有鑑於近年來國人生育率之日趨低落，除了生育意願的下降之外，因為壓力或其他因素造成之不孕症使得許多夫妻一子難求。但隨著科技的發展，現已有不只一種的人工輔助生殖技術(Assisted Reproductive Technology, ART)造福這些想要求子的夫妻，例如體外人工受精的IVF(In vitro fertilization)，若仍無法成功則可選擇更精密的單一精蟲顯微注射法(IntraCytoplasmic Sperm Injection, ICSI)。
上述提到之技術都需要高品質、高活動力的精蟲來進行受孕，甚至是能發育成較強壯之胎兒。本研究之目的即為透過PDMS微流道晶片，以層流的方式區隔較差的精蟲，並藉由精蟲之泳動性篩選收集高品質的精蟲，以提供人工受孕之用。
本研究使用微尺度層流篩選精蟲的控制微流體系統，進行原液檢體之篩選、固定黏滯度之檢體篩選及稀釋寡精濃度之檢體篩選等實驗。此實驗設計包含晶片設計、幫浦參數設定、流式細胞儀分析以及各篩選結果的分析。
從原液檢體篩選的結果可以看到，在四組實驗中藉由調整過的流速(11:1~20:1)明顯地提升了活蟲率，相對於8:1的篩選效果已是大有改善，證實了本流道對於原液檢體只要適當的調整流速，仍然具有篩選活精蟲的能力。
為了改善原液檢體篩選效率較差之問題，將檢體固定稀釋至黏滯度2mPa×s，並一律以8:1 (稀釋液:精液)之流速進行篩選，無論是正常檢體或寡精檢體都能提升10%以上的活蟲率。
在稀釋寡精檢體篩選實驗中，各種濃度之檢體經過本晶片篩選過後平均活蟲率都至少能提升10%以上，篩選效率也平均都超過10%，證實本晶片確實兼具提升寡精檢體的活蟲率和收集活精蟲之能力。
透過流式細胞儀的分析確認收集的活精子比例有明顯的提升，證實微流道晶片的確具有篩選能力後，本研究最終以雙染方式驗證，經過此晶片篩選之精蟲其細胞皆保持良好的完整性。

About the decreasing birth rate of the people in recent years, except for the decline of the desires to have children, many couples are infertile due to stress or other factors. However, with the development of science and technology, there are more than one assisted reproductive technologies (ART) bring benefits to these couples, such as in vitro fertilization (IVF), or more precise IntraCytoplasmic Sperm Injection (ICSI).
The above techniques require high-quality and high-motility sperms for pregnancy, and even growing to a healthier fetus. The purpose of this study is to use PDMS microfluidic chips to separate poor sperms by laminar flow and collect high quality sperms by motility to provide to the artificial fertilization process.
In this study, micro-scale laminar flow control system is used for live sperms separation of the stock sample, fixed-viscosity sperm sorting experiments and diluted oligozoospermia concentration sorting. This research includes structural design, pump settings, flow cytometry analysis and the analysis of the results.
From the results of the stock sample sorting, the viability was obviously improved by the modified flow rate, and the separation with respect to 8: 1 has been greatly improved. Confirming that by appropriate adjustment of the flow rate, the chip is still able to acquire live sperms.
In order to improve the low sorting efficiency of stock sample sorting, the semen was fixedly diluted to 2mPa×s viscosity, and sorted by the flow rate 8: 1 (buffer: semen). This chip can enhance the viability for more than 10% on both normal and oligozoospermia samples.
In the experiment of diluted oligozoospermia sample sorting, the average viability can be increased at least 10% after separation by this chip for any concentrations, and the sorting efficiency is higher than 10% on average.
By flow cytometry analysis, we confirmed that the proportions of live sperms are significantly improved, which shows that the microfluidic chip is actually able to acquire live sperms. Finally, we use double-dye staining to verify the cell membrane of sperm are integral after collection.

目錄 6
圖目錄 9
表目錄 14
第一章 緒論 15
1.1 前言 15
1.2 研究背景 16
1.3 研究動機 16
1.4 本文架構 20
第二章 文獻回顧 21
2.1 傳統精蟲收集法 21
2.1.1 離心洗精法 21
2.1.2 直接上泳法 22
2.1.3 非連續密度梯度離心法 23
2.2 現有之細胞篩選方式 23
2.2.1微流道分選精蟲與表皮細胞 23
2.2.2 結合層流與介電泳力篩選活死細胞 24
2.2.3 結合磁珠篩選活精蟲(MACs) 26
2.3 本研究之相關參考文獻 27
2.3.1 微流道晶片精子篩選與其游速的關係 27
2.3.2 緩慢流速篩選精蟲 28
2.4文獻比較 30
2.5 前代實驗結果回顧與研究目標 31
第三章 晶片設計 32
3.1 精子篩選之微流道晶片 32
3.2 微流體之主導參數 33
3.3 數值模擬 34
3.3.1 流道精液濃度分布模擬 36
第四章 實驗製程與步驟 40
4.1 晶片製程 40
4.1.1 SU8-3050微流道結構製作 41
4.1.2 PDMS翻模 43
4.1.3 氧電漿晶片接合 44
4.2 儀器與設備 46
4.3 Syringe pump操作及應用 47
4.3.1 流速計算 48
4.3.2 幫浦設定 49
4.4 檢體來源及前處理 50
4.4.1 豬精 50
4.4.2 人精 51
4.5 實驗操作流程 53
4.5.1 原液檢體篩選 53
4.5.2 固定黏滯度之稀釋精液篩選 56
4.5.3 稀釋為寡精濃度之人精篩選 60
4.6 精蟲螢光染色 63
4.6.1 螢光染劑對精子的影響 64
4.7 Flow cytometer分析 65
4.8 流道組與離心組精蟲細胞膜完整性檢測 68
第五章 實驗結果 70
5.1 精蟲層流篩選過程 70
5.1.1 呈現穩定的層流 70
5.1.2 活動力的精蟲穿越層流現象 70
5.2 Flow cytometric分析結果 71
5.2.1 以活蟲率和篩選效率討論分離效果 73
5.2.2 模擬注入隻數並比較收集隻數 74
5.3 原液檢體篩選 77
5.4 固定黏滯度之稀釋精液篩選 79
5.5 稀釋為寡精濃度之人精 82
5.5.1 稀釋輕度寡精檢體應用於微流道晶片 83
5.5.2 稀釋中度寡精檢體應用於微流道晶片 84
5.5.3 稀釋重度寡精檢體應用於微流道晶片 84
5.5.4 各濃度之篩選效率比較 85
5.6 流道組與離心組精蟲細胞膜完整性檢測 86
第六章 結論 89
6.1 結論 89
第七章 問題討論與未來展望 91
7.1 問題與討論 91
7.1.1 晶片插管方式 91
7.1.2 精蟲濃度與精液黏滯度之相關性 93
7.2 未來展望 94
7.2.1 廢液槽流道加寬 94
7.2.2 晶片材質變更以及量產 95
參考文獻 97

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