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研究生:謝嘉濬
研究生(外文):Chia-Chun Hsieh
論文名稱:多管道連續式細胞培養平台於奈米藥物觀測之應用
論文名稱(外文):Multi-channel Continuous Cell Culture Platform and Its Applications for Continuous Nano-drug Monitoring
指導教授:李國賓李國賓引用關係
指導教授(外文):Gwo-Bin Lee
學位類別:碩士
校院名稱:國立成功大學
系所名稱:工程科學系碩博士班
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:101
中文關鍵詞:及時監測奈米藥物連續式細胞培養多重管道微機電系統微流體
外文關鍵詞:MicrofluidicMEMSReal-time monitoringMulti-channelContinuous cell cultureNano-drug
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細胞培養系統在疾病檢測及新藥物篩檢及開發的過程中,扮演著極其重要的角色,不只是研究初期的材料提供者,也是研究後期的成果驗收者。傳統上,細胞培養是一個需要大量手動、反覆且耗時的過程,且研究人員必須經過嚴格訓練及具備相當的操作熟練度,在培養過程中必須小心地全程控制培養環境,包括培養箱的溫度、二氧化碳含量及培養液的酸鹼值等,以確保每次培養成果的一致性。為了全程提供適當的培養環境並避免可能的人為疏失,以期獲得大量且一致的培養成果,大部分的研究機構及藥廠皆已採用大型自動化的細胞培養系統,但其體積龐大且價格昂貴仍造成諸多不便。本研究提出以微流體(Microfluidic)理論設計並應用微機電系統(Micro-electro-mechanical-systems, MEMS)技術建構出新型連續式細胞培養(Continuous cell culture)平台,並將此平台應用於及時監測(Real-time monitoring)奈米藥物(Nano-drug)對癌細胞之影響。此平台提供一個適合細胞生長且更精確而直接的觀測方式,平台的微型化也有效減少培養區化學梯度的問題以及實驗資源的浪費。自動化傳輸系統也經特別設計,不同的培養基或藥品可經濟並有效率地經由包含了常閉式微型止回閥(Micro-check-valve)的多重管道(Multi-channel)並藉由低流速微幫浦傳送到培養區,其中多重管道及止回閥的設計可以有效減少管道間不同液體交互汙染的問題,而低流速微幫浦可以降低連續式培養時產生的剪應力對細胞的不良影響。換言之,大幅減少了費力且不穩定的人為的操作。此外,為了得到更高倍率的觀測影像,培養晶片也特別經過設計,使培養區的厚度控制在180 μm 以下,以符合一般光學顯微鏡100 倍物油鏡的工作區限制,進而達到約1000 倍的高放大倍率。於本研究中,該新式細胞培養平台不僅在大型培養箱外提供一個合適、穩定且自動的培養環境,更可應用在需要及時且高倍率的觀測上。透過實驗,成功地於培養箱外,完成人類口腔癌細胞(Oral cancer cell, OC2)的培養,並於培養末期給予藥物的刺激而得到及時且高倍率的細胞破裂影像。
A cell culture system plays an important role on evaluating drug discovery, disease study and many biological applications. Traditionally, cell culturing is a labor-intensive process that requires long hours of repetitive routine work. Well-trained personnel have to be attendant during entire cell culturing process including cell growth, harvesting, re-seeding and analysis if expensive, automatic culture systems are not available. In order to maintain viable and consistent cell lines, cell culturing also requires precise control of micro-environment such as temperature, concentration of carbon dioxide, pH. value of medium and medium flow rate. In order to improve the consistency of the cell culturing process and reduce the operating error from human intervention, large-scale automated cell culture systems have been used by most of pharmaceutical companies and research centers. However, those equipments are not only bulky but also expensive. Therefore, it still remains a need to develop a compact, flexible cell culture system. In this study, a new cell culture platform based on microfluidic technology for monitoring cancer cells response to nano-drug in real-time format was demonstrated. Continuous culture environments are main features of this developed platform. The miniaturization of the culture platform contributes favorably to a low chemical gradient culture environment and reduces consumption of samples and reagents. An automatic delivery system with a layout for achieving a lower flow rate provides an efficient and economical transportation mechanism and causes relatively low effect upon the growing of cells. In addition, the design of the multi-channel layout with normally closed micro-check-valve can deliver different liquids respectively and lower the chances of cross-contamination. As a result, it can alleviate laborious works and human error caused by manual loading process. A special design of the culture area also enables 1000X high-magnification observation during cell culture process. As a whole, not only does the new cell culture platform provide a satisfactory, steady and automatic cell culture environment, but it also allows for real-time observation of cell culture process. We have successfully cultured human oral cancer cell (OC2) and obtained the real-time images of cells response to the nano-drug by using the developed culture platform.
中文摘要 I
Abstract III
致謝 V
目錄 VII
表目錄 X
圖目錄 XI
縮寫及符號說明 XIII

第一章 緒論
1.1 微機電系統簡介 1
1.2 生醫微流體晶片 2
1.3 文獻回顧 3
1.3.1 微流體細胞培養平台 3
1.3.2 微幫浦 9
1.3.3 微閥門 12
1.4 研究動機與目的 14
1.5 論文架構 15
第二章 理論與設計
2.1 微流體傳輸模組原理與設計 19
2.2 多微流管道及培養區設計 22
2.3 微型溫控晶片控制模組原理與設計 24
第三章 製程與實驗方法
3.1 材料選擇 34
3.2 光罩製作 35
3.3 晶片製程 36
3.3.1 晶片表面清潔 37
3.3.2 微影製程 39
3.3.3 ITO 導電玻璃蝕刻 42
3.3.4 金屬薄膜沉積及圖案定義製程 42
3.3.5 微注模 44
3.3.6 氧電漿接合 45
3.4 溫控晶片製作流程 46
3.5 微流體細胞培養晶片製作流程 48
3.6 實驗架設 50
第四章 結果與討論
4.1 流體傳輸進樣系統 66
4.2 自動化多管道連續式細胞培養平台 68
第五章 結論與未來展望
5.1 結論 83
5.2 未來工作 84
參考文獻 86
自述 100
著作 101
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