Title Page
Abstract
Contents
1. Introduction 10
1.1. Microenvironment of the BBB 12
1.2. In vitro 3D BBB model 14
1.2.1. 3D bioprinting limitations 16
1.2.2. Organ on a chip limitations 18
1.2.3. Rapid liquid printing 20
1.3. Polymer crosslinking 22
1.3.1. Ionic crosslinking and polymer library 23
2. Materials and Methods 25
2.1. Cell culture 25
2.2. Preparation of cell pellet-embedded alginate-collagen-Matrigel matrix phases 26
2.3. Ink phase material for 3D printing 27
2.4. Sulfo-SANPAH coating 27
2.5. ATPP systems 28
2.6. Cell viability 28
2.7. Vascular permeability 29
2.8. Immunofluorescence staining 29
3. Results and Discussion 30
3.1. 3D BBB on a chip technology overview 30
3.2. Experimental design 33
3.3. Fabrication and system configuration 35
3.3.1. Fabricating additional assistive systems 40
3.4. Effects of flow rate variation 46
3.5. Effects of ink phase type variation 49
3.6. Selection process for a 3D BBB matrix phase 52
3.6.1. Alginate-gelatin 54
3.6.2. Alginate-Matrigel 56
3.6.3. Alginate-collagen 58
3.6.4. Alginate-collagen-gelatin 60
3.6.5. Alginate-collagen-Matrigel 62
3.7. Effect of calcium chloride treatment 64
3.8. Immunofluorescence analysis 68
3.9. BBB permeability test 70
4. Conclusion 72
5. Reference 74
국문 요약 84
Table 1. Steps to select matrix phase for 3D BBB formation using ATPP 53
Fig. 1. Schematics diagram of BBB's structures 13
Fig. 2. In vitro 3D BBB models 15
Fig. 3. Illustration of 3D bioprinting technologies 17
Fig. 4. 3D BBB organ on a chip models 19
Fig. 5. Micro-scale rapid liquid printing methods 21
Fig. 6. A representation of an alginate crosslinking chain formed after CaCl₂ addition 24
Fig. 7. Overview of the ATPP-based 3D BBB on a chip 32
Fig. 8. Flow chart summarizing the 3D BBB on a chip experiment protocol 34
Fig. 9. Schematic of the planned 3D BBB on a chip design change 37
Fig. 10. Schematic representation of additional parts required for 3D BBB on a chip 38
Fig. 11. Detailed schematic of the 3D BBB on a chip 39
Fig. 12. Illustration of the cooling plate component and surface temperature analysis 42
Fig. 13. Photographs of the device that holds the metal microneedle and the liquid printing of channels based on microneedle size 43
Fig. 14. Photograph of the full ATPP system configuration 44
Fig. 15. Schematic representation of the time and control conditions to drive the Y and Z-axis motors of an ATPP system 45
Fig. 16. The relationship between flow rate and ink phase types 47
Fig. 17. Comparison graph of channel width by ink phase flow rate 48
Fig. 18. Comparison images of microchannel width by ink phase types 50
Fig. 19. Comparison graph of microchannel width by ink phase types 51
Fig. 20. Cell morphology and viability of HA and HBVP cells co-embedded in the 1st matrix phase; Cell viability was examined using calcein-AM (green)/PI (red) staining...[이미지참조] 55
Fig. 21. Cell morphology and viability of HA and HBVP cells co-embedded in the 2nd matrix phases; Cell viability was examined using calcein-AM (green)/PI (red) staining... 57
Fig. 22. Cell morphology and viability of HAs and HBVPs embedded in the 3rd matrix phases[이미지참조] 59
Fig. 23. Cell morphology and viability of HAs and HBVPs embedded in 4th matrix phases[이미지참조] 61
Fig. 24. Cell morphology and viability of HAs and HBVPs embedded in the 5th matrix phases[이미지참조] 63
Fig. 25. Dose test of CaCl₂ dissolved in different cell culture media; HA and HBVP cell morphology and viability in matrix phase according to the concentration of DMEM... 66
Fig. 26. Dose test of CaCl₂ dissolved in different cell culture media; HA and HBVP cell morphology and viability in matrix phase according to the concentration of EGM-2... 67
Fig. 27. 3D BBB structure formed through ATPP 69
Fig. 28. Quantitative measurement of 3D BBB permeability 71