隨著工業革命、科技業興起等等人類文明的高度發展，讓現代人生活在惡劣的環境下，像是汙染、壓力、飲食等等，都是可能造成現今文明病的原因，在21世紀中，不孕的人口大大提升，生殖醫學也正致力於解決懷孕的問題，而主要治療方法多往體外受精胚胎培養發展，但提高胚胎品質的控制變因有限，且需要繁複的人工操作，因此近幾年發現微流體系統特性相當符合仿生應用，便大量發展於生殖醫學方面，而本論文研究主要為利用微機電技術與微流體理論整合自動化抓取與定位追蹤功能並應用於生醫晶片，以改良現今胚胎傳統培養方式，不但取代繁複的人工操作，更使用體外仿生環境培養，提高胚胎體外培養的品質。
重建體外仿子宮微環境，共培養胚胎與子宮內膜細胞，透過子宮內膜細胞分泌的生長因子提供養分，讓胚胎有更良好的發育環境，並藉由液壓流阻類比電路概念設計自動抓取定位結構，再以流體分析軟體ANSYS CFX最佳化流場設計，可減少人工操作對於胚胎之傷害，也便利於往後培養之觀察追蹤，培養方式則採用動態灌養培養，可排除細胞代謝之廢棄物且又可提供新鮮之培養基，為目前胚胎主要發展培養方式。
本研究論文整合自動化定位、動態灌養、共培養等多功能之體外子宮生醫晶片以提高胚胎培養品質，並於實驗中比較共培養、單養、靜態培養、動態灌養與自動定位等等不同變因之實驗，結果可發現，晶片提供良好的培養環境，有和傳統培養大致相同的囊胚比例(60% vs. 57.8%)，而生長速率方面則是較傳統方式稍快，並於實驗最後將培養之胚胎植回母體進行著床且生產出小鼠，證實本研究晶片之前瞻性與可行性。

Infertility is a worldwide important issue. Assisted Reproductive Technology (ART) becomes one of the most important studies in the 21st Century. The reasons of infertility are most from the modern plague, such as poor nutrition, obesity, alcohol, cigarette, stress and drugs. Recently, in vitro fertilization (IVF), which holds the highest success rate, is a major treatment for infertility.
In this research, the microfluidic devices made by a simple fabrication were developed. We provided the method and operation by integrating both microfluidic techniques and coculture of stromal cells with embryos to mimic the uterus in vitro. The culturing by the dynamic system with the fresh medium also enhances the embryo development. Furthermore, the embryos were automatically and individually trapped via the flow resistance design. The design was simulated and analyzed via the commercial software, CFD and ANSYS CFX, to optimize the flow channel design as well as reduce the manual operation, which resulted in the possibility of embryos damaged. And the most important thing is that the chip is easy to track and manage individual embryos.
The integrated Labchip functions include automatic trapping, dynamic perfusion and coculture to mimic the uterus in vitro. The experimental results show that this Labchip could provide better environment compared with some different conditions such as monoculture and static culture. The embryos could be cultured in comparable blastocyst rate versus the traditional method (60% vs. 57.8%). Besides, the embryos cultured on the chip have the faster growth rate than the traditional method. All of these mean that our Labchip provides a bionic environment for embryo developing. Finally, we transfer the embryos cultured from our Labchip into the maternal uterus and born the mice. It verifies the feasibility and prospective of this Labchip.

Abstract I
摘要 II
致謝 III
圖目錄 X
第一章 緒論 1
1.1 研究背景 1
1.1.1 不孕因素 1
1.1.2 輔助生殖技術(ART) 3
1.1.3 生物微機電與實驗室晶片 4
1.1.4 胚胎體內發展 5
1.1.5 子宮內膜細胞與胚胎共培養 6
1.2 動機與目的 7
1.3 文獻回顧 8
1.3.1 傳統體外培養方式 8
1.3.2 體外微流體胚胎培養裝置 9
1.3.3 共培養微流體晶片 14
1.3.4 追蹤單一胚胎發展 16
第二章 晶片設計與原理 19
2.1 設計基礎與理論 19
2.1.1 微流體流阻分析 19
2.2 設計概念 20
2.2.1 胚胎抓取定位之微結構設計 23
2.2.2 晶片操作 24
2.2.3 動態流阻抓取系統 25
2.2.4 胚胎抓取流場模擬 28
第三章 晶片製程 35
3.1 晶片製作流程 35
3.1.1 微流道母模製程 35
3.1.2 微流道晶片製程 38
3.2 製程結果 40
3.2.1 微流道結構 41
3.2.2 製程過程問題與討論 41
第四章 材料與架設 42
4.1 材料準備 42
4.1.1 子宮內膜細胞(基質細胞) 42
4.1.2 晶片前處理 43
4.1.3 聚苯乙烯乳膠微粒 43
4.1.4 小鼠胚胎 44
4.1.5 培養基 46
4.1.6 石蠟油 47
4.2 實驗架設 48
第五章 結果與討論 49
5.1 實驗流程 49
5.2 抓取定位結果與討論 50
5.3 晶片共培養結果與討論 57
5.3.1 子宮內膜細胞培養結果與討論 57
5.3.2 胚胎培養結果與討論 58
5.3.3 胚胎單培養比較 59
5.3.4 胚胎共培養比較 60
5.3.5 晶片與傳統培養比較 61
5.3.6 胚胎植回母體結果 63
第六章 結論 64
參考文獻 65

[1] Gnoth, C., Godehardt, E., Frank-Herrmann, P., Friol, K., Tigges, J., and Freundl, G., Definition and prevalence of subfertility and infertility. Hum Reprod, 2005. 20(5): p. 1144-7.
[2] Gurunath, S., Pandian, Z., Anderson, R. A., and Bhattacharya, S., Defining infertility--a systematic review of prevalence studies. Hum Reprod Update, 2011. 17(5): p. 575-88.
[3] O'Connor, K. A., Holman, D. J., and Wood, J. W., Declining fecundity and ovarian ageing in natural fertility populations. Maturitas, 1998. 30(2): p. 127-36.
[4] Dunson, D. B., Colombo, B., and Baird, D. D., Changes with age in the level and duration of fertility in the menstrual cycle. Hum Reprod, 2002. 17(5): p. 1399-403.
[5] Shady Grove Fertility Center.
[6] Dr. Bond: Total Health & Care with A Human Touch.
[7] Infertility Center of St. Louis.
[8] Drews, U., Taschenatlas der Embryologie. 1993: Stuttgart New York Thieme.
[9] Barmat, L. I., Liu, H. C., Spandorfer, S. D., Kowalik, A., Mele, C., Xu, K., Veeck, L., Damario, M., and Rosenwaks, Z., Autologous endometrial co-culture in patients with repeated failures of implantation after in vitro fertilization-embryo transfer. J Assist Reprod Genet, 1999. 16(3): p. 121-7.
[10] Deachapunya, C. and O'Grady, S. M., Epidermal growth factor regulates the transition from basal sodium absorption to anion secretion in cultured endometrial epithelial cells. J Cell Physiol, 2001. 186(2): p. 243-50.
[11] Dominguez, F., Gadea, B., Mercader, A., Esteban, F. J., Pellicer, A., and Simon, C., Embryologic outcome and secretome profile of implanted blastocysts obtained after coculture in human endometrial epithelial cells versus the sequential system. Fertil Steril, 2010. 93(3): p. 774-782.
[12] Yoshizawa, M., Mitsui, A.,Fukui, E., A Successful Method in Mouse in vitro Fertilization for Beginners. Journal of Mammalian Ova Research, 2005. 22(4): p. 246-250.
[13] Kimura, H., Yamamoto, T., Sakai, H., Sakai, Y., and Fujii, T., An integrated microfluidic system for long-term perfusion culture and on-line monitoring of intestinal tissue models. Lab Chip, 2008. 8(5): p. 741-6.
[14] Sommer, AP., Haddad, MKh. and Fecht, HJ., It is Time for a Change: Petri Dishes Weaken Cells. Journal of Bionic Engineering, 2012. 9(3): p. 353-357.
[15] Swain, J. E. and Smith, G. D., Advances in embryo culture platforms: novel approaches to improve preimplantation embryo development through modifications of the microenvironment. Hum Reprod Update, 2011. 17(4): p. 541-57.
[16] Kolahi, K. S., Donjacour, A., Liu, X., Lin, W., Simbulan, R. K., Bloise, E., Maltepe, E., and Rinaudo, P., Effect of substrate stiffness on early mouse embryo development. PLoS One, 2012. 7(7): p. e41717.
[17] Rappolee, D. A., Basilico, C., Patel, Y., and Werb, Z., Expression and function of FGF-4 in peri-implantation development in mouse embryos. Development, 1994. 120(8): p. 2259-69.
[18] Raty, S., Walters, E. M., Davis, J., Zeringue, H, Beebe, D. J., Rodriguez-Zas, S. L. , and Wheeler, M. B., Embryonic development in the mouse is enhanced via microchannel culture. Lab Chip, 2004. 4(3): p. 186-90.
[19] Walters, E. M., Clark, S. G., Roseman, H. M., Beebe, D. J., and Wheeler, M. B., Production of live piglets following in vitro embryo culture in a microfluidic environment. Theriogenology, 2003. 59(1): p. 353.
[20] Hickman, D. L., Beebe, D. J., Rodriguez-Zas, S. L., and Wheeler, M. B., Comparison of static and dynamic medium environments for culturing of pre-implantation mouse embryos. Comp Med, 2002. 52(2): p. 122-6.
[21] Xie, Y., Wang, F., Zhong, W., Puscheck, E., Shen, H., and Rappolee, D. A., Shear stress induces preimplantation embryo death that is delayed by the zona pellucida and associated with stress-activated protein kinase-mediated apoptosis. Biol Reprod, 2006. 75(1): p. 45-55.
[22] Matsuura, K., Hayashi, N., Kuroda, Y., Takiue, C., Hirata, R., Takenami, M., Aoi, Y., Yoshioka, N., Habara, T., Mukaida, T., and Naruse, K., Improved development of mouse and human embryos using a tilting embryo culture system. Reprod Biomed Online, 2010. 20(3): p. 358-64.
[23] Heo, Y. S., Cabrera, L. M., Bormann, C. L., Shah, C. T., Takayama, S., and Smith, G. D., Dynamic microfunnel culture enhances mouse embryo development and pregnancy rates. Hum Reprod, 2010. 25(3): p. 613-22.
[24] Cabrera, L. M., Heo, Y. S., Ding, J. , Takayama, S. , and Smith, G. D., Improved blastocyst development with microfluidics and Braille pin actuator enabled dynamic culture. Fertil. Steril., 2006. 86(3): p. S43.
[25] Alegretti, J.R. , Rocha, A.M., Barros, B.C., Serafini, P., Motta, E.L.A., and Smith, G. D., Microfluidic dynamic embryo culture increases the production of top quality human embryos through reduction in embryo fragmentation. Fertil Steril, 2011. 96(3): p. S58-S59.
[26] Kim, M. S., Bae, C. Y., Wee, G., Han, Y. M., and Park, J. K., A microfluidic in vitro cultivation system for mechanical stimulation of bovine embryos. Electrophoresis, 2009. 30(18): p. 3276-82.
[27] Bae, C. Y., Kim, M. S., and Park, J. K., Mechanical stimulation of bovine embryos in a microfluidic culture platform. BioChip, 2011. 5(2): p. 106-113.
[28] Isachenko, V., Maettner, R., Sterzik, K., Strehler, E., Kreinberg, R., Hancke, K., Roth, S., and Isachenko, E., In-vitro culture of human embryos with mechanical micro-vibration increases implantation rates. Reprod Biomed Online, 2011. 22(6): p. 536-44.
[29] Isachenko, E., Maettner, R., Isachenko, V., Roth, S., Kreienberg, R., and Sterzik, K., Mechanical agitation during the in vitro culture of human pre-implantation embryos drastically increases the pregnancy rate. Clin Lab, 2010. 56(11-12): p. 569-76.
[30] Xie, Y., Wang, F., Puscheck, E. E., and Rappolee, D. A., Pipetting causes shear stress and elevation of phosphorylated stress-activated protein kinase/jun kinase in preimplantation embryos. Mol Reprod Dev, 2007. 74(10): p. 1287-94.
[31] Jayot, S., Parneix, I., Verdaguer, S., Discamps, G., Audebert, A., and Emperaire, J. C., Coculture of embryos on homologous endometrial cells in patients with repeated failures of implantation. Fertil Steril, 1995. 63(1): p. 109-114.
[32] Eyheremendy, V., Raffo, F. G., Papayannis, M., Barnes, J., Granados, C., and Blaquier, J., Beneficial effect of autologous endometrial cell coculture in patients with repeated implantation failure. Fertil Steril, 2010. 93(3): p. 769-773.
[33] Kimura, H., Nakamura, H., Akai, T., Yamamoto, T., Hattori, H., Sakai, Y., and Fujii, T., On-chip single embryo coculture with microporous-membrane-supported endometrial cells. IEEE Trans Nanobioscience, 2009. 8(4): p. 318-24.
[34] Mizuno, J., Ostrovidov, S., Sakai, Y., Fujii, T., Nakamura, H., and Inui, H., Human ART on chip: improved human blastocyst development and quality with IVF-chip. Fertil Steril, 2007. 88(1): p. S101.
[35] Li, W. X., Liang, G. T., Yan, W., Zhang, Q. L., Wang, W., Zhou, X. M., and Liu, D. Y., Artificial Uterus on a Microfluidic Chip. Chinese Journal of Analytical Chemistry, 2013. 41(4): p. 467-472.
[36] Krisher, R. L. and Wheeler, M. B., Towards the use of microfluidics for individual embryo culture. Reprod Fertil Dev, 2010. 22(1): p. 32-39.
[37] Vajta, G., Peura, T. T., Holm, P., Paldi, A., Greve, T., Trounson, A. O., and Callesen, H., New method for culture of zona-included or zona-free embryos: the Well of the Well (WOW) system. Mol Reprod Dev, 2000. 55(3): p. 256-64.
[38] Sugimura, S., Akai, T., Somfai, T., Hirayama, M., Aikawa, Y., Ohtake, M., Hattori, H., Kobayashi, S., Hashiyada, Y., Konishi, K., and Imai, K., Time-lapse cinematography-compatible polystyrene-based microwell culture system: a novel tool for tracking the development of individual bovine embryos. Biol Reprod, 2010. 83(6): p. 970-8.
[39] Ma, R., Xie, L., Han, C., Su, K., Qiu, T., Wang, L., Huang, G., Xing, W., Qiao, J., Wang, J., and Cheng, J., In vitro fertilization on a single-oocyte positioning system integrated with motile sperm selection and early embryo development. Anal Chem, 2011. 83(8): p. 2964-70.
[40] Tan, W. H. and Takeuchi, S., A trap-and-release integrated microfluidic system for dynamic microarray applications. Proc Natl Acad Sci U S A, 2007. 104(4): p. 1146-51.
[41] Di Carlo, D. and Lee, L. P., Dynamic single-cell analysis for quantitative biology. Anal Chem, 2006. 78(23): p. 7918-25.
[42] Teshima, T., Ishihara, H., Iwai, K., Adachi, A., and Takeuchi, S., A dynamic microarray device for paired bead-based analysis. Lab Chip, 2010. 10(18): p. 2443-8.
[43] Chung, J., Kim, Y. J., and Yoon, E., Highly-efficient single-cell capture in microfluidic array chips using differential hydrodynamic guiding structures. Applied Physics Letters, 2011. 98(12): p. 123701-3.
[44] Saiz, N. and Plusa, B., Early cell fate decisions in the mouse embryo. Reproduction, 2013. 145(3): p. R65-80.
[45] Otsuki, J., Nagai, Y., and Chiba, K., Peroxidation of mineral oil used in droplet culture is detrimental to fertilization and embryo development. Fertil Steril, 2007. 88(3): p. 741-3.
[46] Tae, J. C., Kim, E. Y., Lee, W. D., Park, S. P., and Lim, J. H., Sterile filtered paraffin oil supports in vitro developmental competence in bovine embryos comparable to co-culture. J Assist Reprod Genet, 2006. 23(3): p. 121-7.