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研究生:柏夏拉
研究生(外文):Bhosale, Shalaka Shahajirao
論文名稱:具有自動抓取和子宮內膜細胞共培養功能之體外受卵培育晶片
論文名稱(外文):In-vitro development of the embryo in a microfluidic device for automatic embryo trapping and co-culture with endometrial cells
指導教授:劉承賢劉承賢引用關係
指導教授(外文):Liu, Cheng-Hsien
口試委員:饒達仁徐文祥
口試委員(外文):Yao, Da-JengHsu, Wensyang
口試日期:2018-11-29
學位類別:碩士
校院名稱:國立清華大學
系所名稱:奈米工程與微系統研究所
學門:工程學門
學類:材料工程學類
論文種類:學術論文
論文出版年:2018
畢業學年度:107
語文別:英文
論文頁數:79
中文關鍵詞:共培養體外培養生殖醫學細胞抓取流體動力抓取3T3細胞
外文關鍵詞:Co-cultureIn-vitro CultureReproductive TechnologyCell TrappingHydrodynamic Trapping3T3 Cells
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不孕症狀是目前許多不同國家的夫妻同時面對的問題,雖然不同的區域人們不孕的症狀會因地理或是經濟因素有些許的差異;現今每年全世界大概估計有6000到8000萬的不孕夫妻正在為了無法生育自己的孩子苦惱著. 為了能夠幫助改善這個問題,輔助生育醫學相關技術(Assisted Reproductive Technology,簡稱ART)在21世紀躍升成頗為熱門的研究議題.目前體外人工受孕(IVF)是其中一項成功率高達%30到%40的重要且能夠改善不孕症的方法.
在本論文中所設計的微流體晶片同時整合微流體操控技術和子宮內膜細胞共同培養技術,以期能夠建構一種可以模仿人體子宮內部的環境.首先受精卵可以自動且個別地被抓取到晶片裡面和子宮內膜細胞一起共培養的腔室,這種自動抓取的方式跟傳統的培養皿培養方式的不同在於能夠降低人力在操作上的消耗. 另外此晶片也具有動態培養的功能,也就是培養過程中受精卵和子宮內膜細胞的代謝物不但可以被清除,同時新鮮的培養液體也是時時刻刻都在滋養著受精卵和子宮內膜細胞.具實驗統計,一開始進入沒有子宮內膜細胞的晶片的受精卵是二細胞的狀態,最後動態培養的結果發現受精卵能夠在晶片內個別發育成囊胚的比例是66.67%,而傳統培養的比例則是35.29%. 而進入有子宮內膜細胞共培養晶片的二細胞狀態受精卵則有62.5%的比例可以發育成囊胚.有子宮內膜細胞共培養的晶片可以提升受精卵發育成囊胚的機率,這在IVF的技術中是個可以考慮的手法.另外晶片也能夠單獨地採取所有受精卵的代泄物質,以期之後能夠個別分析其成分來推測每個受精卵的個別狀況.
Infertility is a worldwide problem affecting 60-80 million couples every year. The cause and magnitude may vary with geographical location and social and economic status. Now situation is 1 in 8 couple suffering from infertility. To find a solution for this, Assisted Reproductive Technology (ART) becomes one of the most critical studies in the 21st Century. Currently, in-vitro fertilization (IVF), which holds the highest success rate around 30-40%, is a significant treatment for infertility. In this research, our microfluidic device is developed by integrating both microfluidic techniques and co-culture of endometrial cells with embryos to mimick the environment just like in the uterus. The embryos were automatically and individually trapped in the chip with the help of hydrodynamic flow resistance, resulting in reducing the labours operation required in traditional method. Furthermore, the dynamic perfusion was used to circulate fresh medium around the embryos, which help to wash out the metabolism waste produced by endometrial cells.
The proposed microfluidic chip is able to trap and culture individual embryo inside the culture chamber. Here two-cell stage mouse embryos were used for the experiments. The results showed that the Blastocyst development rates for the mono-culture in traditional method and device are 35.29% and 66.67%, respectively. Whereas for the co-culture experiments, the Blastocyst development rate in the microfluidic chip is 62.5%, which suggests the feasibility of the chip. The co-culture platform mimick the microenvironment for embryo like in-vivo to enhance its overall development, which will be a very promising culture platform in in-vitro. As well the proposed device able to collect the medium around the embryos from individual chamber. This suggests the use of the chip for further new IVF technologies.
Table of contents
Abstract 2
摘要 3
Acknowledgement 4
Table of contents 5
List of Figures 7
List of Tables 11
1. Introduction 12
1.1 Background: 12
1.1.1 Causes of Infertility: 12
1.1.2 Assisted Reproductive Technology (ART) 14
1.1.3 Bio-MEMS and Lab on a chip (LOC) 16
1.1.4 Embryo culture in vivo 18
1.1.5 Autologous co-culture of endometrial cells and embryo 19
1.2 Motivation and objective 20
1.3 Literature review 22
1.3.1 Traditional IVC method 22
1.3.2 A microfluidic device for embryo culture in-vitro 23
1.3.3 Hydraulic trapping 33
2. Device development 35
2.1 Microfluidic flow resistance analysis 35
2.2 Design Concept 39
2.2.1 Working procedure 41
2.2.2 Hydrodynamic Trapping System 43
2.2.3 Numerical simulation of the trapping mechanism 46
3. Fabrication of microfluidic chip 51
3.1 Wafer production process 51
3.1.1 Microchannel master Molding process 51
3.1.2 Microchannel wafer process 52
3.2 Fabrication results 54
4. Experimental setup and methods 55
4.1 Materials: 55
4.1.1 SU-8 Negative photoresist 55
4.1.2 PDMS 55
4.1.3 Collagen 56
4.1.4 Paraffin oil 56
4.2 Cell material 57
4.2.1 NIH 3T3 cells 57
4.2.2 Embryo 58
4.3 Cleaning and surface modification of microchannel 58
4.4 Experimental setup 59
5. Experimental Results 61
5.1 Experimental procedure 61
5.2 Embryo trapping results 62
5.2.1 Single layer and double layer structure 62
5.2.2 Final Chip design 64
5.3 NIH 3T3 cell culture 65
5.4 Embryo culture Results 68
5.4.1 Monoculture of embryos on chip versus in dish 68
5.4.2 Comparison between traditional method and co-culture method 70
5.5 Medium collection from individual culture chamber 72
6. Conclusion 74
References 76
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