自動抓取小鼠胚胎並與人類子宮內膜基質細胞共培養之微流體裝置

A Microfluidic Device for Automatic Trapping and Co-culture of Mice Embryos with Human Endometrial Stromal Cell

宋宜蓉(Yi-jung Sung)

指導教授 ： 劉承賢

國立清華大學/工學院/動力機械工程學系/碩士(2016年)

摘要

近年來由於台灣教育程度提高、都市化發展迅速、女權意識提升、女性普遍經濟獨立以及台灣經濟景氣波動等原因，大部分的人結婚年齡較晚，晚婚的人越來越多。隨著結婚年齡提升，高齡生育的情況亦隨之成長，但是生育力卻會隨年齡增長而下降。除了年齡上升，生育力下降的問題之外，還有先天遺傳上的缺失、內分泌失調、生殖系統異常等導致不孕症。
　　目前治療不孕症的方法主要是體外人工受精，也就是所謂的試管嬰兒。但此方法需要大量且複雜的人工操作程序，還需要訓練有素的技術人員，且提升胚胎品質的因素有限。民國一百年統計之台灣試管嬰兒活展率約27.4%，如何提升胚胎品質為未來主要的研究方向。
　　在生醫工程領域方面，微流體可以達到許多仿生的應用，因此近幾年的生殖醫學研究方向便往微流體發展。本論文之研究為：利用微機電的技術結合自動化抓取以及動態培養系統之晶片，應用於生殖醫學領域，以改良現有的傳統體外培養方式。除了可以取代繁複的人工操作以外，更可以建造仿生的培養環境，以提高胚胎培養的品質。
　　本研究方法為利用胚胎與子宮內膜細胞共培養，以及多孔性薄膜動態灌流，建造體外仿生子宮的培養環境。藉由子宮內膜細胞會分泌胚胎生長因子的特性，提供胚胎良好的成長環境，來促進胚胎的發育。動態灌流培養除了可以帶走代謝廢物以外，還可以提供新鮮的培養基，使胚胎的酸鹼值、滲透壓為持在穩定的狀態。結合液壓流阻類比電路的概念，設計自動化抓取胚胎並定位之結構。除了可以減少人為操作對胚胎之傷害以外，也方便實驗上的觀察。
　　根據實驗結果，受精卵可藉由調整流道高度至140微米而成功被捕捉。子宮內膜細胞在多孔性薄膜上培養分化速度較快，2.5天候可達7成。用傳統培養皿方式比較單養及共培養之結果顯示，2.5天後發展至囊胚期的比率單養為15.79%共培養為57.89%，代表共培養之結果優於單養。比較單養於晶片和培養皿之結果顯示，發展至囊胚期的比率晶片為57.10%培養皿為57.58%，兩者之間並無明顯差異。比較共培養於晶片和培養皿之結果顯示，發展至囊胚期的比率晶片為58.33%培養皿為26.67。共培養於培養皿是無法運作的，另一方面，藉由微流道系統共培養於晶片上可成功運作。胚胎移植機率為66.67%。

關鍵字

胚胎共培養 ； 生殖醫學 ； 動態灌流 ； 胚胎抓取

並列摘要

Because of the high education, rapid urbanization, enhancement of feminism, financial independence of women and fluctuating economy in Taiwan, most of people marry late. Hence, population of fertility in advanced age grows obviously. The fertility rate would decrease as marriage age. Except for age factors, DNA damage, genetic factor, abnormality of reproductive system and hormone imbalance also result in infertility.
The mainstream therapy is in-vitro fertilization (IVF), namely test-tube baby. This method requires complicated operating sequences and well-trained technicians. The quality of IVF is also limited. According to statistical data in 2011, The average fertility rate of IVF is about 27.4% in Taiwan. How to enhance the embryo quality is a crucial target for IVF studies in the future.
The microfluidics could reach lots of bionic applications. Therefore, recent research tends to develop microfluidics for various biomedical applications. In this study, we developed microfluidic Labchip system combining automatic trapping and dynamic culture system to improve traditional IVF method for reproductive biology application. In addition to replacing complicated artificial operation, a bionic culture environment was developed in this research to enhance embryo quality.
In this research, we co-cultured embryos with endometrial stromal cells and used dynamic perfusion system with porous membrane to build in-vitro uterus environment by the means of growth factors which stromal cells secrets to provide a uterus-mimicking growth environment for embryonic development. Dynamic culture can not only carry metabolic waste off but also supply fresh medium to maintain stable osmotic pressure and pH value. We also implemented to the automatic trapping system via fluidic resistance design and manipulation to reduce embryo damage resulting from delicate artificial operation.
According to the experimental results, we successfully captured embryos by adjusting the height of channel to 140μm. The stromal cells grew faster in porous membrane and reached to 70% after 2.5 days culturing. We compared monoculture and coculture in petri dish after culturing 2.5 days. The blastocyst developing rate (BDR) of monoculture is 15.79%, and the BDR of cocultrue is 57.89%. It means coculure with stromal cells grew much better and faster. In comparison with monoculture in chip and petri dish, BDR of the former is 57.10% and BDR the latter is 57.58%. Therefore, there is no significant difference between culturing in chip or in petri dish. We compared the coculture in chip and in petri dish. The BDR of coculture in chip is 58.33%, and the BDR of cocultrue in petri dish is 26.67%. Coculture in petri dish is not able to work. On the other hand, coculture in chip via the microfluidic system did successfully work. The embryo transfer rate is 66.67%.

並列關鍵字

Embryo co-culture ； reproductive medicine ； dynamic perfusion ； embryo trapping

參考文獻

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