黃榮山臺灣大學：應用力學研究所鄧伊筌Teng, Yi-ChiuanYi-ChiuanTeng2007-11-292018-06-282007-11-292018-06-282007http://ntur.lib.ntu.edu.tw//handle/246246/62379為了促進生物學與醫學的發展，幹細胞分化過程與各種應用的研究正熱烈進行中。在發展自身成體幹細胞取得方法及細胞治療方法的同時，也促進了個人化細胞篩選與細胞生物反應晶片的研發。 　　由於以群體細胞平均行為表現的傳統實驗方式，具有無法得知單一細胞在處於某週期狀態下的行為表現之缺點，也無法滿足對環境刺激反應敏感且罕見的幹細胞研究需求。若能利用微機電技術發展微小化的細胞晶片系統，對單顆幹細胞進行研究觀察，而獲得單細胞生物資訊，將更有助於揭開幹細胞的奧秘。 　　本研究目的在建立一套結合培養與即時觀測功能的陣列化生物反應晶片系統，並將其應用於單顆幹細胞的動態培養研究。研究方法可分為細胞晶片設計與製造、細胞培養與監測系統架設、晶片測試與幹細胞培養實驗進行三部份。在細胞晶片設計與製造方面，本研究中利用微流體分離並固定多組單細胞樣本在設計的微堰結構上。細胞培養與監測系統架設方面，結合了微細胞培養室、CytovivaTM150 DMF顯微技術與晶片內的溫度監測。在晶片測試與幹細胞培養實驗進行方面，以骨髓幹細胞(Bone Marrow Mesenchymal Stem Cells， BMSC)進行晶片上之培養測試，同時取得生物影像。 有別於其他單細胞培養研究具有共用培養液之缺點，本研究中的晶片設計可同時提供每組單細胞樣本獨立的培養環境，避免了細胞間分泌物與代謝物之交互影響。並在晶片系統內，培養單顆骨髓幹細胞超過72小時且重複實驗超過三次以上。也成功拍攝了單顆骨髓幹細胞在培養區內成長時的各種生長情況，包括：細胞貼附、細胞凋零、細胞分裂及細胞變形及遷移等。同時在晶片內進行細胞免疫化學鑑定法，於目標細胞上染上生物標記，測試細胞鑑別功能，驗證了本系統未來運用於幹細胞分化研究以及發展應用於細胞治療之個人化幹細胞快速檢測晶片之可行性。Multiple single human bone marrow stem cells with dynamic control of perfusion have been demonstrated in an array of individually isolated microenvironments of a microfluidic device and its associated system. In addition to micro device fabrication, the present study investigates living cells, and monitors the real-time living cell processes in a micro device such as expansion and differentiation of single or several human bone marrow stem cells in individual microenvironments, thus providing a quantitative description of live stem cellular behavior. Such a work, however, has been hampered by the difficulty in data collection using traditional techniques. There is much interest in quantifying the range of biological responses of individual cells to various physiologically-relevant stimuli as opposed to bulk averages. Particularly useful information can be acquired if the environmental factors that contribute to variable responses for individual cells are controlled. Bulk experiments fall short of providing adequate data in that the results elucidate the mean value of a parameter with further experiments providing information about the standard error of the mean. In contrast, single-cell experiments reveal the critical underlying distribution of parameters. This contrast is especially apparent in time-dependent processes such as expansion and differentiation of stem cells in which by averaging over a population. A smooth transition is observed from one state to another, which used to obscure the underlying transition occurring at the cell level. Perfusion culture has been previously used and been able to reduce the effects of diffusible elements on cell behavior by convecting away produced substances that may provide autocrine or paracrine signals. Microfluidics approaches have been developed to allow more precise control of cell positioning and reagent introduction in analyzing single or rare cells. KP-hMSCs (stem cell line derived from human bone marrow) were used in the experiments. Freshly suspended cells were introduced into previously PBS filled microfluidic devices by a syringe connected to a three way valve. Single KP-hMSC was trapped and maintained in individual micro-well by using microfluidic perfusion for dynamic cell culture and differentiation induction. Cells were both cultured in an incubator between images, and were also maintained at 37℃ on a microscope stage for time-lapse experiments. We demonstrate the culture of single KP-hMSC under constant perfusion of media + 20% FBS. In a flow rate of 5μL/min time-lapsed images were taken every minutes of an isolated micro-well of single KP-hMSC on an incubated microscope stage. After one hour, slight changes in morphology were observed away from a spherical morphology towards an adherent morphology. Also, cell division was found in a few cases. After 12 hours, a majority of single cells displayed adherent morphology, and crawled out of the microenviroment by extending the filopodia. We have demonstrated that quantitative analysis of the dynamics of cell adhesion, death, division, and escape from traps were performed in the time length of 72 hours. It was also found that 60% of cells displayed adherent morphology after 24 hours. In 48 hours, 20% of cells showed characteristics of apoptosis, while 10% had escaped from the vicinity of the initial trapping site and 10% of cells had undergone cell division.中文摘要 I Abstract II 謝誌 IV 目錄 V 圖目錄 VII 表目錄 X 第一章 緒論 1 1-1 前言 1 1-2 研究動機 3 1-3文獻探討 5 1-3-1 細胞實驗室(The lab in a cell) 5 1-3-2 細胞操控與定位 7 1-3-3 微小化全分析系統與細胞晶片 12 1-4 研究方法 14 1-5 論文架構 15 第二章 理論 16 2-1 單細胞操控與定位 16 2-2 細胞培養微環境控制 18 2-2-3 傳統細胞培養技術 18 2-2-2 微流道培養環境對細胞生長之影響 21 2-2-3 光照對細胞培養之影響 24 2-3 CytovivaTM150 DMF雙模式螢光模組光學顯微原理 25 2-3-1 光學原理 25 2-3-2 解析度之增強 29 第三章 實驗步驟 32 3-1 微流道單細胞培養陣列晶片設計與製作 32 3-1-1 晶片設計 33 3-1-2 微流道上蓋製作 37 3-1-3 底層電路晶片製作 40 3-1-4 晶片整合 41 3-2 實驗系統架設 44 3-2-1 微培養系統（micro-incubator）架設 44 3-3-3 CytovivaTM150 DMF雙模式螢光模組架設 46 3-2-3 微環境晶片溫度監測 47 3-3 細胞樣本與藥品準備 50 3-4 系統操作與單顆細胞陣列培養及觀測 50 3-4-1 細胞定位操作 53 3-4-2 細胞培養、給藥與即時觀測之操作 54 第四章 實驗結果 55 4-1 微流道晶片流場測試與分析 55 4-2 微培養環境群體細胞照光環境培養測試 57 4-3 微流道晶片細胞定位操作結果與效率 59 4-4 微流道晶片細胞培養結果與單細胞成長情形觀測 63 4-5 單細胞辨識與觀測 70 第五章 結論與未來展望 73 5-1 結論 73 5-2 未來展望 74 參考文獻 766358369 bytesapplication/pdfen-US幹細胞細胞晶片微流道單細胞培養Stem cellCell chipMicrofluidicSingle cell culturethesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/62379/1/ntu-96-R94543051-1.pdf