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研究生:賴奕翰
研究生(外文):Yi-Han Lai
論文名稱:電控微流體微影技術於生醫材料圖案化與三維細胞共養研究
論文名稱(外文):Electromicrofluidic Lithography for BiomaterialsPatterning and Three Dimensional Cell Co-culture
指導教授:范士岡
指導教授(外文):Shih-Kang Fan
口試委員:賴君亮王興華盧彥文
口試日期:2015-07-14
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:機械工程學研究所
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:101
中文關鍵詞:三維結構介電濕潤電微流微影水膠細胞共養
外文關鍵詞:3D scaffoldselectrowettingelectro-microfluidic lithographyhydrogelco-culturing
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二維的細胞培養雖然已經相當成熟,然而卻與體內生存環境有許多差異。三維細胞培養可建立更接近體內的細胞生長環境,而三維單一種細胞微環境已藉由介電泳操控、模具成型、表面親疏水處理、針織技術、生物列印等技術實現。然而兩種以上的細胞同時形成三維複雜的結構,目前仍難以達成。本研究主要利用電控微流體微影(Electr-omicrofluidic lithography)技術完成建立三維微結構,使用介電濕潤現象為主要驅動液體的力量,藉由已設計好的電極,操控多種不同的生物材料、水膠、以及細胞/螢光顆粒,建立三維微結構。我們測試了不同濃度的PEGDA (聚乙二醇二丙烯酸酯)與GelMA (甲基丙烯酸甲酯接枝共聚物)等可交聯水膠溶液,並藉由理論及實驗結果討論在不同環境(空氣、矽油1 cst)中,操控與形成不同寬度 (100-400 m)、高度 (40-100 m)微結構所需要的操作電壓 (40-120 Vpp)。以適當條件(60-80 Vpp, 1 kHz) 同時驅動含有細胞/螢光顆粒的多種水膠溶液,並以UV將其交聯後形成三維微結構。含有細胞(Fibroblasts NIH-3T3與Hepatocytes HepG2)的水膠(5% GelMA與0.5%光起始劑),在電微流晶片上被拉伸成已設計好的電極形狀並固化,隨後進行細胞培養或共養7-9天,觀察其生長情形(遷移、增生、貼附)等生物特徵,並以螢光標記其骨架與細胞核,相較於傳統培養,細胞會三維生長以及對準水膠邊界,更趨近體內細胞生長環境。我們成功提出一個利用電控微流體建立微結構取代傳統微影的方法,可以同時在一

2D cell culturing has been well developed, which provides an in vitro environment for specific cells culture. Alternatively, 3D cell culture techniques, including lectricphoresis manipulation, template molding, surface hydrophobicity treatment, textile technology and bio-printing, provide an in-vivo like realistic environment. However, co-cultivating multiple cells in 3D scaffolds with complex structures is still challenging. This research proposed an electro-microfluidic lithography technique to establish 3D scaffolds. By using electrowetting on a dielectric (EWOD), droplets were driven and 3D structures were deformed by designed electrode patterns with precisely-controlled amount of bio-compatible hydrogels materials containing suspended cells/fluorescent particles. Furthermore, manipulating multiple pre-polymer hydrogel solutions and forming 3D scaffolds were investigated. Different hydrogel solutions, PEGDA (poly(ethylene glycol)diacrylate) and GelMA (gelatin methacrylated) were tested. We compared the theory with experimental results in different ambients (air, silicone oil 1 cst), with varied 3D structure width (100-400 m), height (40-100 m) and voltage (40-120 Vpp). With appropriate operating parameters (60-80 Vpp, 1 kHz), hydrogel microstructures containing cells/fluorescent particles were formed after UV curing. Bio-compatible hydrogel (GelMA 5%, photo initiator 0.5%) solutions containing cells (fibroblast NIH-3T3 and hepatocyte HepG2) were deformed and cured on a chip. Subsequently, cells were incubated for 7-9 days. Cells phenotyping functions (migrating, proliferating and spreading) and fluorescent bio-marking on actin and nucleus of cells were observed. Compared with convectional 2D cell culture, cells grew and aligned with the boundary of the 3D hydrogel structure. We proposed an electro-microfluidic lithography technique to simultaneously manipulate multiple pre-polymer solutions for constructing cell laden hydrogels and 3D cell co-culture.

致謝 ii
中文摘要 iii
ABSTRACT iv
目錄 vi
圖目錄 ix
Chapter 1 緒論 1
1.1 研究背景 1
1.2 文獻回顧 2
1.2.1 微流體系統 2
1.2.2 生物組織工程 6
1.3 研究方法及目的 18
Chapter 2 介電潤濕及介電泳理論介紹 19
2.1 介電潤濕理論 (Electrocwetting on dielectric,EWOD) 19
2.2 介電泳之理論 20
2.3 電控微流體應用 22
Chapter 3 實驗製程、儀器及系統介紹 30
3.1 微流體晶片製程 30
3.1.1 清洗晶片 30
3.1.2 附著層塗佈 30
3.1.3 正光阻旋塗與圖形定義 31
3.1.4 濕蝕刻 (wet etching) 33
3.1.5 介電層旋佈 33
3.1.6 疏水層塗佈 35
3.2 實驗儀器與材料 35
3.3 實驗系統介紹 54
3.3.1 電控微流體晶片操作系統 54
3.3.2 細胞培養系統 55
3.3.3 水膠製備 58
3.3.4 水膠曝光系統 59
3.3.5 實驗水膠溶液製備 60
3.3.6 實驗細胞溶液製備 60
3.3.7 細胞染色實驗 61
3.4 實驗設計及目的 62
3.4.1 光罩設計 62
3.4.2 實驗流程示意圖 64
3.5 總實驗流程圖 66
Chapter 4 結果與討論 67
4.1 微結構極限尺寸 67
4.1.1 操作電壓 (環境與線寬) 67
4.1.2 操作電壓 (高度與線寬) 69
4.2 三維微建構建立 71
4.2.1 水膠操控建立維結構 71
4.2.2 三維微結構 73
4.3 細胞培養於微流體晶片 76
4.3.1 細胞存活率測試 76
4.3.2 細胞圖案化共養 77
4.4 細胞生長 82
4.4.1 分泌物測試 82
4.4.2 細胞活性測試 87
4.5 總結 88
Chapter 5 未來展望 90
5.1 電性與材料考量 90
5.1.1 疏水層材料 90
5.1.2 三維圖案化細胞培養 94
參考文獻 97


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