隨著微機電產業的蓬勃發展，實驗室晶片技術已變成近期的熱門領域。由於微機電元件符合生物分子尺度，因此在過去數十年間，吸引了許多工程與生物研究者的興趣，投入許多心血與努力，促成跨領域的結合。人工肝臟的建立是近年來在組織工程上一個重要的趨勢，但是希望大量且快速的建立並仿造出肝小葉的圖形，仍有一定的難度
。此外，觀察肝臟細胞在不同濃度的藥物下，活性會如何的變化，也是一個被重視的議題。本論文希望重建肝小葉組織，並研究肝小葉結構在不同濃度藥物下的活性反應行為。
本研究為設計一實驗室晶片，透過晶片上電極的設計，藉由施予適當頻率的交流電壓，使細胞表面被極化成電偶極的基礎下，利用介電泳概念排列細胞，建立肝小葉的圖形。在此過程中，存活率是一個重要的關鍵，因此我們利用FDA/EtBr試劑來完成存活率實驗。在肝小葉結構排列完成後，再經由簡單的微流道設計來提升液體擴散效率，產生一個穩定且連續的藥物濃度梯度，最後流至四個生物反應器，產生不同濃度的藥物，並觀察肝臟細胞活性反應。以上所有功能皆以簡單的製程整合於一晶片上。

With the development of Micro-Electro-Mechanical Systems (MEMS) industry, the Lab-on-a-chip technology becomes a popular field in recent days. Because the
scale of micro-device matches with cellular molecule,
the interdisciplinary combinations of biological and engineering fields have been attracting a significant
attention in the past few decades. The establishment
of artificial liver is a trend in tissue engineering.
But it is still difficult to build and mimic a hepatic lobule rapidly. Besides, it is an important issue to observe the liver cells activity differences in different concentrations of drug. Rebuilding hepatic lobule and observing the actions of liver cells are the main targets in this thesis.
This research presents a Lab-on-a-chip device to achieve the cell-patterning function. Based on the
polarity difference within cells caused by applying
suitable frequency of the input voltage, we could use
the dielectrophoresis (DEP) to manipulate cells and
form the pattern of the hepatic lobule. During the patterning process, the viability of cells is a key
issue, so we use FDA/EtBr assay to accomplish the
viability tests. After the cell-patterning is finished, stable and continuous concentration gradient of drug is generated by simple microchannel design. Then, different concentrations of culture media are injected into four bioreactors so that the concentrations of culture media
are different in four bioreactors. Finally, we observe
the effect for liver cells by different concentrations
of culture media.

[1] R. C. Anderson, G. J. Bogdan, A. Puski, and X. Su,
“Genetic analysis systems:Improvements and methods,”
In Proc. Solid-State Sens. Actuator Workshop, Hilton
Head, SC, 7–10, 1998.
[2] C. Bisson, J. Campbell, R. Cheadle, M. Chomiak, J. Lee,
C. Miller, C. Milley, P. Pialis, S. Shaw,W. Weiss, and
C. Widrig, “A microanalytical device for the
assessment of coagulation parameters in whole blood,”
In Proc. Solid-State Sens. Actuator Workshop, Hilton
Head, SC, 1–6, 1998.
[3] N. Chiem, C. Colyer, and J. D. Harrison, “Microfluidic
systems for clinical diagnostics,” In Proc. Int. Solid-
State Sens. Actuators Conf., Chicago, IL, 183–186,
1997.
[4] Branebjerg J et al., “Fast mixing by lamination,”
MEMS’96, 9th IEEE Int. Workshop Micro
Electromechanical System (San Diego, CA), 441-6, 1996.
[5] Ali Asgar S Bhagat, Erik T K Peterson and Ian
Papautsky, “A passive planar micromixer with
obstructions for mixing at low Reynolds numbers,” J.
Micromech. Microeng, 17, 1017–1024, 2007.
[6] Park et al., “Rapid three-dimensional passive rotation
micromixer using the breakup process,” J. Micromech.
Microeng, 14, 6–14, 2004.
[7] Noo Li Jeon et al., “Generation of Solution and
Surface Gradients Using Microfluidic Systens,”
Langmuir, 16, 8311-8316, 2000.
[8] L. Zhu, Q. Zhang, H.H. Feng, S. Ang, F.S. Chau, W.T.
Liu, Lab Chip, 4, 337, 2004.
[9] H. Mohamed, L.D. McCurdy, D.H. Szarowski, S. Duva, J.N.
Turner, M. Caggana, IEEE Trans. Nanobiosci, 3, 251,
2004.
[10] J. Moorthy, D.J. Beebe, Lab Chip, 3, 62, 2003.
[11] L.R. Huang, E.C. Cox, R.H. Austin, J.C. Sturm,
Science, 304, 987, 2004.
[12] A. Khademhosseini, J. Yeh, S. Jon, G Eng, K.Y. Suh,
J.A. Burdick, R. Langer, Lab Chip, 4, 425, 2004.
[13] H. Tani, K. Maehana, T. Kamidate, Anal. Chem, 76,
6693, 2004.
[14] A. Revzin, R.G. Tompkins, M. Toner, Langmuir, 19,
9855, 2003.
[15] N. Chronis, L.P. Lee, J. Microelectromech S, 14, 857,
2005.
[16] J. Lahann, M. Balcells, H. Lu, T. Rondon, K.F. Jensen,
R. Lange, Anal. Chem, 75, 2117, 2003.
[17] B.J Kirby, A.R. Wheeler, R.N. Zare, J.A. Fruetel, T.J.
Shepodd, Lab Chip, 3, 5, 2003.
[18] J.D. Cox, M.S. Curry, S.K. Skirboll, P.L. Gourley,
D.Y. Sasaki, Biomaterials, 23, 929, 2002.
[19] C.J. Kim, A. P. Pisano, R. S. Muller, M. G. Lim,
“Polysilicon Microgripper,”Tech. Dig., IEEE Solid-
State Sensor and Actuator Workshop, 48-51, Jun. 1990.
[20] C. J. Kim, A. P. Pisano, R. S. Muller, M. G. Lim,
“Silicon-Processed Overhanging Microgripper,”
J. MEMS, 1(3), 31-36, Mar. 1992.
[21] Nikolas Chronis and Luke P. Lee, “POLYMER MEMS-BASED
MICROGRIPPER FOR SINGLE CELL MANIPULATION,” Maastricht
MEMS 2004 Technical Digest, 17-20, 2004.
[22] A. Ashkin, J. Dziedzic et al., “Observation of single-
beam gradient force optical trap for dielectric
particles,” Opt. Lett, 11, 288-290, 1986.
[23] A. Ashkin, “Optical trapping and manipulation of
neutral particles using lasers,” Proc Nat Acad Sci
USA, 94, 4853–4860, 1997.
[24] Ciprian Iliescu, Francis E.H. Taya, Guolin Xua and
Liming Yu, “Cell separation technique in
dilectrophoretic chip with bulk electrode,” Proc. of
SPIE, 6036, 60360E, 2006.
[25] C.C. Chen and S. Zappe, et al., “Microfluidic Switch
for Embryo and Cell Sorting,”Transducers, p.1.
2E65.P, 2003.
[26] Ruei-Zeng Lin, Chen-Ta Ho, Cheng-Hsien Liu,
and Hwan-You Chang, “Dielectrophoresis based-cell
patterning for tissue engineering,” Biotechnol. J,
1, 949–957, 2006.
[27] Langer, R., and Vacanti, J. P.,
“Tissue engineering,” Science, 260(5110), 920-6, 1993.
[28] Griffith, L. G., “Emerging design principles in
biomaterials and scaffolds for tissue engineering,”
Ann N Y Acad Sci, 961, 83-95, 2002.
[29] Kulig, K. M., and Vacanti, J. P., “Hepatic tissue
engineering,” Transpl Immunol, 12(3-4), 303-10, 2004.
[30] Brey, E. M., Uriel, S., Greisler, H. P., and McIntire,
L. V., “Therapeutic neovascularization: contributions
from bioengineering,” Tissue Eng, 11(3-4), 567-84,
2005.
[31] Kannan, R. Y., Salacinski, H. J., Sales, K.,
Butler, P., et al., “The roles of tissue
engineering and vascularisation in the development of
micro-vascular networks: a review,” Biomaterials,
26(14), 1857-75, 2005.
[32] Khademhosseini, A., Langer, R., Borenstein, J., and
Vacanti, J. P., “Microscale technologies for tissue
engineering and biology,” Proc Natl Acad Sci U S A,
103(8), 2480-7, 2006.
[33] Andersson, H., and van den Berg, A.,
“Microfabrication and microfluidics for tissue
engineering: state of the art and future
opportunities,” Lab Chip, 4(2), 98-103, 2004.
[34] Kikuchi, A., and Okano, T., “Nanostructured designs
of biomedical materials:applications of cell sheet
engineering to functional regenerative tissues and
organs,” J Control Release, 101(1-3), 69-84, 2005.
[35] Bhatia, S. N., Yarmush, M. L., and Toner, M.,
“Controlling cell interactions by
micropatterning in co-cultures: hepatocytes and 3T3
fibroblasts,” J Biomed Mater Res, 34(2), 189-99, 1997.
[36] Mrksich, M., Dike, L. E., Tien, J., Ingber, D. E.,
et al., “Using microcontact printing to pattern the
attachment of mammalian cells to self-assembled
monolayers of alkanethiolates on transparent films of
gold and silver,” Exp Cell Res, 235(2), 305-13, 1997.
[37] Fukuda, J., Khademhosseini, A., Yeh, J., Eng, G.,
et al., “Micropatterned cell co-cultures using layer- by-layer deposition of extracellular matrix
components,” Biomaterials, 27(8), 1479-86, 2006.
[38] Zhang, S., Yan, L., Altman, M., Lassle, M., et al.,
“Biological surface engineering: a simple system for
cell pattern formation,” Biomaterials, 20(13),
1213-20, 1999.
[39] Chiu, D. T., Jeon, N. L., Huang, S., Kane, R. S.,
et al., “Patterned deposition of cells and proteins
onto surfaces by using three-dimensional microfluidic
systems,” Proc Natl Acad Sci U S A, 97(6), 2408-13,
2000.
[40] Ozkan, M., Pisanic, T., Scheel, J., Barlow, C.,
et al., “Electro-Optical Platform for the
Manipulation of Live Cells,” Langmuir, 19(5),
1532-1538, 2003.
[41] Chiou, P. Y., Ohta, A. T., and Wu, M. C., “Massively
parallel manipulation of single cells and
microparticles using optical images,” Nature,
436(7049), 370-2, 2005.
[42] Ashkin, A., Dziedzic, J. M., and Yamane, T., “Optical
trapping and manipulation of single cells using
infrared laser beams,” Nature, 330(6150), 769-71,
1987.
[43] Grier, D. G., “A revolution in optical
manipulation,” Nature, 424(6950), 810-6, 2003.
[44] Nahmias, Y., Schwartz, R. E., Verfaillie, C. M., and
Odde, D. J., “Laser-guided direct writing for three-
dimensional tissue engineering,” Biotechnol Bioeng,
92(2), 129-36, 2005.
[45] Lee, H., Purdon, A. M., and Westervelta, R. M.,
“Manipulation of biological cells using a
microelectromagnet matrix,” Applied Physics Letters,
85(6), 1063-1065, 2004.
[46] Yellen, B. B., and Friedman, G., “Programmable
assembly of colloidal particles using magnetic
microwell templates,” Langmuir, 20(7), 2553-9, 2004.
[47] Deng, T., and Whitesides, G. M., “Manipulation of
magnetic microbeads in suspension using micromagnetic
systems fabricated with soft lithography,”
Applied Physics Letters, 78(12), 1775-1777, 2001.
[48] Tanase, M., Felton, E. J., Gray, D. S., Hultgren, A.,
et al., “Assembly of multicellular constructs and
microarrays of cells using magnetic nanowires,”
Lab Chip, 5(6), 598-605, 2005.
[49] Gray, D. S., Tan, J. L., Voldman, J., and Chen, C. S.,
“Dielectrophoretic registration of living cells to a
microelectrode array,” Biosens Bioelectron,
19(12), 1765-74, 2004.
[50] Suzuki, M., Yasukawa, T., Mase, Y., Oyamatsu, D.,
et al., “Dielectrophoretic micropatterning with
microparticle monolayers covalently linked to glass
surfaces,” Langmuir, 20(25), 11005-11, 2004.
[51] Auerswald, J., Widmer, D., de Rooij, N. F., Sigrist,
A., et al., “Fast immobilization of probe beads by
dielectrophoresis-controlled adhesion in a
versatile microfluidic platform for affinity assay,”
Electrophoresis, 26(19), 3697-705, 2005.
[52] Davis MW, Vacanti JP., “Toward development of an
implantable tissue-engineered liver,” Biomaterials,
17(3), 365 –372, 1996.
[53] Anje A. te Velde AA, Ladiges NCJJ, Flendrig LM,
Chamuleau RAFM, “Functional activity of isolated pig
hepatocytes attached to different extracellular matrix
substances. Implication for application of pig
hepatocytes in a bioartificial liver,” J Hepatol,
23(2), 184 –192, 1995.
[54] Ho, C. T., Lin, R. Z., Chang, W. Y., Chang, H. Y.,
et al., “Rapid heterogeneous liver-cell on-chip
patterning via the enhanced field-induced
dielectrophoresis trap,” Lab Chip, 6(6), 724-34, 2006.
[55] Strom SC: Isolation of fetal hepatocytes for clinical
transplantation. Liver Transplant, 7: abstract C-16,
2001.
[56] Naoya Kobayashi, Teru Okitsu and Noriaki Tanaka,
“Cell choice for bioartificial livers,” Keio J Med,
52(3), 151–157, September 2003.
[57] Hui T, Rozga J, Demetriou AA., “Bioartificial liver
support,” J Hepatobiliary Pancreat Surg, 8, 1–15,
2001.
[58] Hirose M, Yamato M, and Kwon OH et al., “Temperature-
response surface for novel co-culture systems of
hepatocytes with endothelial cells: 2-D patterned
and double layered co-cultures,” Yonsei Med J, 41(6),
803 –813, 2000.
[59] Eric Leclerc, Yasuyuki Sakai, Teruo Fujii,
“Microfluidic PDMS (Polydimethylsiloxane) Bioreactor
for Large-Scale Culture of Hepatocytes,”
Biotechnol. Prog, 20, 750-755, 2004.
[60] Paul J. Hung, Philip J. Lee, Poorya Sabounchi, Nima
Aghdam, Robert Lin and Luke P. Lee, “A novel high
aspect ratio microfluidic design to provide a stable
and uniform microenvironment for cell growth in a high
throughput mammalian cell culture array,” Lab Chip,
5, 44–48, 2005.
[61] Heida, T., Wagenaar, J.B., Rutten, W.L., Marani, E.,
“Investigating membrane breakdown of neuronal cells
exposed to nonuniform electric fields by finite-
element modeling and experiments,” IEEE Trans.
Biomed. Eng, 49(10), 1195–1203, 2002.
[62] Xujing Wang, Jun Yang, Peter R.C. Gascoyne, “Role of
peroxide in AC electrical field exposure effects on
Friend murine erythroleukemia cells during
dielectrophoretic manipulations,” Biochimica et
Biophysica Acta, 1426, 53-68, 1999.
[63] C. Holzapfel, J. Vienken, and U. Zimmermann,
“Rotation of cells in an alternating electric field:
Theory and experimental proof,” J. Membr. Biol., 67,
13–26, 1982.
[64] Ulrich Zimmermann, Uwe Friedrich, Heiko Mussauer,
Petra Gessner, Katja Hämel, and Vladimir Sukhorukov,
“Electromanipulation of Mammalian Cells:
Fundamentals and Application,” IEEE TRANSACTIONS ON
PLASMA SCIENCE, 28, NO. 1, 2000.
[65] U. Zimmermann and G. A. Neil, “The effect of high
intensity electric field pulses on eukaryotic cell
membranes: Fundamentals and applications, in
Electromanipulation of Cells,” Eds. Boca Raton, FL:
CRC, 1996.
[66] Kuo-Chuan Huang and Cheng-Hsien Liu, “Passive
gradient generator and cell manipulation integrated
with dielectrophoresis for chemotaxis Study,”
Master Thesis, Department of PME, NTHU, 2008.
[67] Tohru Sawanobori, Hideo Takanashi, Masayasu Hiraoka,
Yoshitaka Iida, Kazuaki Kamisaka, Hidenori Maezawa,
“Electrophysiological properties of isolated rat liver
cells,” Journal of Cellular Physiology, 139(3),
580-585, 1989.
[68] J Graf and O H Petersen, “Cell membrane potential and
resistance in liver,” J. Physiol, 284, 105-126, 1978.
[69] Okey A.B., Roberts E.A., Harper P.A. and Denison M.S.,
“Induction of drug-metabolizing enzyme: mechanisms and
consequences,” Clin. Biochem, 19, 132–141, 1986.
[70] Taku Matsushita, Kohji Nakano, Yasufumi Nishikura,
Kenta Higuchi, Akifumi Kiyota and Ryuichi Ueoka,
“Spheroid formation and functional restoration of
human fetal hepatocytes on poly-L-amino acid-coated
dishes after serial proliferation,” Cytotechnology,
42, 57–66, 2003.
[71] Jin Jun Chen, Guo Sheng Chen, Nigel J. Bunce,
“Inhibition of CYP 1A2–Dependent MROD Activity in Rat
Liver Microsomes: An Explanation of the Hepatic
Sequestration of a Limited Subset of Halogenated
Aromatic Hydrocarbons,” Wiley Periodicals, Inc.
Environ Toxicol, 18, 115–119, 2003.
[72] Lubet R.A., Nims R.W., Mayer R.T., Cameron J.W. and
Schechtman L.M., “Measurement of cytochrome P-450
dependent dealkylation of alkoxyphenoxazones in
hepatic S9s and hepatocyte homogenates: effects of
dicumarol,” Mutat. Res, 142, 127–131, 1985.
[73] Cretton E.M. and Sommadossi J.P., “Modulation of
3’-Azido-3’-deoxythymidine catabolism by probenecid
and acetaminophen in freshly isolated rat
hepatocytes,” Biochem. Pharmacol, 42, 1475–1480,
1991.
[74] Chen-Ta Ho, Ruei-Zeng Lin, Hwan-You Chang, and Cheng-
Hsien Liu, “In-vitro rapid centimeter-scale
reconstruction of lobule-mimetic liver tissue
employing dielectrophoresis-based cell patterning,”
The 14th International Conference on Solid-State
Sensors, Actuators and Microsystems, 2007.