人工受孕之技術近年來在全世界備受重視，其核心概念為模擬體內胚胎的培養環境。隨著生物科技以及微奈米科技的發展，整合技術對於人工受孕科技之進步有重要的影響力。許多三維細胞的培養平台被研發，如凝膠、微井或是懸吊式液珠等，本實驗選擇微井作為培養及觀察小鼠胚胎早期發育的平台。藉由聚二甲基矽氧烷製成的微井有許多優點，包括操作方便、低製作成本和生物相容性等。由於微井晶片表面覆蓋一層礦物油，因此每一顆小鼠受精卵皆能夠在微井中被獨立培養，並配合活體細胞顯微鏡觀察受精卵的完整發育過程。本實驗測試5種體積不同之微井，選擇體積393 nL之微井作為後續追蹤發育的結構。人工受孕技術的第一步為體外受精，本實驗利用傳統液珠進行體外受精有67.9%的受精率；在393 nL的微井中培養受精卵從二細胞發育至囊胚後期，目前為79.4%的囊胚率。受精卵在微井中72小時後，將22顆囊胚移植回小鼠母體，經過19天後產下15隻小鼠。本研究根據小鼠受精卵是否能夠發育至囊胚期作為預測受精卵發育品質之依據，歸納出其分裂至四細胞或八細胞的時間點有顯著差異，期望藉由追縱受精卵在微井中的發育過程作為胚胎品質之依據。

Assisted reproductive technologies (ART) are of increasing importance and impact worldwide. A crucial aspect of ART is to control and mimic the in vitro environment of mammalian to the in vivo one. Integration biotechnology with micro- and nanotechnology is one of the necessary ways to improve ART. There are many platforms capable of nurturing cells in 3D, including using gel, microwells or hanging drop techniques. Microwells were chosen for the culture and investigation of early mouse embryos in this study, since microwells made of polydimethylsiloxane (PDMS) are low-cost, easy to operate, and biocompatible. Each mouse early embryo could be maintained in an individual microwell, allowing for high-resolution time-lapse microscopy and collecting the data of developmental process from every single embryo without confounding factors. In addition, the fluidic environment of each microwell could be secluded from each other by layering oil on top, preventing the communication of soluble factors between embryos cultured in individual microwells. The initial step of ART involves in vitro fertilization (IVF), and we demonstrated a successful fertilization rate of 67.9% in this research. We successfully cultured mouse embryos from the two-cell stage to blastocyst stage inside different volume of microwells with a ~80% successful rate. Among the five different volumes of microwells evaluated, we chose 393 nL microwells as the cultured platform to tracing the development process of mouse embryos. After cultured in microwells for 72 hours, 22 embryos at blastocyst stage were transferred into a recipient female mouse, and 15 mice were successfully born after 19 days. The development timings of mouse embryos that developed into blastocysts were statistically different to those of embryos that failed to form blastocysts (p–value < 10-10), and could be robust indicators of the quality of the embryo with >93% sensitivity and 100% specificity. This microwell platform, which supports the development of pre-implant embryos and is low-cost, easy to fabricate and operate, we believe, opens opportunities for a wide range of applications in reproductive medicine and cell biology.

致謝 i
中文摘要 ii
Abstract iii
目錄 iv
圖目錄 vi
表目錄 ix
第一章 緒論 2
1.1 前言 2
1.2 研究動機與目的 3
第二章 文獻回顧 5
2.1 小鼠受精卵(Mouse fertilized eggs) 5
2.2 微流道(Microfluidics)和微井(Microwells) 7
第三章 實驗設計與方法 17
3.1 實驗架構 17
3.2 微井(Microwells)晶片 17
3.2.1 晶片結構設計 18
3.2.2 黃光微影製程 19
3.2.3 PDMS翻模 20
3.2.4 晶片裝置操作流程 21
3.3 實驗晶片測試 22
3.4 實驗樣品對象 23
3.3.1 ICR老鼠之卵母細胞 23
3.3.2 ICR老鼠之精蟲細胞 24
3.5 精卵體外受精之流程 25
3.6 受精卵培養環境與條件 28
第四章 實驗結果 29
4.1傳統體外受精與液珠培養胚胎之結果 29
4.2微井晶片培養受精卵之結果 32
4.3受精卵在微井中的追蹤發育 37
4.4受精卵在微井培養後移植回ICR老鼠 41
第五章 問題討論 44
5.1傳統液珠培養方法 44
5.1.1體外受精條件之比較 44
5.1.2受精卵培養之比較 45
5.2 微井晶片培養方法 47
5.3 微井中受精卵之追蹤 49
第六章 未來計畫 51
第七章 參考文獻 52

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