臺灣博碩士論文加值系統

能將神經細胞培養在印有幾何圖案上的基材表面，這對於觀察神經細胞行為來說是很必須的。近期的研究，藉由微米壓印技術已能控制神經細胞位置及生長方向。然而目前現有的設備不是太貴就是不容易去裝卸。本研究目的在於設計出一個不昂貴的顯微鏡式微米壓印裝置，用來將圖案對準於基材上並引導神經細胞生長。首先，發展出製作母模和印章(由polydimethylsiloxane構成)的方法，然後印章去沾染一種細胞黏著因子(poly-D-lysine (PDL)),來定位出細胞吸引的區塊。接著，壓印過的基材浸泡在胎牛血清蛋白，以界定出細胞排斥區塊。為了將印章可以對準基材上的微電極，在此應用影像處理來調整印章的傾斜度以及對準。此外，將細胞株和皮質神經元培養在壓印過的基材，而基材上的壓印圖案是由一些已研究出指導方針且確定能養活細胞的。這些指導方針包括（1）神經細胞無法良好生長在小的細胞吸引區塊，且細胞吸引和細胞排斥的區塊比例不能小於0.0017；（2）圖案的線寬越寬，細胞的生長狀況越好；（3）不同細胞株和神經細胞對於細胞排斥區塊的反應也不同，例如：一般常用的胎牛血清蛋白對於纖維母細胞的排斥效力並不好。本研究所研發的新型顯微鏡式微米壓印裝置提供有效但不昂貴的方法來對準。未來研究可以遵循指導方針而有系統的發展出能養活細胞的微米壓印技術，生長在有圖案基材的細胞可以提供研究體外神經網路而應用於神經生理學的探討。

It is essential to culture neuronal cells on the geometric patterned substrate to observe the behavior of neuronal cells. Recent studies have made it possible to control the positioning and outgrowth of neuronal cells by using the microcontact printing (μCP) technique. However, currently available devices are either too expensive or not easy to dismantle. The aim of this study is to design an inexpensive microscopy-based μCP device for aligning the pattern to the substrate to guide neuronal growth. First, procedures for fabrication of the master mold and formation of polydimethylsiloxane (PDMS) stamp were developed. The stamp was inked with the solution of poly-D-lysine (PDL), a cell adhesion factor, to define the cytophilic region. Meanwhile, the stamped substrate was immersed to bovine serum albumin (BSA) for defining the cytophobic region. To align the stamp to microelectrodes in the substrate, image processing techniques were applied on-line to adjust the tilting of stamp and alignment of pattern. Moreover, cell lines and cortical neurons were cultured on the patterned substrates from which general guidelines for viable patterned culturing was investigated. These guidelines found include (1) neurons and cells cannot grow well in a small cytophilic region, e.g., in a cytophilic / cytophobic ratio less than 0.0017; (2) the larger the width of pattern line is, the better the cells can grow; (3) varied cell lines and neurons respond differently to cytophobic reagents, e.g., common use of BSA is proved not effective to repel fibroblasts. Our novel design of microscope-based μCP device provides an effective but inexpensive tool to precisely align the patterns. Further works are needed to follow the general guidelines for systematically developing viable μCP techniques for varied cells which would become a precious tool for studying in-vitro patterns of neuronal networks for neuroelectrophysiological studies.

Content
Abstract………………………………………………………...........…….....I
中文摘要…………………………………………………………................II
Acknowledage…………………………….................................................. III
Content..........................................................................................................IV
List of Tables…………………………………………..…………...............VI
List of Figures………………………..........................................................VII
Chapter 1 Introduction……………………………………………….............1
1.1 Applications of in-vitro cell culture……………….………….............1
1.2 Microcontact printing （μCP）…………………….………..............2
1.3 Key elements of μCP to culturing neuronal network……....................4
1.3.1 Elastic stamp…………………….................................................4
1.3.2 Inking methods…………………………………………..............6
1.3.3 Reagents for defining cytophilic and cytophobic area...........…...7
1.4 Alignment device for μCP………………………………………..…...8
1.5 The aims of this research………………...............................................9
Chapter 2 Materials and Methods……………………………………...........11
2.1 Soft lithography......................................……………………...............11
2.1.1 Master mold………………………………………....................11
2.1.2 PDMS stamp…………………………………………...............12
2.2 A novel design of μCP device…………………………..…….…….....12
2.3 Surface modification for stamps and substrates……….........…….…..13
2.4 Image approaches for tilting and alignment of μCP device................15
2.5 Procedures of microcontact printing…………...…………..................16
2.6 Cell culture………………………………………….........…...…..…..17
Chapter 3 Results…………………………………………………….............18
3.1 Fabrication of master molds and stamp..……………………..........….18
3.2 Hydrophilic surface modification for the substrates…..………...........20
3.3 Novel microcontact printing (μCP) device............………………........21
3.4 Correcting tilting with the aid of image processing..............................23
3.5 Alignment procedures via image processing.....................................…25
3.6 General guidelines for culturing cells on the patterned substrates........27
3.5.1 Adhesive area for different cells………..…..................................27
3.5.2 Time for coating cytophilic and cytophobic reagents..............…28
3.5.3 The efficiency of cytophobic solution…………..……...........…..30
3.5.4 Cells grow on the patterned substrates…………….…….............31
Chapter 4 Discussion ……………………………………..…………............33
4.1 Fabrication and evaluation of molds and stamps......................................33
4.2 Hydrophilic substrates...............................................................................33
4.3 A novel μCP system for patterning neuronal network...............................34
4.4 Image-aided for tilting correction and alignment processes.....................34
4.5 Guidelines for cell patterning....................................................................35
4.5.1 Selection of cytophilic and cytophobic solution...............................35
4.5.2 Ratio between cytophilic and cytophobic area..................................36
4.5.3 Pattern design for neurons.................................................................36
Chapter 5 Conclusion and Future Developments ……………………...........37
References……………………………………………………………...........38

[1] Y. Nam , J. Chang, D. Khatami, G.J. Brewer , B.C. Wheeler, “Patterning to enhance activity of cultured neuronal Networks”, IEE Proc.-Nanobiotechnol, 151 (2004), P109-115
[2] T. H. Park, M. L. Shuler, “Integration of Cell Culture and Microfabrication Technology”, Biotechnology Progress, 19 (2003), P243-253
[3] R. S. Kane, S. Takayama, E. Ostuni, D. E. Ingber, G.M. Whitesides, “Patterning proteins and cells using soft lithography” , Biomaterials, 20 (1999), P2363-2376
[4] Y. Xia, G.M. Whitesides, “SOFT LITHOGRAPHY”, Annual Review of Materials Science, 28 (1998), P153–84
[5] S. Nagaoka, K. Ashiba, H. Kawakami, “Interaction between biocomponents and surface modified f luorinated polyimide”, Materials Science and Engineering C, 20 (2002), P181–185
[6] J. C. Chang, G. J. Brewer, B. C. Wheeler, “A modified microstamping technique enhances polylysine transfer and neuronal cell patterning”, Biomaterials, 24 (2003), P2863–2870
[7] L.Griscom, P. Degenaar, B. LePioufle, E. Tamiya, H. Fujita, “Techniques for Patterning and Guidance of Primary Culture Neurons on Micro-electrode Array”, Sensors and Actuators B, 83 (2002), P15-21
[8] H. Sorribas, D. Braun, L. Leder, P. Sonderegger, L. Tiefenauer, “ Adhesion proteins for a tight neuron–electrode contact”, Journal of Neuroscience Methods, 104 (2001), P133–141
[9] P. Clark, S.Britland, P. Connolly, “Growth cone guidance and neuron morphology on micropatterned laminin surfaces”, Journal of Cell Science, 105 (1993), P203-212
[10] M. Mrksich, L. E. Dike, J. Tien, D. E. Ingber, G. M. Whitesides, “Using Microcontact Printing to Pattern the Attachment of Mammalian Cells to Self-Assembled Monolayers of Alkanethiolates on Transparent Films of Gold and Silver”, Experimental Cell Research, 235 (1997), P305–313
[11] B. Michel, A. Bernard, A. Bietsch, E. Delamarche, “Printing meets lithography: Soft approaches to high-resolution printing”, IBM Journal of Research and Development, 45 (2001), P697-719
[12] F. Turcu, K.T. Nitz, S. Thanos, W. Schuhmann, P. Heiduschka, “Ink-jet printing for micropattern generation of laminin for neuronal adhesion”, Journal of Neuroscience Methods , 131 (2003), P141–148
[13] G. Csucs, R. Michel, J.W. Lussi, M. Textor, G. Danuser, “Microcontact printing of novel co-polymers in combination with proteins for cell-biological applications”, Biomaterials, 24 (2003), P1713–1720
[14] C. Donzel, M. Geissler, A. Bernard, H. Wolf, B. Michel, J. Hilborn, E.Delamarche, “Hydrophilic Poly (dimethylsiloxane) Stamps for Microcontact Printing”, Advanced Materials ,13 (2001), P1164-1167
[15] Y. Nam, J.C. Chang, B.C. Wheeler, G. J. Brewer, “Gold-Coated Microelectrode Array With Thiol Linked Self-Assembled Monolayers for Engineering Neuronal Cultures ”, IEEE Transactions on Biomedical Engineering, 51 (2004), P158-165
[16] M. Mrksich, L. E. Dike, J. Tien, D. E. Ingber, and G. M. Whitesides, “Using Microcontact Printing to Pattern the Attachment of Mammalian Cells to Self-Assembled Monolayers of Alkanethiolates on Transparent Films of Gold and Silver”, Experimental Cell Research, 235 (1997), P305–313
[17] D. C. Trimbach, M. A.Hussein, W. H. Jeu, , M.Decre, D. J. Broer, C. W. M. Bastiaansen, “Hydrophilic Elastomers for Microcontact Printing of Polar Inks” , Langmuir, 20 (2004), P4738-4742
[18] K. Y. Suh, R.Langer, J. Lahann, “Fabrication of elastomeric stamps with polymer-reinforced sidewalls via chemically selective vapor deposition polymerization of poly (p-xylylene)”, Applied Physics Letters, 83 (2003), P 4250-4253
[19] T. Pompe, A. Fery, S. Herminghaus, A. Kriele, H. Lorenz, J. P. Kotthaus, “Submicron Contact Printing on Silicon Using Stamp Pads ”, Langmuir,15 (1999), P 2398-2401
[20] E. Pavlovic, A. P. Quist, L. Nyholm, A. Pallin, U. Gelius, S. Oscarsson , “Patterned Generation of Reactive Thiolsulfinates / Thiolsulfonates on Silicon Oxide by Electrooxidation Using Electromicrocontact Printing”, Langmuir, 19 (2003), P 10267-10270
[21] C. Y. Hui , A. Jagota , Y. Y. Lin , E. J. Kramer , “Constraints on Microcontact Printing Imposed by Stamp Deformation”, Langmuir , 18 (2002), P 1394-1407
[22] L.Lauer, S. Ingebrandt, M. Scholl, A. Offenhäusser, “Aligned Microcontact Printing of Biomolecules on Microelectronic Device Surfaces”, IEEE Transactions on Biomedical Engineering, 48 (2001), P 838-842
[23] C. D. James, R. Davis, M. Meyer, A. Turner, S. Turner, G. Withers, L. Kam, G. Banker, H. Craighead, M. Isaacson, J. Turner, W. Shain, “Aligned Microcontact Printing of Micrometer-Scale Poly-L-Lysine Structures for Controlled Growth of Cultured Neurons on Planar Microelectrode Arrays”, IEEE Transactions on Biomedical Engineering, 47 (2000), P17-21
[24] Y Xia, G. M. Whitesides, “Soft Lithography”, Angewandte chemie-international edition, 37(1998), p550-575.
[25] D. W. Branch, B. C. Wheeler, G. J. Brewer, and D. E. Leckband, “Long-Term Maintenance of Patterns of Hippocampal Pyramidal Cells on Substrates of Polyethylene Glycol and Microstamped Polylysine ”, IEEE Transactions on Biomedical Engineering, 47 (2000), p290-300
[26] R Singhvi , A. Kumar, G..M.Whitesides, D. E. Ingber, G. P. Lopez, D. I. Wang, G. N. Stephanopoulos, “Adhering cells to cytophilic islands separated by cytophobic regions to form patterns and manipulate cells”, Experimental Cell Reaearch , 260 (2000), P306-317
[27] J. D. Mendelsohn, S. Y. Yang, J.A. Hiller, A. I. Hochbaum, and M. F. Rubner, “Rational Design of Cytophilic and Cytophobic Polyelectrolyte Multilayer Thin Films”, Biomacromolecules, 4 (2003), P96-106
[28] J. M. Corey, B. C. Wheeler, and G. J. Brewer, “Micrometer resolution silane-based patterning of hippocampal neurons: Critical variables in photoresist and laser ablation processes for substrate fabrication,” IEEE Transactions on Biomedical Engineering, 43 (1996), P944-955
[29] L. Lauer, C. Klein, A. Offenhäusser, “Spot compliant neuronal networks by structure optimized micro-contact printing”, Biomaterial, 22 (2001), P1925-1932