以可逆加成斷裂鏈轉移方式合成溫度敏感性嵌段共聚物用於細胞脫附上之評估

簡易檢索 / 詳目顯示

回結果列表

研究生：	莊舜皓 Shun-Hao Chuang
論文名稱：	以可逆加成斷裂鏈轉移方式合成溫度敏感性嵌段共聚物用於細胞脫附上之評估 Synthesis Thermo-responsive Block Copolymer by Using Reversible Addition-Fragmentation Chain Transfer Polymerization for Cell detachment
指導教授：	蔡協致 Hsieh-Chih Tsai
口試委員:	蔡協致 Hsieh-Chih Tsai 謝達斌 Dar-Bin Shieh 洪維松 Wei-Song Hung 林宣因 Suian-Yin Lin 陳玉暄 Yu-Shuan Chen
學位類別：	碩士 Master
系所名稱：	應用科技學院 - 應用科技研究所 Graduate Institute of Applied Science and Technology
論文出版年：	2020
畢業學年度：	108
語文別：	英文
論文頁數：	110
中文關鍵詞：	溫度敏感性高分子、低臨界溶液溫度、高臨界溶解溫度、微載體、Cytodex 3 、細胞分離試劑
外文關鍵詞：	Thermo-responsive polymer, LCST, UCST, Microcarrier, Cytodex 3, Cell dissociation reagent
相關次數：	點閱：260 下載：0
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

上一筆

細胞培養是細胞生物學和分子生物學的重要技術。細胞分離試劑在細胞培養過程中是必須的。胰蛋白酶是常見的細胞分離試劑，但是其會破壞細胞和細胞外基質。還有一些較冷門的細胞分離試劑，其中一些需要花費更多時間才能從表面分離細胞，另一些則會破壞細胞和細胞外基質。研究表示，在培養容器底部鋪一層薄的Poly(N-isopropylacrylamide) (pNIPAm)薄膜可以用來幫助細胞脫付。pNIPAm是一種具有良好熱反應性的常見生物材料並且可用於許多領域。我們成功的合成了低臨界溶液溫度在37度的溫度敏感性嵌段共聚物，並透過核磁共振氫譜與全反射傅立葉轉換紅外線光譜儀來鑑定所合成的共聚高分子結構，並利用紫外光/ 可見光光譜儀來觀測其低臨界溶液溫度。另外，還進行了體外細胞毒性測試。嵌段共聚物溶液可以塗佈在不同的細胞培養基質上，例如蓋玻片和微載體。測量塗佈在蓋玻片膜的厚度與接觸角，膜厚小於1微米，接觸角接近55°，屬於親水性並且適合細胞培養。細胞可以貼附及生長在高分子薄膜表面，當溫度冷卻低於低臨界溶液溫度時，細胞可以脫附。初步的研究結果證實我們所合成的pNIPAm嵌段共聚物在細胞培養技術上具有做為溫感型細胞脫附底物的潛力。

Cell culture is an important technique for cellular and molecular biology. In the cell culture process, cell dissociation reagent is necessary for detachment of cells. Trypsin is a common cell dissociation reagent but it would damage cells and the extracellular matrix. There are several types of cell dissociation reagents. Among them, some may take time to detach cells from the surface and could still damage the cell and the extracellular matrix. Studies have shown that thin film of Poly(N-isopropylacrylamide) (pNIPAm) on the substrates can help cell to detach form the surface. pNIPAm is a commonly used polymer for thermo-responsive applications in a wide range of fields. We successfully synthesized a thermo-responsive block copolymer which have lower critical temperature solution temperature (LCST) close to 37°C. To examine the properties of the thermo-responsive block copolymer, we analyzed its 1H NMR, FT-IR and UV-Vis properties. Additionally, we also conducted in vitro cytotoxicity test in the material. The block copolymer solution can be coated on different cell culture substrates such as coverslip and microcarrier. The film coated on coverslip was measured for the thickness and the contact angles. The film was discovered be thinner than 1μm and the contact angle is approximately 55°, presenting its hydrophilic thus is ideal for cell culture. The cells can attach and grow on the surface of the copolymer and can be detached from the surface once the temperature was lower than LCST. In summary, our preliminary results indicated that the pNIPAm copolymer could be a potential matrix for cell detachment from the dish moderated by temperature.

誌謝    I
中文摘要    IV
ABSTRACT    V
CONTENTS    VII
LIST OF FIGURES    X
LIST OF TABLES    XIV
Chapter 1    Motivation and purpose    1
Chapter 2    Introduction    3
1    Cell culture technique    3
1.1    Introduction    3
1.2    Cell dissociation reagent    3
1.3    Thermo-responsive hydrogel for cell detachment    4
1.4    Microcarrier    5
2    Thermo-responsive polymer    8
2.1    Introduction    8
2.2    Stimuli-responsive polymers    10
2.3    Thermo-responsive polymers    11
2.4    Atom Transfer Radical Polymerization (ATRP)    13
2.5    Activators ReGenerated by Electron Transfer Atom Transfer Radical Polymerization (ARGET ATRP)    14
2.6    Reversible Addition-Fragmentation chain Transfer (RAFT)    15
2.7    Poly(N-isopropylacrylamide) (pNIPAm)    17
2.8    Poly[2-(Methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl) ammonium hydroxide (DMAPS)    19
3    Hydrogels    20
3.1    Introduction    20
3.2    Stimuli-responsive hydrogels    21
3.3    Thermo-responsive hydrogels    22
Chapter 3    Materials and methods    25
1    Materials    25
1.1    Preparation of the PEG macroinitiator    28
1.2    Preparation of the pDMAPS-PEG-pDMAPS triblock copolymer    28
1.3    Preparation of the poly(NIPAm-co-AAc) macroRAFT agent    29
1.4    Preparation of the poly(NIPAm-co-AAc)-block-PS copolymer    30
1.5    Preparation of the poly(NIPAm-co-AAm) macroRAFT agent    31
1.6    Preparation of the poly(NIPAm-co-AAm)-block-PS copolymer    32
2    Methods    33
2.1    Equipments    33
2.2    1H nuclear magnetic resonance spectroscopy (1H NMR)    37
2.3    Fourier-transform infrared spectroscopy (FT-IR)    37
2.4    Gel permeation chromatography (GPC)    37
2.5    Ultraviolet–visible spectroscopy (UV-Vis)    38
2.6    Thin film formation by spin coating    38
2.7    Alpha-step (α-step)    39
2.8    Scanning Electron Microscope (SEM)    40
2.9    Contact angle    40
2.10    Cytodex 3 prepared    40
2.11    Cytodex 3 coated    41
3    In vitro technology    42
3.1    Cell culture and cell harvesting    42
3.2    Cytotoxicity assays    42
3.3    Cell culture on film    43
3.4    Cell culture by Cytodex 3 microcarrier    44
Chapter 4    Result and discussion    45
1    Characterization of polymers    45
2    Molecular weight    53
3    UCST turbidity measurement    55
4    LCST turbidity measurement    57
5    Film characterization    60
5.1    Film thickness measured by alpha step    60
5.2    Film thickness measured by SEM    61
5.3    Contact angle    66
6    In vitro studies    70
6.1    Cytotoxicity    70
6.2    Cell culture on film    74
6.3    Characterization of coated microcarrier    85
6.4    Cell culture on microcarrier    87
Chapter 5    Conclusion    90
REFERENCE    92


                                

1. Yang, H. S.; Jeon, O.; Bhang, S. H.; Lee, S.-H.; Kim, B.-S., Suspension Culture of Mammalian Cells Using Thermosensitive Microcarrier that Allows Cell Detachment without Proteolytic Enzyme Treatment. Cell Transplantation 2010, 19 (9), 1123-1132.
2. Frantz, C.; Stewart, K. M.; Weaver, V. M., The extracellular matrix at a glance. Journal of Cell Science 2010, 123 (24), 4195-4200.
3. Omidian, H.; Park, K., Introduction to Hydrogels. In Biomedical Applications of Hydrogels Handbook, Ottenbrite, R. M.; Park, K.; Okano, T., Eds. Springer New York: New York, NY, 2010; pp 1-16.
4. Rizwan, M.; Yahya, R.; Hassan, A.; Yar, M.; Azzahari, A. D.; Selvanathan, V.; Sonsudin, F.; Abouloula, C. N., pH Sensitive Hydrogels in Drug Delivery: Brief History, Properties, Swelling, and Release Mechanism, Material Selection and Applications. Polymers 2017, 9 (4), 137.
5. Lanzalaco, S.; Armelin, E., Poly(N-isopropylacrylamide) and Copolymers: A Review on Recent Progresses in Biomedical Applications. Gels 2017, 3 (4), 36.
6. Tsuji, K.; Ojima, M.; Otabe, K.; Horie, M.; Koga, H.; Sekiya, I.; Muneta, T., Effects of Different Cell-Detaching Methods on the Viability and Cell Surface Antigen Expression of Synovial Mesenchymal Stem Cells. Cell Transplantation 2017, 26 (6), 1089-1102.
7. Huang, H.-L.; Hsing, H.-W.; Lai, T.-C.; Chen, Y.-W.; Lee, T.-R.; Chan, H.-T.; Lyu, P.-C.; Wu, C.-L.; Lu, Y.-C.; Lin, S.-T.; Lin, C.-W.; Lai, C.-H.; Chang, H.-T.; Chou, H.-C.; Chan, H.-L., Trypsin-induced proteome alteration during cell subculture in mammalian cells. Journal of Biomedical Science 2010, 17 (1), 36.
8. Waymouth, C., To disaggregate or not to disaggregate, injury and cell disaggregation, transient or permanent? In Vitro 1974, 10 (1), 97-111.
9. Okano, T.; Yamada, N.; Okuhara, M.; Sakai, H.; Sakurai, Y., Mechanism of cell detachment from temperature-modulated, hydrophilic–hydrophobic polymer surfaces. In The Biomaterials: Silver Jubilee Compendium, Williams, D. F., Ed. Elsevier Science: Oxford, 1995; pp 109-115.
10. Masuda, S.; Shimizu, T., Three-dimensional cardiac tissue fabrication based on cell sheet technology. Advanced Drug Delivery Reviews 2016, 96, 103-109.
11. Van Wezel, A. L., Growth of Cell-strains and Primary Cells on Micro-carriers in Homogeneous Culture. Nature 1967, 216 (5110), 64-65.
12. Kato, D.; Takeuchi, M.; Sakurai, T.; Furukawa, S.-i.; Mizokami, H.; Sakata, M.; Hirayama, C.; Kunitake, M., The design of polymer microcarrier surfaces for enhanced cell growth. Biomaterials 2003, 24 (23), 4253-4264.
13. Ravve, A., Introduction and Nomenclature. In Principles of Polymer Chemistry, Springer New York: New York, NY, 2012; pp 1-15.
14. Huang, J.; Turner, S. R., Recent advances in alternating copolymers: The synthesis, modification, and applications of precision polymers. Polymer 2017, 116, 572-586.
15. Bossion, A.; Heifferon, K. V.; Meabe, L.; Zivic, N.; Taton, D.; Hedrick, J. L.; Long, T. E.; Sardon, H., Opportunities for organocatalysis in polymer synthesis via step-growth methods. Progress in Polymer Science 2019, 90, 164-210.
16. Meng, H.; Hu, J., A Brief Review of Stimulus-active Polymers Responsive to Thermal, Light, Magnetic, Electric, and Water/Solvent Stimuli. Journal of Intelligent Material Systems and Structures 2010, 21 (9), 859-885.
17. Liu, F.; Urban, M. W., Recent advances and challenges in designing stimuli-responsive polymers. Progress in Polymer Science 2010, 35 (1), 3-23.
18. Reineke, T. M., Stimuli-Responsive Polymers for Biological Detection and Delivery. ACS Macro Letters 2016, 5 (1), 14-18.
19. Gooch, J. W., Critical Solution Temperature. In Encyclopedic Dictionary of Polymers, Gooch, J. W., Ed. Springer New York: New York, NY, 2011; pp 180-180.
20. Wang, J.-S.; Matyjaszewski, K., Controlled/"living" radical polymerization. atom transfer radical polymerization in the presence of transition-metal complexes. Journal of the American Chemical Society 1995, 117 (20), 5614-5615.
21. Kato, M.; Kamigaito, M.; Sawamoto, M.; Higashimura, T., Polymerization of Methyl Methacrylate with the Carbon Tetrachloride/Dichlorotris- (triphenylphosphine)ruthenium(II)/Methylaluminum Bis(2,6-di-tert-butylphenoxide) Initiating System: Possibility of Living Radical Polymerization. Macromolecules 1995, 28 (5), 1721-1723.
22. Matyjaszewski, K.; Xia, J., Atom Transfer Radical Polymerization. Chemical Reviews 2001, 101 (9), 2921-2990.
23. Matyjaszewski, K.; Jakubowski, W.; Min, K.; Tang, W.; Huang, J.; Braunecker, W. A.; Tsarevsky, N. V., Diminishing catalyst concentration in atom transfer radical polymerization with reducing agents. Proceedings of the National Academy of Sciences 2006, 103 (42), 15309-15314.
24. Jakubowski, W.; Matyjaszewski, K., Activators Regenerated by Electron Transfer for Atom-Transfer Radical Polymerization of (Meth)acrylates and Related Block Copolymers. Angewandte Chemie International Edition 2006, 45 (27), 4482-4486.
25. Chiefari, J.; Chong, Y. K.; Ercole, F.; Krstina, J.; Jeffery, J.; Le, T. P. T.; Mayadunne, R. T. A.; Meijs, G. F.; Moad, C. L.; Moad, G.; Rizzardo, E.; Thang, S. H., Living Free-Radical Polymerization by Reversible Addition−Fragmentation Chain Transfer: The RAFT Process. Macromolecules 1998, 31 (16), 5559-5562.
26. van der Linden, H. J.; Herber, S.; Olthuis, W.; Bergveld, P., Stimulus-sensitive hydrogels and their applications in chemical (micro)analysis. Analyst 2003, 128 (4), 325-331.
27. Katsumoto, Y.; Tanaka, T.; Sato, H.; Ozaki, Y., Conformational Change of Poly(N-isopropylacrylamide) during the Coil−Globule Transition Investigated by Attenuated Total Reflection/Infrared Spectroscopy and Density Functional Theory Calculation. The Journal of Physical Chemistry A 2002, 106 (14), 3429-3435.
28. Chang, Y.; Liao, S.-C.; Higuchi, A.; Ruaan, R.-C.; Chu, C.-W.; Chen, W.-Y., A Highly Stable Nonbiofouling Surface with Well-Packed Grafted Zwitterionic Polysulfobetaine for Plasma Protein Repulsion. Langmuir 2008, 24 (10), 5453-5458.
29. Dai, F.; Wang, P.; Wang, Y.; Tang, L.; Yang, J.; Liu, W.; Li, H.; Wang, G., Double thermoresponsive polybetaine-based ABA triblock copolymers with capability to condense DNA. Polymer 2008, 49 (24), 5322-5328.
30. Varaprasad, K.; Raghavendra, G. M.; Jayaramudu, T.; Yallapu, M. M.; Sadiku, R., A mini review on hydrogels classification and recent developments in miscellaneous applications. Materials Science and Engineering: C 2017, 79, 958-971.
31. Shi, Q.; Liu, H.; Tang, D.; Li, Y.; Li, X.; Xu, F., Bioactuators based on stimulus-responsive hydrogels and their emerging biomedical applications. NPG Asia Materials 2019, 11 (1), 64.
32. Schmaljohann, D., Thermo- and pH-responsive polymers in drug delivery. Advanced Drug Delivery Reviews 2006, 58 (15), 1655-1670.
33. Phillips, D. J.; Gibson, M. I., Towards being genuinely smart: ‘isothermally-responsive’ polymers as versatile, programmable scaffolds for biologically-adaptable materials. Polymer Chemistry 2015, 6 (7), 1033-1043.
34. Schild, H. G., Poly(N-isopropylacrylamide): experiment, theory and application. Progress in Polymer Science 1992, 17 (2), 163-249.
35. Shibayama, M.; Norisuye, T.; Nomura, S., Cross-link Density Dependence of Spatial Inhomogeneities and Dynamic Fluctuations of Poly(N-isopropylacrylamide) Gels. Macromolecules 1996, 29 (27), 8746-8750.
36. Idziak, I.; Avoce, D.; Lessard, D.; Gravel, D.; Zhu, X. X., Thermosensitivity of Aqueous Solutions of Poly(N,N-diethylacrylamide). Macromolecules 1999, 32 (4), 1260-1263.
37. Mikheeva, L. M.; Grinberg, N. V.; Mashkevich, A. Y.; Grinberg, V. Y.; Thanh, L. T. M.; Makhaeva, E. E.; Khokhlov, A. R., Microcalorimetric Study of Thermal Cooperative Transitions in Poly(N-vinylcaprolactam) Hydrogels. Macromolecules 1997, 30 (9), 2693-2699.
38. Van Durme, K.; Verbrugghe, S.; Du Prez, F. E.; Van Mele, B., Influence of Poly(ethylene oxide) Grafts on Kinetics of LCST Behavior in Aqueous Poly(N-vinylcaprolactam) Solutions and Networks Studied by Modulated Temperature DSC. Macromolecules 2004, 37 (3), 1054-1061.
39. Makhaeva, E. E.; Tenhu, H.; Khokhlov, A. R., Conformational Changes of Poly(vinylcaprolactam) Macromolecules and Their Complexes with Ionic Surfactants in Aqueous Solution. Macromolecules 1998, 31 (18), 6112-6118.
40. Kwon, Y.; Kim, S., Thermosensitive biodegradable hydrogels for the delivery of therapeutic agents. Polymeric Drug Delivery Systems 2005, 251.
41. Lee, J.; Macosko, C. W.; Urry, D. W., Swelling Behavior of γ-Irradiation Cross-Linked Elastomeric Polypentapeptide-Based Hydrogels. Macromolecules 2001, 34 (12), 4114-4123.
42. Aoki, T.; Kawashima, M.; Katono, H.; Sanui, K.; Ogata, N.; Okano, T.; Sakurai, Y., Temperature-Responsive Interpenetrating Polymer Networks Constructed with Poly(acrylic acid) and Poly(N,N-dimethylacrylamide). Macromolecules 1994, 27 (4), 947-952.
43. Mosmann, T., Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods 1983, 65 (1), 55-63.
44. Elbert, D. L.; Hubbell, J. A., Surface Treatments of Polymers for Biocompatibility. Annual Review of Materials Science 1996, 26 (1), 365-394.
45. Lee, J. H.; Khang, G.; Lee, J. W.; Lee, H. B., Interaction of Different Types of Cells on Polymer Surfaces with Wettability Gradient. Journal of Colloid and Interface Science 1998, 205 (2), 323-330.
46. Xi, T. F.; Fan, C. X.; Feng, X. M.; Wan, Z. Y.; Wang, C. R.; Chou, L. L., Cytotoxicity and altered c-myc gene expression by medical polyacrylamide hydrogel. Journal of Biomedical Materials Research Part A 2006, 78A (2), 283-290.

全文公開日期 2025/08/17 (校內網路)
全文公開日期本全文未授權公開 (校外網路)
全文公開日期本全文未授權公開 (國家圖書館：臺灣博碩士論文系統)

簡易檢索 / 詳目顯示

相關論文