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研究生：	周宛璇 Chou, Wan-Hsuan
論文名稱：	微米/奈米膠球三維細胞成長基的製備與表面改質效應研究 Fabrication and Surface Modification of Micro/Nano Spherical Hydrogel Scaffolds for 3D Cell Culture
指導教授：	劉瑞祥 Liu, Jui-Hsiang
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2021
畢業學年度：	109
語文別：	英文
論文頁數：	83
中文關鍵詞：	三維細胞培養、微米球、奈米顆粒、陽離子單體、明膠
外文關鍵詞：	3D cell culture, microspheres, nanoparticles, cationic monomer, gelatin
相關次數：	點閱：81 下載：0
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本研究展示了聚（2-甲基丙烯酸羥乙酯）(PHEMA)微米與奈米膠球的合成，以用作在體外構建三維人工組織的細胞支架。微米球和奈米球分別使用懸浮聚合與油包水乳化聚合方法來製備，過程中使用無毒的介面活性劑。聚合的微米球直徑大約為 500 μm，另外，奈米球的平均粒徑為 450 nm。本實驗研究了微粒和奈米顆粒在三維細胞組織培養中的應用，微米球支架用於人類真皮纖維細胞(NHDF)的培養，而奈米顆粒用於小鼠纖維細胞(L929)的培養。為了促進細胞黏附能力，具有陽離子電荷的單體3-(甲基丙烯醯丙基)三甲基氯化銨(MPTC)共聚合於微米球中，結果顯示MPTC 含量除了增強熱穩定性外也改善細胞附著力。另外，研究了使用明膠和殼聚醣塗佈於微米球的表面是否對細胞生長有增益效果，結果指出適當的陽離子電荷含量和明膠塗層都促進了人類真皮纖維細胞在微米球上的生長，相較於其他結果，具有2% MPTC含量的PHEMA 微米球在 3D支架中表現出最好的細胞存活率和增殖能力。至於與微米球支架概念不同的奈米粒子，其結果顯現出通過與奈米粒子的混合，多層結構的L929細胞可通過與奈米粒子的混合來實現。根據結果，含有微量MPTC與明膠塗層的 PHEMA 微米球成功應用在體外NHDF細胞在三維空間的培養，此外，也證明了PHEMA奈米顆粒具有三維組織工程的可能性。

This study demonstrates the synthesis of poly(2-hydroxyethyl methacrylate) (PHEMA) micro-/nano-particles using as scaffolds to construct three-dimensional (3D) artificial tissues in vitro. Microsphere and nanosphere hydrogel scaffolds were synthesized via suspension and inverse emulsion polymerization. The synthesized average diameter of microspheres were around 500 μm, and the average size of nanoparticles was 450 nm. Applications of both microparticles and nanoparticles on 3D cell culture were investigated. Normal human dermal fibroblast (NHDF) cells were seeded onto microsphere scaffolds, whereas mouse fibroblast L929 cells were cultured with nanoparticles. To promote cell adhesion, cationic charged monomer [3-(methacryloylamino)propyl]trimethyl- ammonium chloride (MPTC) was introduced into the microspheres. In addition, enhancement of cell growing via the surface coating of gelatin and chitosan onto the microspheres was investigated. Increase of MPTC content enhances the thermal stability and cell attachment obviously. The fluorescent images indicated that both cationic charge content and gelatin-coating elevated the growth of NHDF cells on microparticles. In terms of results, PHEMA microspheres with an optimal positive charge content of 2% MPTC demonstrate high cell viability and proliferation rate in 3D scaffolds. As for PHEMA nanoparticles, fabrication of the multilayer of L929 cells was achieved by sedimentation of the blending with nanoparticles. Based on the results, NHDF cell culture in 3D space was achieved using PHEMA microsphere scaffolds in vitro. Furthermore, the possibility of 3D tissue engineering using nanoparticles is also evidenced.

Abstract	I
中文摘要	II
誌謝	III
Contents	IV
List of Figures	VII
List of Tables	XII
1.	Introduction	1
1-1	Preface	1
1-2	Research motivation	2
2.	Literature Review	3
2-1 Heterogeneous Free-Radical Polymerization Techniques	3
2-1-1 Suspension Polymerization	4
2-1-2 Emulsion Polymerization	5
2-1-3 Inverse Emulsion Polymerization	8
2-2 Tissue Engineering	8
2-2-1 Introduction	9
2-2-2 3D Scaffolds	9
2-2-3 Types of 3D Scaffolds-Based on Their Geometry [14]	12
2-2-4 Scaffolds Fabrication Methods	16
2-3 Microspheres/Microparticles for Cell Culture	17
2-3-1 Introduction	17
2-3-1 3D Cell Culture Technology Using Microcarriers	18
2-3-2 Characteristics of Microparticles	19
2-4 Surface Modification of Microparticle Scaffolds	20
2-4-1 Modification with Charged Groups	20
2-4-2 Surface Modification with Proteins	21
2-4-3 Surface Modification with Bioactive Compounds	22
2-5 Poly(HEMA)	23
2-5-1 Characteristics of Poly(HEMA)	23
2-5-2 Applications of Poly(HEMA)	23
2-6 Gelatin	26
2-6-1 Types of Gelatins	27
2-7 Chitosan	27
3.	Experimental Section	29
3-1 Materials	29
3-2 Instruments	31
3-3 Experimental Section	32
3-3-1 Synthesis of Poly(HEMA) and Poly(HEMA-co-MPTC)) Microparticles	32
3-3-2 Surface Modification of Poly(HEMA) Microparticles	34
3-3-3 Equilibrium Water Content of Poly(HEMA) Microparticles	36
3-3-4 Characterization of Poly(HEMA) Microparticles	36
3-3-5 Synthesis of Poly(HEMA) Nanoparticles	37
3-3-6 Characterization of Poly(HEMA) Nanoparticles	41
3-3-7 Cell Culture Study	41
4.	Results and Discussions	44
4-1 Synthesis of Poly(HEMA) Microparticles	44
4-1-1 Characterization of Poly(HEMA) Microparticles	45
4-1-2 Morphology of Poly(HEMA) Microparticles	46
4-1-3 Thermal Properties of Poly(HEMA) Microparticles	49
4-1-4 Equilibrium Water Content of Poly(HEMA) Microparticles	50
4-2 Surface Modification of Poly(HEMA) Microparticles	51
4-2-1 Morphology of Modified Poly(HEMA) Microparticles	52
4-2-2 Analysis of Surface-Modified Poly(HEMA) Microparticles	53
4-3 Synthesis of Poly(HEMA) Nanoparticles via Emulsion Polymerization	55
4-3-1 Size Distribution of Poly(HEMA) Nanoparticles	55
4-4 Synthesis of Poly(HEMA) Nanoparticles via Inverse Emulsion Polymerization	56
4-4-1 Size Distribution of Poly(HEMA) Nanoparticles	59
4-4-2 Morphology of Poly(HEMA) Nanoparticles	61
4-4-3 Characterization of Poly(HEMA) Nanoparticles	62
4-5 Studies on Microsphere Scaffolds for Cell Culture	64
4-5-1 Cell Growth on poly(HEMA) Microsphere Scaffolds	65
4-5-2 Cell Proliferation on Poly(HEMA) Microsphere Scaffolds	68
4-5-3 Cell Adhesion on Poly(HEMA) Microsphere Scaffolds	72
4-5-4 Cell Viability on Poly(HEMA) Microsphere Scaffolds	73
4-5-5 Cell Growth with poly(HEMA) Nanoparticles	75
5.	Conclusions	77
Reference	78
                                    

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