islet density on in-vivo glycemic control. Illustration of experimental groups (a, left), blood glucose measurement over time (n＝3) (a, right) and IPGTT on day... 64
Figure 28. Comparative in vivo evaluations of islet injection and developed sheet. (a) H&E Staining image (SB: 100 μm) and TUNEL assay result at 2 weeks of subcutaneous transplantation (SB: 100 μm... 65
Figure 29. Assessment of therapeutic potential in xenogeneic transplantation of the developed sheet. (a) experimental group illustration and bright-field images of retrieved Sheets with and without GW2580... 66
Figure 30. Impact of co-printing GW2580 on fibrosis. (a) H&E Staining Images at 2 weeks after subcutaneous transplantation (asterisk denotes the sheet; arrow indicates fibrotic layer; SB: 50 μm). (b)... 67
Figure 31. Control of line width of lattice sheet structure using a bioprinting technology. Microscope images (a) and quantification (b) of controlled line width by adjusting gelatin concentration in the... 68
Figure 32. Effect of control of line width in lattice structure on fibrosis after 2 weeks of subcutaneous transplantation. (a) Photographs of retrieved structures. Comparison of the number of macrophages (b),... 69
초록보기
Cell aggregate is cohesively gathered and adhered cells, enabling to mimic cell-to-cell interaction in tissue. To provide this feature, various generation methods of cell aggregates have been introduced for tissue engineering. However, due to a lack of additional combinations with three-dimensional (3D) fabrication process, creating tissue constructs using cell aggregates has been constrained with these conventional methods. This thesis explores the utilization of extrusion-based 3D bioprinting technology to print cell aggregates, presenting novel approaches in the tissue engineering field.
In session 1, we describe the general overview of cell aggregate. First, we define the terminology of "cell aggregate" in detail. And addressing the architecture of liver tissue as an example, we emphasize the significance of considering cell-to-cell interaction with a cell aggregate in tissue engineering. Furthermore, conventional methods for generating cell aggregates are explained, reviewing their fundamental principles.
Session 2 focuses on our development of a new 3D printing methodology of cell aggregates. Current bioprinting methodologies have successfully facilitated the printing of pre-generated cell aggregates to fabricate 3D tissue constructs. However, these methods face limitations in accurately and precisely patterning multiple types of cell aggregates to mimic complexity in tissue. Our study aims at developing the 3D printing method for precisely patterning of multiple types of cell aggregates. To achieve this, cells with dissolvable materials were embedded-printed into matrix, forming cell aggregates in-situ during culture period. This technique allowed for the successful printing of cell aggregates of multiple cell types with accurate control over diameter and position on a scale of tens of microns.
Session 3 covers another study, developing a novel design of macroencapsulation system at treating type 1 diabetes with 3D printing of pancreatic islets which are naturally cell aggregates. Macroencapsulation systems of islets for transplantation have shown a great potential to treat type 1 diabetes. However, current systems have hurdles for clinically promising subcutaneous transplantation, due to deficiency of interaction between islets and host blood vessels. We demonstrate developed multilayer sheet where islets are specifically positioned in the shell layer, enabling efficient exchange of oxygen and nutrient between islets and host blood vessel.
In session 4, we finalize the thesis by providing suggestions of a further study for the future based on our research findings. We address directions for the further application including organ-on-a-chip platform using our new printing method. Additionally, we open discussion on the translational potential of our developed islet macroencapsulation system for human treatment.MARC 보기
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