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

胚胎共培養 ； 生殖醫學 ； 動態灌流 ； 胚胎抓取 ； Embryo co-culture ； reproductive medicine ； dynamic perfusion ； embryo trapping

清華大學動力機械工程學系學位論文

2016年

碩士

劉承賢

英文

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%。

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%.

1. [4] C. B. Dunson DB, Baird DD., "Changes with age in the level and duration of fertility in the menstrual cycle.," Hum Reprod, pp. 1399-403., May 2002.
   連結：
2. [5] H. D. O'Connor KA, Wood JW., "Declining fecundity and ovarian ageing in natural fertility populations.," Maturitas, pp. 127-36, Oct 1998.
   連結：
3. [6] G. E. Gnoth C, Frank-Herrmann P, Friol K, Tigges J, Freundl G., "Definition and prevalence of subfertility and infertility," Hum Reprod, pp. 1144-1147, 2005 Mar 31.
   連結：
4. [7] P. Z. Gurunath S, Anderson RA, Bhattacharya S., "Defining infertility--a systematic review of prevalence studies.," Hum Reprod Update, pp. 575-88, 2011 Sep-Oct.
   連結：
5. [11] C. B. Francisco Raga, Jose Remohi, Fernando Bonilla-Musoles, Carlos Simo´n and Antonio Pellicer, "Reproductive impact of congenital Mu¨llerian anomalies.," Human Reproduction, pp. 2277-2281, 1997.
   連結：
6. [12] L. Y. Kang S, Li B, Wang N, Zhou RM, Zhao XW., "Genetic variation of the E-cadherin gene is associated with primary infertility in patients with ovarian endometriosis.," Fertil Steril., 2014 Aug 20.
   連結：
7. [13] A. O. García-Ulloa AC, "Tubal occlusion causing infertility due to an excessive inflammatory response in patients with predisposition for keloid formation.," Medical Hypotheses, pp. 908-14, 2005.
   連結：
8. [14] P. E. S. M G Hull, and D R Bromham, "Anovulatory and ovulatory infertility: results with simplified management.," Br Med J (Clin Res Ed), pp. 1681–1685, Jun 5, 1982.
   連結：
9. [16] C. F. Rowe PJ, Hargreave TB, Mahmoud AMA, "WHO Manual for the Standardized Investigation, Diagnosis and Management of the Infertile Male," Cambridge University Press, pp. 5–16, 2000.
   連結：
10. [17] Y. L. Guixiang Ji, Yong Zhou, Cong Huang, Aihua Gu and Xinru Wang, "Common variants in mismatch repair genes associated with increased risk of sperm DNA damage and male infertility.," BMC Medicine, 17 May 2012.
    連結：
11. [18] C. G., "Male idiopathic oligoasthenoteratozoospermia.," Asian J Androl., pp. 143-57, 2006 Mar;8.
    連結：
12. [19] "What is in vitro fertilisation (IVF) and how does it work?," Human fertilisation and embryology authority, Retrieved Aug. 27, 2014.
    連結：
13. [21] M. H. Johnson, "Sir Robert Edwards obituary," Theguardian, 10 April 2013.
    連結：
14. [24] "In Vitro Fertilisation with intracytoplasmic sperm injection (IVF-ICSI) " Clinica eugin, Retrieved Aug. 27, 2014.
    連結：
15. [25] "What is intra-cytoplasmic sperm injection (ICSI) and how does it work?," Human fertilisation and embryology authority, Retrieved Aug. 27, 2014.
    連結：
16. [26] "Stem Cell Reports: Appendix A Early Development," National Institutes of Health, Retrieved Aug. 28, 2014.
    連結：
17. [27] R. J. Cockburn K, "Making the blastocyst: lessons from the mouse.," J Clin Invest, pp. 995-1003, 2010 Apr.
    連結：
18. [30] O. G. S. Deachapunya C, "Epidermal growth factor regulates the transition from basal sodium absorption to anion secretion in cultured endometrial epithelial cells.," J Cell Physiol., pp. 243-50, 2001 Feb.
    連結：
19. [31] G. B. Dominguez F, 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.," Fertil Steril., pp. 774-782, 2010 Feb.
    連結：
20. [32] A. M. Midori Yoshizawa, Emiko Fukui, "A Successful Method in Mouse in vitro Fertilization for Beginners," Journal of Mammalian Ova Research, pp. 246-250, 2005.
    連結：
21. [33] Y. T. Kimura H, Sakai H, Sakai Y, Fujii T., "An integrated microfluidic system for long-term perfusion culture and on-line monitoring of intestinal tissue models.," Lab Chip., pp. 741-6, 2008 May.
    連結：
22. [34] M. K. H. Andrei P. Sommer, Hans-Joerg Fecht, "It is Time for a Change: Petri Dishes Weaken Cells," Bionic Engineering, pp. Pages 353–357, September 2012.
    連結：
23. [35] S. G. Swain JE, "Advances in embryo culture platforms: novel approaches to improve preimplantation embryo development through modifications of the microenvironment.," Hum Reprod Update. , pp. 541-57, 2011 Jul-Aug.
    連結：
24. [37] M. P. Rebollar-Lazaro I, "The culture of human cleavage stage embryos alone or in groups: effect upon blastocyst utilization rates and implantation.," Reprod Biol., pp. 227-34, 2010 Nov.
    連結：
25. [38] G. D. Lane M, "Effect of incubation volume and embryo density on the development and viability of mouse embryos in vitro.," Hum Reprod., pp. 558-62, 1992 Apr.
    連結：
26. [41] W. E. Raty S, Davis J, Zeringue H, Beebe DJ, Rodriguez-Zas SL, Wheeler MB., "Embryonic development in the mouse is enhanced via microchannel culture.," Lab Chip., pp. 186-90, 2004 Jun.
    連結：
27. [42] W. F. Xie Y, Zhong W, Puscheck E, Shen H, Rappolee DA., "Shear stress induces preimplantation embryo death that is delayed by the zona pellucida and associated with stress-activated protein kinase-mediated apoptosis.," Biol Reprod., pp. 45-55, 2006 Jul.
    連結：
28. [43] H. N. Matsuura K, Kuroda Y, Takiue C, Hirata R, Takenami M, Aoi Y, Yoshioka N, Habara T, Mukaida T, Naruse K., "Improved development of mouse and human embryos using a tilting embryo culture system," Reprod Biomed Online, pp. 358-64., 2010 Mar.
    連結：
29. [45] Y. S. Heo, L.M. Cabrera, C.L. Bormann, C.T. Shah, S. Takayama, and G.D. Smith, "Dynamic microfunnel culture enhances mouse embryo development and pregnancy rates," Hum Reprod. , 2010.
    連結：
30. [46] R. A. Alegretti J, Barros B, Serafini P, Motta E, Smith G., "Microfluidic dynamic embryo culture increases the production of top quality human embryos through reduction in embryo fragmentation.," Fertil. Steril., pp. P. S58-S59., 2011. 96(3).
    連結：
31. [47] B. C. Kim MS, Wee G, Han YM, Park JK., "A microfluidic in vitro cultivation system for mechanical stimulation of bovine embryos.," Electrophoresis., pp. 3276-82., 2009 Sep.
    連結：
32. [48] M. S. K. Chae Yun Bae, Je-Kyun Park, "Mechanical stimulation of bovine embryos in a microfluidic culture platform.," BioChip, pp. pp 106-113, 20 Jun 2011.
    連結：
33. [50] M. R. Isachenko V, 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.," Reprod Biomed Online, pp. 536-44., 2011 Jun.
    連結：
34. [51] S. O. J. Mizuno, Y. Sakai, T. Fujii, H. Nakamura, H. Inui, "Human ART on chip: improved human blastocyst development and quality with IVF-chip.," Fertility and Sterility, September 2007.
    連結：
35. [52] N. H. Kimura H, Akai T, Yamamoto T, Hattori H, Sakai Y, Fujii T., "On-chip single embryo coculture with microporous-membrane-supported endometrial cells.," IEEE Trans Nanobioscience, pp. 318-24., 2009 Dec.
    連結：
36. [53] G.-T. L. Wei-Xuan LI, Wei YAN, Qiong ZHANG, Wei WANG, Xiao-Mian ZHOU, Da-Yu LIU, "Artificial Uterus on a Microfluidic Chip.," Chinese Journal of Analytical Chemistry, pp. Pages 467–472, April 2013.
    連結：
37. [54] W. M. Krisher RL, "Towards the use of microfluidics for individual embryo culture.," Reprod Fertil Dev., pp. 32-9, 2010.
    連結：
38. [55] P. T. Vajta G, Holm P, Páldi A, Greve T, Trounson AO, Callesen H., "New method for culture of zona-included or zona-free embryos: the Well of the Well (WOW) system.," Mol Reprod Dev., pp. 256-64., 2000 Mar.
    連結：
39. [56] A. T. Sugimura S, Somfai T, Hirayama M, Aikawa Y, Ohtake M, Hattori H, Kobayashi S, Hashiyada Y, Konishi K, Imai K., "Time-lapse cinematography-compatible polystyrene-based microwell culture system: a novel tool for tracking the development of individual bovine embryos.," Biol Reprod., pp. 970-8, 2010 Dec.
    連結：
40. [57] X. L. Ma R, Han C, Su K, Qiu T, Wang L, Huang G, Xing W, Qiao J, Wang J, Cheng J., "In vitro fertilization on a single-oocyte positioning system integrated with motile sperm selection and early embryo development.," Anal Chem. , pp. 2964-70, 2011 Apr 15.
    連結：
41. [58] L. L. D Carlo D, "Dynamic single-cell analysis for quantitative biology.," Anal Chem., pp. 7918-25., 2006 Dec.
    連結：
42. [59] T. S. Tan W, "A trap-and-release integrated microfluidic system for dynamic microarray applications.," Proceedings of the National Academy of Sciences, pp. 1146–1151, 2007.
    連結：
43. References
44. [1] CDC, "Preventing Smoking and Exposure to Secondhand Smoke Before, During, and After Pregnancy.," factsheet, July 2007.
45. [2] "Infertility definitions and terminology," World heath organization, Retrieved Aug. 21, 2014.
46. [3] "Infertility," World heath organization, Retrieved Aug. 21, 2014, from
47. [8] "How Age Plays A Role In Your Fertility," Shandy Grove Fertility Center, Retrieved Aug. 22, 2014.
48. [9] "IVF Pregnancy Rates & Outcomes," Center for Human Reproduction, Retrieved Aug. 26, 2014.
49. [10] F. B. Evian Annual Reproduction (EVAR) Workshop Group 2010, Diedrich K, Bouchard P, Domínguez F, Matzuk M, Franks S, Hamamah S, Simón C, Devroey P, Ezcurra D, Howles CM., "Contemporary genetic technologies and female reproduction.," Human Reproduction Update, pp. 829-47, 2011 Nov-Dec.
50. [15] E. N. Trevor G. Cooper, Sigrid von Eckardstein, Jacques Auger, H.W. Gordon Baker, Hermann M. Behre, Trine B. Haugen, Thinus Kruger, Christina Wang, Michael T. Mbizvo and Kirsten M. Vogelsong,, "World Health Organization reference values for human semen characteristics. ," Human Reproduction Update, pp. 231–245, November 24, 2009.
51. [20] B. NEWS, "1978: First 'test tube baby' born."
52. [22] "2009 Clinic Summary Report," Society for Reproductive Medicine.
53. [23] "ICSI - What is It?," Baby Med, Retrieved Aug. 27, 2014.
54. [28] U. Drews, "Taschenatlas der Embryologie," 1993: Stuttgart New York Thieme.
55. [29] L. H. Barmat LI, 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.," J Assist Reprod Genet, pp. 121-7, 1999 Mar.
56. [36] K. S. D. Kolahi, Annemarie; Liu, Xiaowei; Lin, Wingka; Simbulan, Rhodel K.; Bloise, Enrrico; Maltepe, Emin; Rinaudo, Paolo, "Effect of substrate stiffness on early mouse embryo development.," PLoS ONE, July 2012.
57. [39] B. C. Rappolee DA, Patel Y, Werb Z., "Expression and function of FGF-4 in peri-implantation development in mouse embryos.," Development, pp. 2259-69, 1994 Aug.
58. [40] B. D. Hickman DL, Rodriguez-Zas SL, Wheeler MB., "Comparison of static and dynamic medium environments for culturing of pre-implantation mouse embryos.," Comp Med., pp. 122-6, 2002 Apr.
59. [44] L. M. Cabrera, Y.S. Heo, J. Ding, S. Takayama, and G.D. Smith, "Improved blastocyst development with microfluidics and Braille pin actuator enabled dynamic culture.," Fertil. Steril., p. P. S43, 2006. 86(3).
60. [49] M. R. Isachenko E, 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. ," Clin Lab. , pp. 569-76., 2010.