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研究生： 王鈺富
Wang, Yu-Fu
論文名稱： 發展具光交聯及生物可相容性的三維細胞支架作為細胞之應用
Development of Photo-Crosslinking Biocompatible Three Dimensional Pattern Scaffold for Cell Application
指導教授： 葉明龍
Yeh, Ming-Long
學位類別： 碩士
Master
系所名稱： 工學院 - 醫學工程研究所
Institute of Biomedical Engineering
論文出版年： 2011
畢業學年度： 99
語文別： 英文
論文頁數： 46
中文關鍵詞： 光交聯 、烯基化聚乙二醇 、水膠 、雙光子雷射掃瞄顯微鏡
外文關鍵詞： Photon Cross-linking, Poly (ethylene) glycol-diacrylate (PEG-DA), Hydrogel, Two Photon Laser Scanning Microscopy
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    • 在三維的支架中可以提供細胞一個更接近真實以及讓細胞生長的環境，同時也提供細胞在多維度下的訊息傳遞。目前，越來越多得文獻回顧也提及在三維空間的細胞培養，相較於二維空間的細胞培養有個明顯的差異性。雙光子雷射掃瞄系統是一個可以深入活體組織，並減少侵入性的雷射掃瞄系統。此系統不僅僅可以作為掃瞄系統，也同時可以作為光交聯聚合物材料之光源，利用此雷射系統可以在基材內準確的製造出三維之光交聯圖形。因此，結合三維的支架細胞培養，以及雙光子雷射掃瞄系統之加工過程，我們希望可以發展出一套引導細胞生長及移動之網路。
    此篇研究目的為找出利用烯基化聚乙二醇作為材料之支架，並配合雙光子雷射掃瞄系統之應用範疇內，最適當之加工參數。結果顯示，利用自製的烯基化聚乙二醇做為支架，其適當的光交聯濃度為百分之十五。同時雙光子雷射掃瞄系統也可適當的作為支架之加工工具。當支架表面含有吸引細胞貼附之胜肽時，可以增加其細胞之貼附狀況。最後，也發現利用纖維蛋白膠混入細胞可以作為支架之中心結構。再建構了整個基本系統後，此系統是具有潛力進一步發展成利用三維、可生物降解支架，來引導細胞之生長和移動。

    The three dimensional (3D) cultures can provide a more realistic environment for cell growth with its resemblance in cell-to-cell interaction in all dimensions. More studies have shown that cells cultured in 3D environment respond differently to those in two dimensions. Two photon laser scanning microscopy can image the living tissue at higher depth and less invasive. The two photon laser can also work as light source for photo-crosslinking polymers to create precise 3D crosslinking pattern. Therefore, by combining 3D cell culture scaffolds with two photon laser microprocessing, the cell guiding networks can be achieved.
    The purpose of this study was to develop the foundation of the poly (ethylene) glycol-diacrylate (PEG-DA) hydrogel with the two photon laser scanning system. Our results showed that the most appropriate concentration of self-made PEG-DA for gelation was 15 % ( w/v). The two photon laser scanning system could be used as a processing tool to the PRG-DA hydrogel. With the cell adhesive ligand, GRGDK, at the surface of the scaffold, it could increase the condition of the cell attachment. Finally, we also developed the core structure with the fibrin gel. With the basic condition established, the further step could be developed. With cells inside this kind of 3D patterned and biodegradable scaffold, cell migration and proliferation could be guided. The cell manipulation and signal recording in the 3D environment could also potentially be achieved.

    摘要 II ABSTRACT III 致謝 IV TABLE OF CONTENTS V LIST OF FIGURES VII LIST OF TABLES IX CHAPTER 1 INTRODUCTION 1 1.1 BACKGROUND 1 1.2 CHOICE OF POLYMER 2 1.3 PHOTO-POLYMERIZATION 3 1.4 DEGRADATION OF SCAFFOLD 3 1.5 TWO PHOTON LASER SCANNING MICROSCOPY 4 1.6 PURPOSE AND SPECIFIC AIMS 6 CHAPTER 2. MATERIALS AND METHODS 7 2.1 FLOW CHART OF THE EXPERIMENT 7 2.2 MATERIALS AND INSTRUMENTS 8 2.3 CELL CULTURE 9 2.4 CHARACTERISTIC OF PEG-DA GEL 10 2.4.1 PEG-DA Synthesis 10 2.4.2 UV Effect to the Cell 11 2.4.3 Swelling Ratio 11 2.4.4 Cell compatibility with PEG-DA Gel 12 2.5 2D HYDROGEL SCAFFOLD 12 2.5.1 PEG-DA gel with R6G 13 2.5.2 PEG-DA gel with 5-FAM 13 2.5.3 PEG-DA gel with 5-FAM-GRGDK 14 2.5.4 PEG-DA gel with 5-FAM-GRGDK and cell 15 2.6 3D HYDROGEL SCAFFOLD 16 2.6.1 3D scaffold with designed pattern 16 2.6.2 Core structure of the 3D hydrogel scaffold 17 2.7 STATISTICAL ANALYSIS 18 CHAPTER 3. RESULTS 19 3.1 PEG-DA SYNTHESIS 19 3.2 UV EFFECT TO THE CELLS 20 3.3 SWELLING RATIO 21 3.4 CELL COMPATIBILITY WITH PEG-DA GEL 22 3.5 PEG-DA GEL WITH R6G 23 3.6 PEG-DA GEL WITH 5-FAM 24 3.7 PEG-DA GEL WITH 5-FAM-GRGDK 25 3.8 PEG-DA GEL WITH 5-FAM-GRGDK AND CELLS 27 3.9 3D SCAFFOLD WITH DESIGNED PATTERN 29 3.10 CORE STRUCTURE OF THE 3D HYDROGEL SCAFFOLD 30 CHAPTER 4. DISCUSSION 32 4.1 CHARACTERISTIC OF PEG-DA GEL 32 4.2 2D HYDROGEL SCAFFOLD 34 4.3 3D HYDROGEL SCAFFOLD 37 4.4 EXPERIMENT LIMITATION 37 4.5 FUTURE PROSPECTION 38 CHAPTER 5. CONCLUSION 39 REFERENCES 40 CURRICULUM VITAE 45

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