自工業革命以來，科技的發展日新月異，促使人們生活型態急速變遷，卻也同時造就各種文明病的產生，其中，不孕症則是一個顯著的隱憂。為改善不斷攀升的不孕比例，生殖醫學結合了微機電與微流體技術，期望藉由微流體晶片的方式來突破以往傳統培養上的限制，本研究主要為利用微流體系統與原理來達成自動化的胚胎抓取與追蹤，並使胚胎與子宮內膜細胞進行循環式的共培養，藉此提高體外胚胎發育的品質與成功率。
　　實驗初步設計微流體結構並安排各部分的相應功能，可藉由液壓流阻類比電路的概念來設計自動胚胎抓取結構，並以COMSOL分析軟體來進行流場分析，達成最佳化數值，以便之後抓取胚胎。接著為建立合適的仿子宮環境來進行胚胎培養，會將子宮內膜細胞培養至成熟貼附底部，利用其分泌的生長因子與激素提供胚胎發育所需的養分。以結構抓取到胚胎後，會以流場操控胚胎的方式將其送入仿子宮環境之共培養槽進行共培養，於此期間會以動態灌流方式不斷替換培養基，並使胚胎在循環流動的情況下不斷運動，不僅可將細胞代謝物與凋亡細胞帶走，運動提供的物理性刺激也能夠加速胚胎發育。
　　本篇研究預期開發多功能整合之體外仿子宮微流體晶片，包含自動抓取、動態灌流、胚胎操控與循環共培養等功能，並比較胚胎單培養、共培養以及傳統培養法之囊胚成長比例及胚胎成長速率。實驗最終結果顯示，單培養與共培養組皆為晶片組囊胚比例佔優勢，證明晶片確實能提供合適的生長環境，但針對子宮內膜細胞之共培養則由於實驗操作因素並無帶來顯著效益。實驗結尾將胚胎植回母體並成功達成著床之目的，證實本研究之可行性與前瞻性。

There are many reasons for infertility, including fallopian tube disease, irregular menstruation and the age. The ability of fertilization for women over thirty years old decreases significantly. In order to solve the growing proportion of infertility, In Vitro Fertilization (IVF) serves as the main treatment nowadays which also plays a critical role in the development of reproductive medicine.
In recent years, the application of biomimetic microfluidic system to IVF has been paid much attention. To optimize the quality of embryos culture, many factors are used to improve the quality of embryos.
In this study, this microfluidic chip integrates the various functions, such as the trapping mechanism via dynamic flow resistance, and co-culturing embryos with human stromal cells providing the growth factors. In my Labchip design, embryos can be manipulated to get into the G-shape co-culture chamber by fluidic field, which enhances embryos growth by the rolling mechanism induced via co-culture.
In this research, we developed the multifunctional microfluidic chip. It is expected that the quality and successful rate of embryos development could be improved due to providing the physical stimulus to construct the biomimicking micro-environment. The experimental results show that the embryos development in our microfluidic chip is higher than the control, which proves our Labchip could provide a suitable micro-environment for the embryos to grow. But the experiment of co-culture with stromal cells doesn’t provide the significant benefits for the embryos due to the experimental operation. At the end of the experiments, the mature embryos can hatch after implanted into the uterus of the mice. Our experimental results demonstrate the feasibility of our multifunctional embryos culture chip.

Abstract 2
摘要 3
致謝 4
目錄 6
圖目錄 10
第一章 緒論 14
1.1 研究背景 14
1.1.1 不孕因素 14
1.1.2 輔助生殖技術(Assisted Reproductive Technology，ART) 15
1.1.3 生醫微機電與實驗室晶片 17
1.1.4 胚胎體內發展 18
1.1.5 自體子宮內膜細胞與受精卵共培養 19
1.2 動機與目的 20
1.3 文獻回顧 21
1.3.1 傳統體外培養方式 21
1.3.2 傳統培養材料 21
1.3.3 體外微流體胚胎培養裝置 22
1.3.4 共養微流體晶片 27
1.3.5 追蹤單一胚胎發展 29
第二章 晶片設計與原理 33
2.1 設計基礎與理論 33
2.1.1 微流體流阻分析 33
2.2 設計概念 34
2.2.1 動態流阻抓取之微結構設計 37
2.2.2 動態流阻抓取分析與模擬 38
2.2.3 G型共培養槽之微結構設計 40
2.2.4 G型共培養槽之操作流程 42
第三章 晶片製程 44
3.1 晶片製作流程 44
3.1.1 晶圓母模製程 44
3.1.2 上下層晶片製程 46
3.1.3 薄膜製程與晶片接合 47
3.2 製程結果 49
3.2.1 微流道實體結構 49
3.3 製程問題與討論 50
第四章 材料與實驗架設 51
4.1 材料準備 51
4.1.1 子宮內膜細胞 51
4.1.2 聚苯乙烯乳膠微粒 52
4.1.3 小鼠胚胎 53
4.1.4 培養基 54
4.2 實驗架設 55
4.2.1 儀器架設 55
第五章 結果與討論 56
5.1 晶片測試結果與討論 56
5.1.1 動態流阻系統微粒與胚胎抓取測試 56
5.1.2 多孔薄膜流場染色擴散測試 57
5.1.3 胚胎操控移入測試 58
5.1.4 胚胎操控取出測試 59
5.2 晶片培養結果與討論 60
5.2.1 子宮內膜細胞培養結果與討論 60
5.2.2 胚胎單培養結果與討論 61
5.2.3 胚胎共培養結果與討論 63
5.2.4 控制組與晶片組之生長階段比較 65
5.2.5 胚胎植回母體著床結果 69
5.3 未來改進與討論 70
第六章 結論 71
參考文獻 72

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