傳統的體外二維培養具方便易觀察的優點，但二維模型往往無法模擬體內器官的立體結構和微環境。體外三維模型提供了一種新方法，可以彌補傳統 2D 培養和動物模型之間的差距。大腦是人類最複雜的器官之一，且神經退化性疾病已成為未來老年化社會的重要問題。水膠載體的開發有助於神經組織工程的應用；而三維體外大腦模型的建構將能有助於探討疾病問題、輔助藥物篩選、以及新療法的開發。三維生物列印是近年熱門的體外模型建構工具之一，但模擬大腦的三維生物列印仍有許多待突破的困境。為了能夠更真實模擬神經網絡在三維結構下生長的微環境，需要建構可以準確模擬適合神經細胞生長的微環境並貼近人體內生理環境組成的三維結構。本研究旨在開發適合神經生長環境的生物墨水、建構體外模擬3D大腦的微環境，並探討其應用於3D大腦列印的可行性。
為了開發可以應用於模擬真實大腦組織的三維列印仿生墨水，本研究萃取蠶絲蛋白並經經過GMA(Glycidyl Methacrylate)化學修飾改質為光交聯甲基丙烯醯化蠶絲蛋白 (Sil-MA)，並添加取自水果果皮的Pectin以製備系列Sil-MA-Pectin生物墨水。Sil-MA是一種具有高生物相容性與機械性質的水膠，而藉由添加不同濃度之Pectin可調整水膠的軟硬度並增加其可列印性。GMA對蠶絲蛋白的修飾可以通過在傅立葉變換紅外光譜 (FT-IR) 和核磁共振光譜 (H-NMR)中檢視GMA以及SF 的特徵相關峰，並證明GMA對蠶絲蛋白的修飾。此外，藉由量測Sil-MA-Pectin水膠的機械和流變性質，發現可以透過改變 Sil-MA 或Pectin的濃度來調整水膠的機械性質。隨著Sil-MA濃度的增加，水膠的壓縮模量會增加，但反之Pectin的加入越多，水膠會變得越來越軟。此系統可利用兩種交聯方式成膠，Sil-MA是透過光交聯成膠，Pectin則是透過離子交聯。Sil-MA-Pectin水膠的掃描電子顯微照片（SEM）顯示出水膠微觀結構，表現出適合細胞生長或延伸的多孔型態。細胞活性測試以及毒性測試證明系列Sil-MA-Pectin水膠均具有良好的生物相容性。為了探討此系列水膠應用於體外大腦模型建構與神經組織工程的可行性，接著將取自懷孕16天的母鼠胚胎的神經幹細胞球體包覆在系列Sil-MA-Pectin水膠中以觀察神經幹細胞在三維模型中的分化情形，並藉由染色結果進行量化分析。結果顯示，在未添加任何生長因子的環境中，神經幹細胞在系列Sil-MA-Pectin水膠均展現良好的分化性質，其中神經元的分化百分比均高於80%。而可列印性測試證實15%SilMA-0.5%Pectin具有最佳的可列印性。因此最後使用此生物墨水以擠出式3D列印機對包埋神經幹細胞的生物墨水進行列印，細胞死活與免疫染色結果均顯示，神經幹細胞通過列印噴嘴後仍然可以保持高活力狀態並發現Sil-MA-Pectin生物墨水依然展現出良好的分化性質，其中神經元的分化百分比均高於80%，成功建構出模擬大腦的3D體外模型。
本研究使用以甲基丙烯醯化蠶絲蛋白與果膠結合製備出的3D水膠模擬體外大腦微環境，能夠有效搭載神經幹細胞並促進神經幹細胞的分化與生長，未來可能開發出更複雜的3D體外大腦模型或建立其他疾病模型並應用於神經組織工程中。

Generally, conventional in vitro cell-based evaluations are implemented under two-dimensional culture (2D) conditions, however, Common 2D models easy to observe but lack the proper recapitulation of organ structure and environment. In vitro 3D model provides a new approach which bridges the gap between traditional 2D culture and animal model. Brain is one of the most complex organs in human beings, and neurodegenerative diseases have become an important issue in the future. The development of hydrogel carriers is conducive to the application of neural tissue engineering; and the construction of 3D in vitro brain models will help to explore disease problems, assist drug screening, and develop new therapies. 3D bioprinting is one of the popular in vitro model building tools in recent years, but there are still many difficulties to be overcome in 3D bioprinting simulating the brain. Therefore, it is necessary to create a platform consist of cell models that accurately simulate the cells microenvironment, along with flexibly prototyped cell handling structures that mimic the in vivo environment.
Hydrogels are promising biomedical materials because they resemble biological tissues with regard to their morphology, high degree of flexibility and water holding capacity.
To develop a 3D in vitro model that can mimic the real tissue, photo-crosslinkable acid silk fibroin (Sil-MA) and Pectin, an inexpensive and negatively charged polysaccharide that is found in the cell walls of terrestrial plants, were used to prepare the Sil-MA/Pectin hydrogels. Sil-MA is a hydrogel with high biocompatibility and mechanical properties. By adding different concentrations of Pectin, the hardness of the hydrogel can be adjusted and its printability can be increased.
Modification of Glycidyl Methacrylate (GMA) on silk fibroin was confirmed through identification of GMA-related peaks and Silk Fibroin(SF)-related peaks by Fourier-transform infrared spectroscopy (FT-IR) and Nuclear Magnetic Resonance spectroscopy (H-NMR). Besides, the mechanical and rheological properties of Sil-MA/Pectin hydrogel were determined and it is revealed that the mechanical properties were modulated by varying the Sil-MA or pectin contents, With the increase of Sil-MA concentration, the compressive modulus increase of the hydrogel were observed. In contrast, when more Pectin is added, the hydrogel become softer. There are two kinds of crosslinking mechanism were used in this study, Sil-MA is cross-linked by UV and Pectin is through ionic cross-linking. Scanning electron micrographs photographs of Sil-MA-Pectin hydrogels show that hydrogel demonstrated porous morphology which is suitable for cell growth, and cell viability test also proved that Sil-MA/Pectin hydrogel showed good biocompatibility.
In order to evaluate the feasibility of the application of the series of Silk fibroin based bioinks on neural engineering and the in vitro model, Neural stem cells (NSCs) spheroids obtained from pregnant ED 16 Wistar rat embryos were encapsulated in a series of Sil-MA-Pectin hydrogels to observe the differentiation of neural stem cells in a 3D model. In addition, the differentiation percentages of neuron/astrocyte were analyzed by immunostaining. The results showed that NSCs exhibited good differentiation properties in the series of Sil-MA-Pectin hydrogels, and the percentage of neuron differentiation was higher than 80%.
The printability test confirmed that 15% SilMA-0.5% Pectin showed the best printability. Therefore, this hydrogel was finally used on NSCs spheroid laden 3D bioprinting by an extrusion 3D printer. The results of Live&Dead and immunostaining all showed that the NSCs spheroids can still maintain a high vitality state after passing through the printing nozzle.
In this study, 3D printed used bio-inks prepared by Sil-MA and pectin was used to simulate the brain in vitro microenvironment, which can effectively carry NSCs and promote the differentiation and growth. It is considered that series of adjustable Sil-MA/pectin bio-inks provides the alternative on 3D bio-printing for in vitro biological tissue model development. In the future, it is possible to develop more complex 3D in vitro Brain models or other disease models and applied in neural tissue engineering.

致謝 ii
中文摘要 iii
Abstract vi
縮寫一覽表 x
目錄 xi
圖目錄 xv
表目錄 xvii
第一章 序論 1
1.1 前言 1
1.2 研究動機與目的 3
1.3 實驗架構 4
第二章 研究背景、原理與文獻回顧 5
2.1 神經幹細胞 5
2.1.1 神經幹細胞之特性 5
2.1.2 微環境對神經幹細胞的影響 5
2.2 體外微環境 6
2.2.1 2D體外微環境模擬 6
2.2.2 3D體外微環境模擬 7
2.2.3 體外大腦模型 9
2.3 生物墨水 10
2.3.1 生物墨水 10
2.3.2 天然來源的生物墨水 10
2.3.3 人工合成的生物墨水 11
2.3.4 水膠交聯機制 12
2.3.5 雙交聯水膠 (Dual crosslinking) 13
2.3.6 蠶絲蛋白(Silk fibroin)與Sil-MA 14
2.3.7 果膠Pectin 15
2.3.8 以蠶絲蛋白為基底的生物墨水及其應用 16
2.4 3D列印 17
2.4.1 3D列印原理 17
2.4.2 3D生物列印及其應用 18
第三章 實驗方法與材料 21
3.1 藥品、藥品製備與儀器 21
3.1.1 藥品 21
3.2 儀器設備 24
3.3 試劑製備 25
3.3.1 L929細胞培養基製備 (for 1 Liter, pH7.4) 25
3.3.2 神經幹細胞培養基製備 (for 1 Liter, pH7.4) 25
3.3.3 PBS製備 (for 0.5 Liter, pH7.4) 26
3.3.4 HBSS製備 (for 1 Liter, pH 7.4) 26
3.3.5 BSA製備 (for 50 ml) 27
3.3.6 MTT assay 製備 (for 50 ml) 27
3.3.7 蠶絲蛋白(Silk Fibroin)合成 27
3.3.8 Sil-MA合成 28
3.3.9 Sil-MA、Sil-MA/Pectin溶液製備 29
3.3.10 PDMS 模具 30
3.3.11 Sil-MA、Sil-MA/Pectin光交聯 31
3.4 細胞培養 31
3.4.1 老鼠纖維母細胞株L929培養 31
3.4.2 老鼠纖維母細胞株L929繼代 31
3.4.3 神經幹細胞分離 32
3.4.4 神經幹細胞繼代培養 33
3.4.5 神經幹細胞之細胞計數 34
3.5 實驗步驟與方法 35
3.5.1 核磁共振光譜儀(1H-NMR) 35
3.5.2 傅立葉變換紅外光譜(FT-IR) 35
3.5.3 高解析熱場發射掃描式電子顯微鏡(SEM) 36
3.5.4 壓縮模量 36
3.5.5 溶脹性質測試(Swelling ratio) 36
3.5.6 流變性質測試 37
3.5.7 頻率掃描(frequency sweep) 38
3.5.8 應變掃描(strain sweep) 38
3.5.9 細胞活性測試 38
3.5.10.1 LDH assay 細胞毒性之生化分析 38
3.5.10.2 MTT assay細胞活性之生化分析 39
3.5.10.3 CCK-8細胞增殖與細胞毒性分析 40
3.5.10.4 Live & Dead細胞活力之生化分析 40
3.5.10 幹細胞分化測試 41
3.5.11 可列印性測試 43
3.5.12 3D列印神經幹細胞株(NE-4C)Live&Dead測試 44
3.5.13 3D列印神經幹細胞分化測試 45
3.5.14 統計分析 46
第四章 結果與討論 47
4.1 Sil-MA合成與取代度分析結果 47
4.1.1 1H-NMR光譜取代度分析結果 47
4.1.2 FTIR比較結果 48
4.2 Sil-MA、Sil-MA-Pectin交聯結果 49
4.3 Sil-MA、Sil-MA-Pectin 機械性質結果 54
4.4.1 Sil-MA、Sil-MA-Pectin膨潤度測試結果 54
4.4.2 Sil-MA、Sil-MA-Pectin壓縮性質結果 56
4.4.3 Sil-MA、Sil-MA-Pectin剪切稀化結果 58
4.4.4 Sil-MA、Sil-MA-Pectin流變性質結果 61
4.4 Sil-MA、Sil-MA-Pectin SEM結果 66
4.5 生物相容性測試結果 68
4.5.1 細胞毒性分析(LDH assay)測試結果 68
4.5.2 細胞存活力分析(MTT assay)測試結果 70
4.5.3 CCK8測試結果 70
4.5.4 Live & Dead測試結果 72
4.6 3D神經幹細胞分化測試結果 74
4.7 3D 列印可列印測試結果 78
4.8 3D列印神經幹細胞株(NE-4C) Live&Dead測試結果 81
4.9 3D列印神經幹細胞測試結果 84
4.10 Sil-MA/Pectin 3D擠出式列印輔助方法 87
第五章 結論 92
第六章 參考文獻 93

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