簡易檢索 / 詳目顯示

回結果列表
研究生： 柏夏拉
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
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
Liu, Cheng-Hsien
口試委員: 饒達仁
Yao, Da-Jeng
徐文祥
Hsu, Wensyang
學位類別： 碩士
Master
系所名稱： 工學院 - 奈米工程與微系統研究所
Institute of NanoEngineering and MicroSystems
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-culture, In-vitro Culture, Reproductive Technology, Cell Trapping, Hydrodynamic Trapping, 3T3 Cells
相關次數： 點閱：2  下載：0 
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報

    • 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

    1. https://ehealthforum.com/health/natural-infertility-treatment-t349094
    2. HonmoreV.M. and M.G. Jadhav, Sclf-esteem and coping resources among fertile and infertile adults. Indian Journal of Health & Wellbeing, 2016. 7(7): p. 742-745.
    3. Zegers-Hochschild, F., et al., International Committee for Monitoring Assisted Reproductive Technology (ICMART) and the World Health Organization (WHO) revised glossary of ART terminology, 2009∗. Fertility and Sterility, 2009. 92(5): p. 1520-1524.
    4. Sudha G. and Reddy K.S.N., Causes of female infertility: a cross-sectional study, International Journal of Latest Research in Science and Technology, 2013, 2, pp.119-123
    5. Healy DL, Trounson AO, Andersen AN., Female infertility: causes and treatment. The Lancet, 1994. 343(8912): p. 1539-1544.
    6. https://www.medicalnewstoday.com/articles/165748.php
    7. http://www.indoreinfertilityclinic.com
    8. https://www.shadygrovefertility.com
    9. https://www.babycentre.co.uk/a6155/your-age-and-fertility
    10. Dyer S, Chambers GM, de Mouzon J, Nygren KG, Zegers-Hochschild F, Mansour R, Ishihara O, Banker M, Adamson GD., International Committee for Monitoring Assisted Reproductive Technologies world report: Assisted Reproductive Technology 2008, 2009 and 2010†. Human Reproduction, 2016. 31(7): p. 1588-1609.
    11. https://commons.wikimedia.org/w/index.php?curid=23260794
    12. https://www.pinkfamilies.com/fertilization/
    13. Magli MC, Gianaroli L, Ferraretti AP, Fortini D, Fiorentino A, D'Errico A., Human embryo co-culture: results of a randomized prospective study. International journal of fertility and menopausal studies, 1995. 40(5): p. 254-259.
    14. Barmat LI, Liu HC, Spandorfer SD, Kowalik A, Mele C, Xu K, Veeck L, Damario M, Rosenwaks Z., Autologous endometrial co-culture in patients with repeated failures of implantation after in vitro fertilization-embryo transfer. Journal of assisted reproduction and genetics, 1999. 16(3): p. 121-127.
    15. Dominguez F, Gadea B, Mercader A, Esteban FJ, Pellicer A, Simón C., Embryologic outcome and secretome profile of implanted blastocysts obtained after coculture in human endometrial epithelial cells versus the sequential system. Fertility and Sterility, 2010. 93(3): p. 774-782.e1.
    16. 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.
    17. F Serhal P, M Ranieri D, Kinis A, Marchant S, Davies M, M Khadum I., Oocyte morphology predicts outcome of intracytoplasmic sperm injection. Vol. 12. 1997. 1267-70.
    18. Lucinda L. Veeck., Abnormal morphology of the human oocyte and conceptus, Atlas of the Human Oocyte and Early Conceptus, 1991. 2, pp. 151-153.
    19. Bongso A, Ng S-C, Fong C-Y, Ratnam S., Cocultures: a new lead in embryo quality improvement for assisted reproduction. Fertility and Sterility, 1991. 56(2): p. 179-191.
    20. Bongso A, Fong C-Y, Ng S-C, Ratnam S., The search for improved in-vitro systems should not be ignored: embryo co-culture may be one of them. Human Reproduction, 1993. 8(8): p. 1155-1160.
    21. Kimura H, Yamamoto T, Sakai H, Sakai Y, Fujii T., An integrated microfluidic system for long-term perfusion culture and on-line monitoring of intestinal tissue models. Lab on a Chip, 2008. 8(5): p. 741-746.
    22. https://www.infertile.com/treating-infertility-improvements-ivf-laboratory
    23. Sommer AP, Haddad MK, Fecht H-J., It is Time for a Change: Petri Dishes Weaken Cells. Journal of Bionic Engineering, 2012. 9(3): p. 353-357.
    24. Swain JE, Smith GD., Advances in embryo culture platforms: novel approaches to improve preimplantation embryo development through modifications of the microenvironment. Human Reproduction Update, 2011. 17(4): p. 541-557.
    25. Kolahi KS, Donjacour A, Liu X, Lin W, Simbulan RK, Bloise E, Maltepe E, Rinaudo P., Effect of Substrate Stiffness on Early Mouse Embryo Development. PLOS ONE, 2012. 7(7): p.1717.
    26. Swain JE, Carrell D, Cobo A, Meseguer M, Rubio C, Smith GD., Optimizing the culture environment and embryo manipulation to help maintain embryo developmental potential. Fertility and Sterility, 2016. 105(3): p. 571-587.
    27. Vajta G, Korösi T, Du Y, Nakata K, Ieda S, Kuwayama M, Nagy ZP., The Well-of-the-Well system: an efficient approach to improve embryo development. Reproductive BioMedicine Online, 2008. 17(1): p. 73-81.
    28. Ma R, Xie L, Han C, Su K, Qiu T, Wang L, Huang G, Xing W, Qiao J, Wang J et al., In Vitro Fertilization on a Single-Oocyte Positioning System Integrated with Motile Sperm Selection and Early Embryo Development. Analytical Chemistry, 2011. 83(8): p. 2964-2970.
    29. Heo YS, Cabrera LM, Bormann CL, Shah CT, Takayama S, Smith GD., Dynamic microfunnel culture enhances mouse embryo development and pregnancy rates. Human reproduction (Oxford, England), 2010. 25(3): p. 613-622.
    30. Kim MS, Bae CY, Wee G, Han Y-M, Park J-K., A microfluidic in vitro cultivation system for mechanical stimulation of bovine embryos. 2009. 30(18): p. 3276-3282.
    31. Esteves TC, van Rossem F, Nordhoff V, Schlatt S, Boiani M, Le Gac S., A microfluidic system supports single mouse embryo culture leading to full-term development. RSC Advances, 2013. 3(48): p. 26451-26458.
    32. Kieslinger DC, Hao Z, Vergouw CG, Kostelijk EH, Lambalk CB, Le Gac S., In vitro development of donated frozen-thawed human embryos in a prototype static microfluidic device: a randomized controlled trial. Fertility and Sterility, 2015. 103(3): p. 680-686.
    33. Mizuno J, Ostrovidov S, Sakai Y, Fujii T, Nakamura H, Inui H., Human ART on chip: improved human blastocyst development and quality with IVF-chip. Fertility and Sterility, 2007. 88: p. S101.
    34. Kimura H, Nakamura H, Akai T, Yamamoto T, Hattori H, Sakai Y, Fujii T., On-Chip Single Embryo Coculture With Microporous-Membrane-Supported Endometrial Cells. IEEE Transactions on NanoBioscience, 2009. 8(4): p. 318-324.

    35. Li W-X, Liang G-T, Yan W, Zhang Q, Wang W, Zhou X-M, Liu D-Y., Artificial Uterus on a Microfluidic Chip. Chinese Journal of Analytical Chemistry, 2013. 41(4): p. 467-472.
    36. Ledda S, Idda A, Kelly J, Ariu F, Bogliolo L, Bebbere D., A novel technique for in vitro maturation of sheep oocytes in a liquid marble microbioreactor. Journal of assisted reproduction and genetics, 2016. 33(4): p. 513-518.
    37. Jingchun L, Qi L, Guosheng W, Shengjun L, Jiabao Z, Yanbing L., Application of a Microfluidic Chip with Mechanical Stimuli for Culturing Mouse Embryos in vitro. International Journal Bioautomation, 2018. 22(2): p. 107-116.
    38. Ferraz MAMM, Rho HS, Hemerich D, Henning HHW, van Tol HTA, Hölker M, Besenfelder U, Mokry M, Vos PLAM, Stout TAE et al., An oviduct-on-a-chip provides an enhanced in vitro environment for zygote genome reprogramming. Nature Communications, 2018. 9(1): p. 4934.
    39. Kumano I, Hosoda K, Suzuki H, Hirata K, Yomo T., Hydrodynamic trapping of Tetrahymena Thermophila for the long-term monitoring of cell behaviors. Vol. 12. 2012. 3451-7.
    40. Novotný J, Foret F., Fluid manipulation on the micro-scale: Basics of fluid behavior in microfluidics, Journal of Separation Science, 2017. 40(1): p. 383-394.
    41. Oh KW, Lee K, Ahn B, Furlani EP., Design of pressure-driven microfluidic networks using electric circuit analogy. Lab on a Chip, 2012. 12(3): p. 515-545.
    42. White MD, Plachta N., Chapter One - How Adhesion Forms the Early Mammalian Embryo, in Current Topics in Developmental Biology, A.S. Yap, Editor. 2015, Academic Press. p. 1-17.
    43. Isachenko E, Maettner R, Isachenko V, Roth S, Kreienberg R, Sterzik K., Mechanical Agitation During the in vitro Culture of Human Pre-Implantation Embryos Drastically Increases the Pregnancy Rate. Vol. 56. 2010. 569-76.
    44. Azadbakht M, Valojerdi MR, Mowla SJ, Development of mouse embryos co-cultured with polarized or non-polarized uterine epithelial cells using sequential culture media. Animal Reproduction Science, 2007. 100(1): p. 141-157.
    45. Isachenko V, Maettner R, Sterzik K, Strehler E, Kreinberg R, Hancke K, Roth S, Isachenko E., In-vitro culture of human embryos with mechanical micro-vibration increases implantation rates. Reproductive BioMedicine Online, 2011. 22(6): p. 536-544.

    QR CODE