Title

電控微流體微影技術於生醫材料圖案化與三維細胞共養研究

Translated Titles

Electromicrofluidic Lithography for Biomaterials Patterning and Three Dimensional Cell Co-culture

DOI

10.6342/NTU.2015.01710

Authors

賴奕翰

Key Words

三維結構 ; 介電濕潤 ; 電微流微影 ; 水膠 ; 細胞共養 ; 3D scaffolds ; electrowetting ; electro-microfluidic lithography ; hydrogel ; co-culturing

PublicationName

臺灣大學機械工程學研究所學位論文

Volume or Term/Year and Month of Publication

2015年

Academic Degree Category

碩士

Advisor

范士岡

Content Language

繁體中文

Chinese Abstract

二維的細胞培養雖然已經相當成熟,然而卻與體內生存環境有許多差異。三維細胞培養可建立更接近體內的細胞生長環境,而三維單一種細胞微環境已藉由介電泳操控、模具成型、表面親疏水處理、針織技術、生物列印等技術實現。然而兩種以上的細胞同時形成三維複雜的結構,目前仍難以達成。本研究主要利用電控微流體微影(Electr-omicrofluidic lithography)技術完成建立三維微結構,使用介電濕潤現象為主要驅動液體的力量,藉由已設計好的電極,操控多種不同的生物材料、水膠、以及細胞/螢光顆粒,建立三維微結構。我們測試了不同濃度的PEGDA (聚乙二醇二丙烯酸酯)與GelMA (甲基丙烯酸甲酯接枝共聚物)等可交聯水膠溶液,並藉由理論及實驗結果討論在不同環境(空氣、矽油1 cst)中,操控與形成不同寬度 (100-400 m)、高度 (40-100 m)微結構所需要的操作電壓 (40-120 Vpp)。以適當條件(60-80 Vpp, 1 kHz) 同時驅動含有細胞/螢光顆粒的多種水膠溶液,並以UV將其交聯後形成三維微結構。含有細胞(Fibroblasts NIH-3T3與Hepatocytes HepG2)的水膠(5% GelMA與0.5%光起始劑),在電微流晶片上被拉伸成已設計好的電極形狀並固化,隨後進行細胞培養或共養7-9天,觀察其生長情形(遷移、增生、貼附)等生物特徵,並以螢光標記其骨架與細胞核,相較於傳統培養,細胞會三維生長以及對準水膠邊界,更趨近體內細胞生長環境。我們成功提出一個利用電控微流體建立微結構取代傳統微影的方法,可以同時在一

English Abstract

2D cell culturing has been well developed, which provides an in vitro environment for specific cells culture. Alternatively, 3D cell culture techniques, including lectricphoresis manipulation, template molding, surface hydrophobicity treatment, textile technology and bio-printing, provide an in-vivo like realistic environment. However, co-cultivating multiple cells in 3D scaffolds with complex structures is still challenging. This research proposed an electro-microfluidic lithography technique to establish 3D scaffolds. By using electrowetting on a dielectric (EWOD), droplets were driven and 3D structures were deformed by designed electrode patterns with precisely-controlled amount of bio-compatible hydrogels materials containing suspended cells/fluorescent particles. Furthermore, manipulating multiple pre-polymer hydrogel solutions and forming 3D scaffolds were investigated. Different hydrogel solutions, PEGDA (poly(ethylene glycol)diacrylate) and GelMA (gelatin methacrylated) were tested. We compared the theory with experimental results in different ambients (air, silicone oil 1 cst), with varied 3D structure width (100-400 m), height (40-100 m) and voltage (40-120 Vpp). With appropriate operating parameters (60-80 Vpp, 1 kHz), hydrogel microstructures containing cells/fluorescent particles were formed after UV curing. Bio-compatible hydrogel (GelMA 5%, photo initiator 0.5%) solutions containing cells (fibroblast NIH-3T3 and hepatocyte HepG2) were deformed and cured on a chip. Subsequently, cells were incubated for 7-9 days. Cells phenotyping functions (migrating, proliferating and spreading) and fluorescent bio-marking on actin and nucleus of cells were observed. Compared with convectional 2D cell culture, cells grew and aligned with the boundary of the 3D hydrogel structure. We proposed an electro-microfluidic lithography technique to simultaneously manipulate multiple pre-polymer solutions for constructing cell laden hydrogels and 3D cell co-culture.

Topic Category 工學院 > 機械工程學研究所
工程學 > 機械工程
Reference
  1. [4] M. G. Pollack, R. B. Fair, and A. D. Shenderov, “Electrowetting-based actuation of liquid droplets for microfluidic applications,” Applied Physics Letters, 2000, Volume 77, 11.
    連結:
  2. [6] S.-K. Fan, C. Hashi, and C.-J Kim, “Manipulation of multiple droplets on N×M grid by cross-reference EWOD driving scheme and pressure-contact packaging,” Micro Electro Mechanical Systems , Kyoto, Jan 19-23, 2002, pp. 694-697.
    連結:
  3. [8] C.-J. Kim, “Portable digital microfluidics platform with active but disposable Lab-On-Chip,” Micro Electro Mechanical Systems, Netherlands, Jan 25-29, 2004, pp. 355-358.
    連結:
  4. [10] S.-K. Cho, H. Moon and C.-J Kim, “Creating,Transporting,Cutting,and Merging Liquid Droplets by Electrowetting-Based Actuation for Digital Microfluidic Circuits,” Microelectromechanical Systems, 2003, Volume 12, pp. 70-80.
    連結:
  5. [11] R. Harrison., “Observations on the living developing nerve fiber,” Anatomical Record, 1907, Volume 1, pp. 116-128.
    連結:
  6. [12] N. Boucard, C. Viton, D. Agay, E. Man, T. Roger, Y. Chancerelle, A. Domard, “The use of physical hydrogels of chitosan for skin regeneration following third-degree burns,” Biomaterials, 2007, Volume 28, pp. 3479-3488.
    連結:
  7. [13] G. Chen, T. Sato, T. Ushida, N. Ochiai, T. Tateishi, “Tissue Engineering of Cartilage Using a Hybrid Scaffold of Synthetic Polymer and Collagen” Tissue Engineering, 2004, Volume 10, pp. 323-330.
    連結:
  8. [14] A. Atala, S. B. Bauer, S. Soker, J. J. Yoo, A. B. Retik, “Tissue-engineered autologous bladders for patients needing cystoplasty,” THE LANCET, 2006, Volume 367, pp. 1241-1246.
    連結:
  9. [15] J. Malda, T. J. Klein, Z. Upton, “The Roles of Hypoxia in the In Vitro Engineering of Tissues,” 2007, Volume 13, pp. 2153-2162.
    連結:
  10. [16] J. Rouwkema, N. C. Rivron, C. A. Bitterswjk, “Vascularization in tissue engineering”, Trends in Biotechnology, 2008, Volume 26, pp. 434-441.
    連結:
  11. [17] K. Nishida, M. Yamato, K. Watanabe, K. Yamamoto, E. Adachi, S. Nagai, A. Kikuchi, N. Maeda, H. Watanabe, T. Okano, Y. Tano, N. Engl, “Corneal Reconstruction with Tissue-Engineered Cell Sheets Composed of Autologous Oral Mucosal Epithelium,” The NEW ENGLAND JOURNAL of MEDICINE, 2004, Volume 16, pp. 1187-1196.
    連結:
  12. [18] F. T. Moutos, L. E. Freed, F. Guilak, “A biomimetic three-dimensional woven composite scaffold for functional tissue engineering of cartilage,” Nature Materials, 2007, Volume 6, pp. 162-167.
    連結:
  13. [19] N. Engl, “Tissue-Engineered Blood Vessel for Adult Arterial Revascularization,” The NEW ENGLAND JOURNAL of MEDICINE, 2007, Volume 357, pp. 1451-1453.
    連結:
  14. [20] S. N. Bhatia, U. J. Balis, M. L. Yarmush, and M. Toner, “Probing heterotypic cell interactions: Hepatocyte function in microfabricated co-cultures,” Biomaterials Science, 1998, Volume 9, pp. 1137-1160.
    連結:
  15. [21] T. Matsue, N. Mastsumoto, and I. Uchida, “Rapid micropatterning of living cells by repulsive dielectrophoretic force,” Electrochimica, 1997, Volume 42, pp. 3251-3256.
    連結:
  16. [22] C. T. Ho, R. Z. Lin, W. Y. Chang, H. Y. Chang, and C. H. Liu, “Rapid heterogeneous liver-cell on-chip patterning via enhanced field-induced dielectrophoresis trap,” Lab On Chip, 2006, Volume 6, pp. 724-734.
    連結:
  17. [24] A. Khademohosseini, and R. Langer, “Microengineered hydrogels for tissue engineering,” Biomaterial, 2007, Volume 28, pp. 8087-8092.
    連結:
  18. [25] M. Nikkhah, N. Eshak, P. Zorlutuna, N. Annabi, M. Castello, K. Kim, A. D. Pirouz, F. Edalat, H. Bae, Y. Yang, and A. Khademohosseini, “Directed endothelial cell morphogenesis in micropatterened gelatin methacrylate hydrogel,” Biomaterial, 2015, Volume 33, pp. 9009-9018.
    連結:
  19. [27] A. Tourovskaia, X. F. Masot, and A. Folch, “Differentiation-on-a-chip: A microfluidic platform for long-term cell culture studies,” Lab on Chip, 2005, Volume 5, pp. 14-19.
    連結:
  20. [29] C. Norotte, F. S. Marga, L. E. Niklason, G. Forgacs, “Scaffold-free vascular tissue engineering using bioprinting,” Biomaterial, 2009, Volume 30, pp. 5910-5917.
    連結:
  21. [30] X. Cui, K. Breitenkamp, M. G. Finn, M. Lotz, D. D. Dlima, “Direct Human Cartilage Repair Using Three-Dimensional Bioprinting Technology,” Tissue Engineering, 2012, Volume 18, pp. 1304-1312.
    連結:
  22. [31] N. G. Durmus, S. Tasoglu, U. Demirci, “Functional droplet networks,” Nature Materials, 2013, Volume 12, pp. 478-479.
    連結:
  23. [32] G. Piret, E. Galopin, Y. Coffinier, R. Boukherroub, D. Legrand, C. Slomianny, “Culture of mammalian cells on patterned superhydrophilic/superhydrophobic silicon nanowire arrays,” Soft Matter, 2011, Volume7, pp. 8642-8649.
    連結:
  24. [33] X. Cui, K. Breitenkamp, M. G. Finn, M. Lotz,, D. D. D’Lima, “Direct Human Cartilage Repair Using Three-Dimensional Bioprinting Technology,” Tissue Engineering, 2012, Volume 18, pp. 1304-1312
    連結:
  25. [34] G. Y. Huang, L. H. Zhou, Q. C. Zhang, Y. M. Chen, W. Sun, F. Xu, T. J. Lu, “Microfluidic hydrogels for tissue engineering,” Biofabrication, Volume 3, No. 1
    連結:
  26. [36] M. Suzuki, T. Yasukawa, H. Shiku, and T. Matsue, “Negative dielectrophoretic patterning with different cell types,” Biosensors and Bioelectronics, 2008, Volume 24, pp. 1043-1047.
    連結:
  27. [38] A. Watanabe, “Investigations of some electric force effects in dielectric Liquid,” Japanese Journal of Applied Physics, 1973, Volume 12, pp. 593.
    連結:
  28. [39] V. Srinivasan, V.K. Pamula, and R.B. Fair, “An intergrated digital microfluidic lab-on-a-chip for clinical diagnostics on human physiological fluids,” Lab On Chip,2004, Volume 4, pp. 310-315.
    連結:
  29. [40] J. R. Melcher, “Dielectrophoretic Liquid Explusion,” Journal of Spacecraft and Rockets, 1969, Volume 6, No. 9.
    連結:
  30. [41] T. B. Jones, M. Gunji, M. Washizu, and M.J. Feldmanm, “Dielectrophoretic liquid actuation and nanodroplet formation,” Journal of Applied Physics., 2001, Voume 89, pp. 1441.
    連結:
  31. [42] J. A. Witkowski, “Experimental pathology and the origins of tissue culture: Leo Loeb's contribution,” Medical History, 1983, Volume 27, pp. 269-288.
    連結:
  32. [43] W. F. Pickard, “Experimental investigation of the Sumoto effect,” Journal of Applied Physics, 1961, Volume 32, pp. 1888-1893
    連結:
  33. [44] C. Zou, Z. Shen, “An optimized in vitro assay for screening compounds that stimulate liver cell glucose utilization with low cytotoxicity,” Journal of Pharmacologival and Toxicological Method, 2007, Volume 56, pp. 58-62.
    連結:
  34. [45] Y. Dong, P. Li, C. B. Chen, Z. H Wang, P. Ma, G. Q. Chen, “The improvement of fibroblast growth on hydrophobic biopolyesters by coating with polyhydroxyalkanoate granule binding protein Phap fused with cell adhesion motif RGD,” Biomaterials, 2010, Volume 31, pp. 8921-8930.
    連結:
  35. [46] I. Y . Y. Bu, S. P Oei, “Hydrophobic vertically aligned carbon nanotubes on Corning glass for self cleaning applications”, Applied Surface Science, 2010, Volume 256, pp. 6699-6704.
    連結:
  36. [47] H. Gu, M. H. F. Duits, F. Muqele, “A hybrid microfluidic chip with electrowetting functionality using ultraviolet (UV)-curable polymer”, LOC, 2010, Volume 10, pp. 1550-1556
    連結:
  37. [48] J. C. Love, L. A. Estroff, J. K Kriebel, R. G. Nuzzo, G. M. Whitesides, “Self-Assembled Monolayers of Thiolates on Metals as a Form of Nanotechnology”, Chem. Rev, 2005, Volume 105, pp. 1103-1169.
    連結:
  38. [49] D. Janssen, R. D. Palma, S. Verlaak, P. Heremans, W. Dehaen, “Static solvent contact angle measurements, surface free energy and wettability determination of various self-assembled monolayers on silicon dioxide”, Thin solid films, 2006, Volume 515, pp. 1433-1438.
    連結:
  39. [50] R. B. Fair, “Digital Microfluidics is a true lab-on-a-chip possible?,” Microfluidics and Nanofluidics, 2007, Volume 3, pp. 245-281.
    連結:
  40. [51] V. Srinivasan, V. K. Pamula, R. B. Fair, “An integrated digital microfluidic lab-on-a-chip for clinical diagnostics on human physiological fluids,” Lab on chip, 2004, Volume 4, pp. 310-315.
    連結:
  41. [52] M. -Y. Chiang, Y. -W. Hsu, H. -Y. Hsieh, S. -Y. Chen, S. -K. Fan, “Microengineered Heterogeneous Substrates for Cell Culture by Electro-Microfluidics,” International Conference on Miniaturized Systems for Chemistry and Life Sciences, Texas, Oct. 26-30, 2014, pp. 96-98.
    連結:
  42. [55] S. Kuiper, L. P. Lee, “Micromachined transmissive scanning confocal microscope,” Applied Physics Letters, 2004, Volume 85, pp. 1128-1130.
    連結:
  43. [56] J. W. Nichol, S. T. Koshy, H. Bae, C. M. Hwang, S. Yamanlar, A. khademhosseini, “Cell-laden microengineered gelatin methacrylate hydrogels,” Biomaterials, 2010, Volume 31, pp. 5536-5544.
    連結:
  44. [57] S. -K. Fan, P. -W. Huang, T. -T. Wang, Y. -H. Peng, “Cross-scale electric manipulations of cells and droplets by frequency-modulated dielectrophoresis and electrowetting,” Lab on chip, 2008, Volume 8, pp. 1325-1331.
    連結:
  45. [1] 中華民國器官捐贈協會,www.organ.org.tw.
  46. [2] J. Lee, “Microactuation by Continuous Electrowetting and Electrowetting: Theory,Fabrication,and Demonstration,” Ph.D. Thesis,University of California,Los Angeles, 2000.
  47. [3] H. J. Lee and C.-J. Kim, “Liquid Micromotor Driven by Continuous Electrowetting,” Proceeding IEEE Micro Electro Mechanical Systems, Heidelberg, Jan 25-28, 1998, pp. 538-543.
  48. [5] J. Gong and C.-J. Kim, “Two-Dimensional Digital Microfluidic System by Multi-Layer Printed Circuit Board,” Proceeding IEEE Conference Micro Electro Mechanical Systems, Fontainebleau Hilton Resort Miami Beach, Jan 30-Feb 3, 2005, pp. 726-729.
  49. [7] C.-J. Kim, “Integrated Digital Microfluidic Circuits Operated by Electrowetting-on-Dielectrics (EWOD) Principle,” granted in 2000 by Defense Advanced Research Projects Agency (DARPA), award number N66001-0130-3664.
  50. [9] M. G. Pollack, “Electrowetting-Based Microactuation of Droplets For Digital Microfluidics,” Ph.D. Thesis, Duke University, 2001.
  51. [23] H. Pellat, and C.R. Seances, Acad. Sci. (Paris), 1894, vol. 119, pp. 675.
  52. [26] D. Huh, G. A. Hamilton, and D. E. Ingber, “From 3D cell culture to organs-on-chips,” Cells paper, 2011, Volume 21, pp. 745-754.
  53. [28] L. E. Bertassoni, J. C. Cardoso, V. Manoharan, A. L. Cristino, N. S. Bhise, W. A. Araujo, P. Zorlutuna, N. E. Vrana, A.M. Ghaemmaghami, M. R. Dokmeci, and A. Khademhosseini, “Direct-write bioprinting of cell-laden methacrylated gelatin hydrogels,” Biofabrication, 2014, Volume 6, No. 2.
  54. [35] I. A. Eydelnant, B. B. Li, and A. R. Wheeler, “Microgels on-demand,” Nature Communications, 2014, Volume 5, No. 3355.
  55. [37] M. Akbari, A. Tamayol, V. Laforte, N. Annabi, A. Hassani Najafabadi, and A. Khademhosseini, D. Junker, “Composite Living Fibers for Creating Tissue Constructs Using Textile Techniques,” Advanced Functional Materials, 2014, Volume 24, pp. 4060-4067.
  56. [53] S. H. Au, M. D. Chamberlain, S. Mahesh, M. V. Sefton, A. R. Wheeler, “Hepatic organoids for microfluidic drug screening,” Lab on chip, 2014, Volume 14, pp. 3290-3299.
  57. [54] P. Y. Chiou, H. Moon, H. Toshiyoshi, C. J. Kim, M. C. Wu, “Light actuation of liquid by Optoelectrowetting,” Sensors & Actuators A-Physical, 2003, Volume 104, pp. 222-228.