傳統細胞培養主要以聚苯乙烯培養皿做為載體進行，此類裝置的使用已有標準的操作程序，也適合持續性大量培養，但在需要微尺度控制與實驗時有著精準程度上的限制。生醫晶片以微機電製程技術達成生物實驗設計需求，透過微流體控制達成動態的培養環境，並能對細胞進行精準操作，同時降低化學試劑的使用量。微流體生醫晶片可以突破傳統培養方法的侷限，提高對生物細胞與微環境及細胞之間交互作用的理解與控制。共培養是一項新的培養理論，透過將二種生物細胞置於同一環境下培養，可以增加相互之間的發展趨勢。胚胎共培養即以此為基礎提升體外受精技術的成功率。微流體晶片可以透過流場操作細胞與胚胎的移動，並以動態方式補充培養基，對應多重細胞加速養分消耗的問題，並帶走已衰老細胞與代謝物，維持環境酸鹼值與穩定度，以胚胎與細胞的交互作用提高胚胎於體外培養的效率。
本研究以黃光微影技術製作用於NIH-3T3細胞培養之微流道晶片，並結合紫外光與氧電漿進行PDMS表面改質修飾，成功在不使用生物蛋白分子之下令細胞貼附於PDMS表面並開始生長，並以動態流體系統持續替換培養基維持環境穩定度，提高細胞生長週期長度，本實驗設計之單培養區晶片可以在注入3×105之細胞數之後於48小時成長到90%以上的細胞覆蓋率，而多培養區晶片的細胞覆蓋率則能在注入9×105之細胞數後於72小時達到90%以上。而透過動態流體也能使3T3細胞適應KSOM培養液並提高存活時間，我們先於晶片內培養細胞至90%以上覆蓋率再進行KSOM的更換與動態流動，使細胞在72小時之內依然能維持70%以上的覆蓋率，提高細胞的存活量，並比起傳統靜態培養法將細胞存活比例從72%提升至85%，提供足夠的時間與穩定性進行共培養。我們以此方法進行晶片內細胞的培養，並結合胚胎的捕捉與分離設計，達到單一胚胎與細胞的共培養，成功在PDMS晶片內以共培養方式將胚胎於體外培養至成熟。

Traditional cell culture major performs on polystyrene culture dish. This method was suit for continuously macroscopic culture. But it has limit when it comes to microscopic control and experiment. Biochips can provide dynamic culture environment and give precise control to cells with microfluidic system, while reduce reagent consumption. Co-culture means by culturing two kinds of cells in the same environment, their interaction can improve the quality of cultivation. Embryo co-culture combines embryonic feeder cells to improve the development of embryos, thus increase the successful rate of in vitro fertilization. Microfluidic chips can operate cell and embryo to move within the designed structure, while exchange medium with dynamic flow. This will maintain the nutrient concentration and pH value in a stable state. The co-culture microenvironment can provide a better development condition for embryos, thus increase the efficiency of in vitro embryo culture.
In this research, we fabricated the microfluidic chips for 3T3 cell culture by microlithography process. We combined ultraviolet exposure and oxygen plasma for PDMS surface modification. It successfully improved cell attachment on PDMS without the aid of cellular proteins. By applying dynamic fluid system, we can achieve 90% cell confluence at 48 hours for single chamber chips, and at 72 hours for multi-chamber chips. Dynamic fluid culture can improve 3T3 cell viability in KSOM. We can maintain 70% confluence for at least 72 hours in KSOM, while having its viability increase from 72% to 85% compare to traditional static culture. We successfully combined these method for cell culture and embryo trapping design and perform in vitro single embryo and 3T3 cell co-culture.

摘要...................................................................................................................................-1-
Abstract............................................................................................................................-2-
第一章　緒論...................................................................................................................-9-
1.1 研究背景與動機................................................................................................-9-
1.2 文獻回顧..........................................................................................................-11-
1.2.1 細胞培養材料.......................................................................................-11-
1.2.2 PDMS細胞培養...................................................................................-12-
1.2.3 PDMS表面修飾...................................................................................-14-
1.2.4 體外細胞培養.......................................................................................-20-
1.2.5 體外胚胎培養.......................................................................................-25-
1.2.6 動態流體培養系統...............................................................................-29-
1.2.7 微流體粒子捕捉...................................................................................-35-
1.2.8 細胞交互作用.......................................................................................-37-
1.2.9 胚胎共培養...........................................................................................-40-
第二章 實驗準備...........................................................................................................-43-
2.1 晶片製程..........................................................................................................-43-
2.2 PDMS表面修飾..............................................................................................-44-
2.3 實驗器材架構..................................................................................................-46-
2.4 實驗樣本準備..................................................................................................-46-
2.2.1 小鼠纖維細胞.......................................................................................-46-
2.2.2 老鼠胚胎...............................................................................................-47-
2.2.3 培養液...................................................................................................-47-
2.5 晶片前處理......................................................................................................-49-

第三章 單培養區晶片細胞培養...................................................................................-50-
3.1 晶片結構設計..................................................................................................-50-
3.2 靜態晶片細胞培養..........................................................................................-53-
3.3 動態晶片細胞培養..........................................................................................-57-
第四章 共培養環境模擬...............................................................................................-59-
4.1 24-well共培養環境模擬.................................................................................-59-
4.2 靜態晶片共培養環境模擬..............................................................................-61-
4.3 動態晶片共培養環境模擬..............................................................................-62-
4.4 細胞存活率分析..............................................................................................-63-
第五章 多培養區晶片細胞培養...................................................................................-66-
5.1 晶片結構設計..................................................................................................-66-
5.2 靜態晶片細胞培養..........................................................................................-69-
5.3 動態晶片細胞培養..........................................................................................-71-
5.4 動態晶片共培養環境模擬..............................................................................-72-
第六章 胚胎培養...........................................................................................................-74-
6.1 胚胎單一培養..................................................................................................-74-
6.2 24-well胚胎共培養.........................................................................................-76-
6.3 動態晶片胚胎共培養......................................................................................-78-
第七章 結論與未來展望...............................................................................................-82-
7.1 結論..................................................................................................................-82-
7.2 晶片與分選捕捉結構改善..............................................................................-83-
7.3 細胞、胚胎注入與培養液更換時間..............................................................-84-
7.4 胚胎捕捉操作時間與成功率..........................................................................-85-
第八章 參考文獻...........................................................................................................-86-

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