2013年在世界各地平均每六對夫婦就有一對受孕困難，患有不孕症，在過去二十年來，生殖醫學致力於解決低懷孕率問題。本研究開發與應用整合微流體技術和共培養子宮內膜細胞以改善傳統方法對胚胎體外發展僅側重於調整靜態培養皿之化學溶液成分，希望提高胚胎在體外培養之效能。過去的文獻顯示，需透過人工繁複的操作來定位胚胎以達到後續觀察之目的，更新培養液與移動胚胎的過程都可能增加損傷胚胎之風險。
此研究欲探討胚胎體外仿生環境在晶片與傳統培養之差別，本論文提出一種簡便的方法利用液壓流阻概念和電路類比分析來設計自動定位單一小鼠胚胎之系統，減少對受精卵的操作次數以減少對生物不利之影響，並與子宮內膜細胞動態共養系統整合去模擬體內胚胎發育之情形，動態灌流系統用以排除廢棄物提供子宮內膜細胞新鮮的培養液，以維持體外小鼠胚胎最佳的體外共養環境，並以導流片結構減少氣泡停留於晶片內，藉以結合三者之優點完成體外發展至囊胚期之高良率和便利性。
本論文已完成了自動化胚胎共養晶片，採用動態微陣列的格式管理哺乳動物之胚胎並在各別的微槽中追蹤各時期之發展，已測試三大功能，抓取100um的粒子與8細胞之小鼠胚胎，與子宮內膜細胞共培養情形與胚胎取出，在比較傳統培養法與晶片上，單養胚胎囊胚期發展速率為55.6±3.4%和61.8±3.3%，共養則為69.8±7.8%和77.7±6.7%，共養晶片組比傳統法可提高發展至囊胚之比例，已建立合適的共培養制式程序與調整最佳化參數，提供更加良好的體外胚胎發展之環境，晶片具有更加便利的操作性且利用仿生的平台從而提高胚胎整體的囊胚期發展速度與胚胎品質，實現自動化的原型，該晶片將開啟體外共培養系統之前瞻研究，強化個別胚胎輔助生殖技之管理，是一個相當具有前景的自動化共養平台。

One in six couples worldwide has difficulty conceiving children in 2013. People have been suffering from infertility for the past two decades. In this research, Reproductive technology has been applied for the goal of improving the low pregnancy rate issues by integrating both microfluidic techniques and coculture of endometrial cells into enhance the embryo development in vitro. Traditional methods towards embryo development in vitro still required the complicated procedures which were all done manually for position the embryo, tracking each development, replacing medium and moving the embryo. The labor operation might increase the risks of cell damage.
In this study, the comparison of embryo coculture with endometrial cells on chip and in dish was investigated. This master study proposed a design of an automated positioning single murine embryo by utilizing a hydraulic concept and electrical circuit analysis to reduce the operation procedure and adverse effects on organism. The coculture of embryo with endometrial cells within a dynamic perfusion system was also applied in this master study to mimic the embryo development in vivo. The dynamic perfusion system was performed to exclude the waste, provide fresh medium and be combined with the designed microchannels of specific microfluidic streamline in the coculture chamber to minimize the trapped bubbles to enhance the coculture environment in vitro for murine embryo culture.
The automated embryo coculture device has been achieved in this study for the management of individual mammalian embryos by using the dynamic microarray format. The developed microsystem can manage and coculture individual embryos in each microchamber. The embryo development in whole culture period could be tracked via this microsystem design. 8-cell-stage murine embryos were used for the starting stage of embryo developments in our experiments. The results showed that the Blastocyst development rates in traditional method and device are 55.6±3.4% and 61.8±3.3%, respectively, for the monoculture group. They are 69.8±7.8% and 77.7±6.7%, respectively, for the coculture group. In addition, the microfluidic device developed in this master research is simple in operation, and is feasible to achieve the higher blastocyst rates. The coculture platform mimics the micro environment of embryo growth in vivo to enhance its overall development. An automated prototype is achieved through this master study. The development of individual embryo could be enhanced via this microsystem, which is a very promising coculture platform in vitro.

Abstract I
中文摘要 II
致謝 III
LIST OF FIGURES VIII
LIST OF TABLES XVII
Chapter 1 Introduction 1
1.1 BACKGROUND 1
1.1.1 Cause of Infertility 1
1.1.2 Assisted Reproductive Technology (ART) 3
1.1.3 Bio-MEMS and Lab on a chip (LOC) 5
1.1.4 Embryo culture in vivo. 6
1.1.5 Autologous coculture of endometrial cells and embry 7
1.2 MOTIVATION AND OBJECTIVE 8
1.3 LITERATURE REVIEW 10
1.3.1 Traditional IVC method 10
1.3.2 Selection of IVC material 10
1.3.3 Microfluidic device for embryo culture in vitro 11
1.3.3.1 Static culture in vitro 11
1.3.3.2 Dynamic culture in vitro 13
1.3.4 Co-culture microfluidic device 19
1.3.5 Technology for single embryo development tracking 21
1.3.5.1 Modified petri dish with Concave and Fence 22
1.3.5.2 Microfluidic device with Barrier and microwell 24
1.3.5.3. Hydraulic trapping 26
Chapter 2 Device development 28
2.1 PHYSICS OF PRESSURE-DRIVEN LAMINA FLOW IN MICROFLUIDIC CHANNELS 28
2.1.1 Reynolds number 29
2.1.2 Poiseuille flow 30
2.1.3 Hagen-Poiseuille’s law 32
2.1.4 Hydraulic resistance 33
2.1.5 Electrical circuits analogy 36
2.2 BRIEF INTRODUCTION OF TRIPLE-LAYER DEVICE 39
2.2.1 Working procedure 40
2.3 THE DESIGN OF UPPER LAYER 44
2.3.1 Rapid embryo trapping microchannel 45
2.3.2 Electrical circuit analogy of the trapping unit 46
2.3.3 Numerical simulation of trapping mechanism 50
2.4 DESIGN OF INTERMEDIATE LAYER 54
2.4.1 Electrical circuit analogy of the simplification moving path of the embryo 55
2.5 DESIGN OF BOTTOM LAYER 57
2.5.1 Multi-branched distribution and meandered microchannels 58
2.5.2 Coculture microchamber 59
2.5.3 Numerical simulation of perfusion system 61
Chapter 3 Fabrication of microchip 63
3.1 FABRICATION OF MASTER MOLD 65
3.1.1 Lithography procedure 65
3.1.2 CNC engraving procedure 67
3.2 PDMS REPLICAS AND BONDING 68
3.3 THE RESULT OF FABRICATION AND DISCUSSION 69
3.3.1 Dyed view of device 69
3.3.2 Measurements of master mold 71
3.3.3 Measurements of PDMS microchannel 73
3.3.4 Alignment precision 75
Chapter 4 Material and Experiment stetup 76
4.1 MATERIAL 76
4.1.1 SU-8 2150 76
4.1.2 PDMS 77
4.1.3 Agarose hydrogel beads 77
4.1.4 Collagen IV 78
4.1.5 Paraffin Oil 78
4.2 CELLS 79
4.2.1 Stromal cells (Endometrial Cells) 79
4.2.2 Embryo 81
4.3. INSTRUMENT SETUP 82
4.3.1 Surface modification 83
4.3.2 Sub culturing and endometrium cells seeding 84
Chapter 5 Results and Discussion 85
5.1 PRELIMINARY TESTS OF DIFFERENT BOTTOM DESIGN FOR COCULTURE 85
5.1.1 Double-layer microwell 86
5.1.2 Circular and Elliptic microchamber 87
5.1.3 Elliptic microchamber with circular array of columns and streamlined barrier 89
5.2 SYSTEMATIC EXPERIMENTAL PROGRAM 92
5.2.1 Dynamic versus static chip 93
5.2.2 Dynamic chip versus dish 94
5.2.3 Selection of coculture medium 96
5.2.4 High efficiency of microparticles trapping demonstration with Cytodex beads 98
5.2.5Automated embryo trapping system 101
5.3 THE RESULT OF EMBRYO CULTURE IN VITRO 104
5.3.1 Monoculture on chip versus in dish 104
5.3.2 Coculture on chip versus in dish 107
CHapter 6 Conclusion 111
Reference 114

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