臺灣博碩士論文加值系統

本研究以介電濕潤晶片做為體外胚胎培養裝置，利用介電濕潤晶片可操控微液滴移動之功能，模仿體內動態流體環境，對胚胎進行動態培養，並改善培養環境，如培養液內濃度梯度、擴散等物理因素，以幫助胚胎分裂之發展。實驗以微機電技術製做出共平面式介電濕潤晶片，選用透光性良好之玻璃作為基板以方便觀察胚胎，晶片設計電極大小為1.5 mm × 0.75 mm，流道高度為260 μm，供應電壓為68.5VRMS且頻率於500Hz之電訊號。以小鼠胚胎作為培育對象在介電濕潤晶片上進行動態培養實驗，驅動1 μl培養液滴內含有單顆2-細胞期胚鼠卵，並包覆4 μl培養油於培養液滴與流道電極上，在96小時中每間隔一段時間後驅動一次液滴移動，其液滴平均移動速率為0.58 mm/s，另外在介電濕潤晶片上進行靜態培養做為對照組，並在胚齡2.5天、3.5天和4.5天時觀察記錄胚胎分裂情況。本實驗成功使用介電濕潤晶片做為培養裝置，在胚齡4.5時胚胎發育至囊胚期機率靜態可達到93％、動態可達到89％，由多次的實驗結果觀察出動態培養之胚胎分裂速度較快，胚胎發育至囊胚孵化期機率靜態為23％、動態為54％，其速度與靜態胚胎相比差約0.5天，證實使用介電濕潤晶片對胚胎進行動態培養可幫助胚胎分裂。

This study presents in vitro embryo culture devices based on Electrowetting on Dielectric (EWOD) devices, which can manipulate micro droplets in order to mimic dynamic fluidics in vivo environment. Physical effects, such as medium concentration gradient and medium diffusion, are improved by dynamic culture for enhancing embryo cleavage and development. A coplanar EWOD chip is fabricated by MEMS technique, the electrodes size are 1.5 mm × 0.75 mm and channel gap between two parallel plates is 260 μm. A 1 μl droplet is driven by EWOD force while applying 68.5VRMS at 500 Hz. A 2-cell-stage mouse embryo is cultured in 1 μl medium droplet covered with 4 μl culture oil in the incubator with 5% CO2 at 37℃. In dynamic culture experiments, an embryo is moved in the driven droplet many times at average velocity 0.58 mm/s during 96 hours. Embryos cleavage and development are recorded in E2.5, E3.5 and E4.5. This study successfully uses EWOD devices as in vitro embryo culture devices to mimic a dynamic environment. The results show that the development blastocyst rates of static and dynamic is 93% and 89%. The cleavage rates of dynamic culture are faster than that of static culture, embryo cleavage to hatching stage rates of static and dynamic is 23% and 54%. Hence, the dynamic culture in EWOD devices enhancing embryo cleavage and development is demonstrated in this study.

誌謝 i
摘要 ii
Abstract iii
目錄 iv
圖目錄 vi
表目錄 viii
第一章 緒論 1
1.1 前言 1
1.2 研究動機與目的 2
1.3 文獻回顧 3
1.3.1 介電濕潤現象與微液珠操控 3
1.3.2 介電層材料之探討 9
1.3.3 體外胚胎培養裝置發展 12
第二章 基本原理 22
2.1 介電濕潤原理 22
2.2 共平面式介電濕潤系統 24
2.3 胚胎培養與發育 25
第三章 研究方法 27
3.1 介電濕潤系統架構 27
3.1.1 共平面式電極設計 27
3.1.2 介電濕潤晶片製程 30
3.1.3 操控系統架設 35
3.2 小鼠胚胎培養實驗設計 37
3.2.1 小鼠胚胎前處理 37
3.2.2 小鼠胚胎培養實驗 38
第四章 實驗結果與討論 40
4.1 介電濕潤系統 40
4.1.1 晶片製程結果 40
4.1.2 晶片品質與微液滴速度 44
4.2 小鼠胚胎培養 47
4.2.1 介電濕潤系統之靜態培養 47
4.2.2 介電濕潤系統之動態培養 50
4.3 結果與討論 57
第五章 結論與未來工作 61
5.1 結論 61
5.2 未來展望 63
第六章 參考文獻 64

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