In recent years, liver failure to cause death is always a critical issue. Particularly in Taiwan, the incidence of liver diseases is higher even more and has been ranked on top in the world. Actually, about the field of (hepatic) tissue engineering, owing to the shortage of liver organ source in surgical reconstruction medicine and the safety problem about infection and autoimmunity, the emphasis of this field has been gradually shifting from the conventional clinical therapies-organ transplants to the way we generate engineered tissue for study of human tissue physiology and pathophysiology in-vitro. Therefore, develop an in-vitro engineered liver tissue here to serve as an excellent model system for the study of liver-related issue, like liver regeneration, ischemia/reperfusion injury, fibrosis, microbial infection, and inflammation, is significant for prolonging patient's lives.
This study will present the design, microfabrication, and implementation of our dielectrophoretic (DEP)-based cell-patterning microfluidic platform, which includes triplicate analysis microfluidic channel design for approaching the requirements of experiment consistency and the biological statistics analysis in our chip, and the microchamber with microelectrodes for in-vitro engineered lobule-mimetic liver tissue reconstruction. The combination of MEMS technology and well-developed DEP-based cell-patterning enable the regeneration of complicated liver tissue to be easier and reproducible. In addition, to evaluate how the stress of DEP manipulation on cells, which is a key issue about this method, we performed FDA/EtBr assay to assess the cell viability and acquired the survival percentage of cell, after DEP operation, is 95 %. Moreover, from the drug test for metabolism studies, we also demonstrated the enhancement of hepatocyte activity, 30 % upgrading, by our engineering arrangement of lobule-mimetic liver tissue, which supports our biomimetic engineering concept about mimicking the cell-cell interactions and genetic architecture for preserving cell activity and the specific and complex functions of complicated tissue.
We anticipate this liver-on-chip engineering to be a starting point and direct for more sophisticated tissue regeneration and a platform for extracting the biology information from engineer tissues in the future.

近年來，肝病致死一直是個棘手的問題，特別在台灣，發生肝臟疾病的比例更是高，位居全球之冠。在肝臟組織工程的領域，過往由於在器官移植的進行上，肝臟來源長期不足和病毒感染、自體免疫排斥等問題，近來的發展重心漸漸朝向於體外的肝臟組織重建和研究，依此模式有助於我們對人體內的行為機制有更進一步的認識，也對於研究肝臟相關的疾病和反應以達到免疫、防治和延長病患的壽命的目標。
本研究，我們提出一新穎的微流體細胞排列晶片，透過微流道的設計，滿足適合生物實驗，多重複及統計分析的需求，同時藉由電極圖形的設計，利用介電泳效應，成功完成仿體內肝小葉(肝臟的基本單元)圖形的工程肝臟組織體外重建。透過整合微機電和介電泳細胞排列技術，我們將複雜的肝臟組織重建變得容易且具再現性。利用介電泳效應做細胞操控，最主要的關鍵在於操作過程中，細胞所受到的傷害程度，這裡我們利用FDA/EtBr試劑對細胞的存活率做評估，結論指出細胞在操控後的存活率依然相當的高，有95 %。另外，透過藥物實驗分析，我們進一步探討了不同條件下細胞的代謝活性，結果證實經過工程操控(仿建肝小葉)後的肝臟細胞，活性提升了30 %，這個結果支持了我們這樣的仿生工程，藉由仿建體內細胞環境來重現組織功能、建構器官的概念。
我們很期待這樣的肝組織晶片技術將來可幫助重現更多、更複雜的組織器官，同時也可用於探討更多生物相關議題的平台。

[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, pp. 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, pp. 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, pp. 183–186, (1997).
[4] L. G. Griffith, and G. Naughton, “Tissue engineering--current challenges and expanding opportunities,” Science. 295(5557), pp. 1009-14, (2002).
[5] L. G. Griffith, and M.A. Swartz, “Capturing complex 3D tissue physiology in vitro,” Nat Rev Mol Cell Biol. 7(3), pp. 211-24, (2006).
[6] Y. Nahmias, F. Berthiaume, and M.L. Yarmush, “Integration of technologies for hepatic tissue engineering,” Adv Biochem Eng Biotechnol. 103, pp. 309-29, (2007).
[7] C. T. Ho, R. Z. Lin, H. Y. Chang, and C. H. Liu, “In-vitro rapid centimeter-scale reconstruction of lobule-mimetic liver tissue employing dielectrophoresis based cell patterning,” in Digest Tech. Papers Transducers‘07 Conference, vol. 1, pp. 351-354, (2007).
[8] WTEC Panel Report on Tissue Engineering Research, International Technology Research Institute, Jan, (2002).
[9] S.J. Hollister, “Porous scaffold design for tissue engineering,” Nat. Mater., 4, pp. 518-524, (2005).
[10] J. L. Drury, and D. J. Mooney, “Hydrogels for tissue engineering: scaffold design variables and applications,” Biomaterials 24, pp. 4337-4351, (2003).
[11] R. Z. Lin, C. T. Ho, C. H. Liu, and H. Y. Chang, “Dielectrophoresis based-cell ptterning for tissue engineering,” Biotechnol. J. 1, pp. 949-957, (2006).
[12] K. M. Kulig, and J. P. Vacanti, “Hepatic tissue engineering,” Transpl Immunol,
12(3-4), pp. 303-10, (2004).
[13] A. Khademhosseini, R. Langer, J. Borenstein, and J. P. Vacanti, “Microscale
technologies for tissue engineering and biology,” Proc Natl Acad Sci U S A,
103(8), pp. 2480-7, (2006).
[14] H. Andersson, and A.van den Berg, “Microfabrication and microfluidics for
tissue engineering: state of the art and future opportunities,” Lab Chip, 4(2), pp. 98-103, (2004).
[15] A. Kikuchi, and T. Okano, “Nanostrutured designs of biomedical materials: applications of cell sheet engineering to functional regenerative tissues and organs,” J Control Release, 101(1-3), pp. 69-84, (2005).
[16] S.N. Bhatia, M. Yarmusch and M. Toner, “Controlling cell interactions by micropatterning in co-cultures:hepotocytes and 3T3 fibroblasts, “ J. Biomed. Mater. Res. 34, pp. 189-199, (1997).
[17] S.N. Bhatia, U.J. Balis, M.L. Yarmush and M. Toner, “Probing heterotypic cell interactions: hepatocyte function in microfabricated co-cultures,” J. Biomater. Sci. Polymer Ed. 9, pp. 1137-60, (1998).
[18] S.N. Bhatia, U.J. Balis, M.L. Yarmush and M. Toner, “Effect of cell–cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells,” FASEB, 13, pp. 1883-1900, (1999).
[19] J. El-Ali, P.K. Sorger and K.F. Jensen, Cells on chips, Nature, 442(27), pp. 403-411, (2006).
[20] K. Bhadriraju and C.S. Chen, “Engineering cellular microenvironments to improve cell-based drug testing,” Drug Discovery Today, 7(11), pp. 612-620, (2002).
[21] D. W. Huntmacher, “Scaffold design and fabrication technologies for engineering tissues-state of the art and future perspectives,” Journal of Biomaterials Science, Polymer Edition, 12(1), pp. 107-124, (2001).
[22] V. L. Tsang and S. N. Bhatia, “Three-dimensional tissue fabrication,” Advanced Drug Delivery Reviews, 56, pp. 1635-1647, (2004).
[23] J. Fukuda, A. Khademhosseini, J. Yeh, G. Eng et al., “Micropatterned cell
co-cultures using layer-by-layer deposition of extracellular matrix
components,” Biomaterials, 27(8), pp. 1479-86, (2006).
[24] D. T. Chiu, N. L. Jeon, S. Huang, R. S. Kane 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), pp. 2408-13, (2000).
[25] P. Matthieu et al., “Comparative study and improvement of current cell micro-patterning techniques,” Lab on a chip, 7, Apr. (2007).
[26] E. Delamarche, A. Bernard, H. Schmid, B. Michel and H. Biebuyck, “Patterned delivery of immunoglobulins to surfaces using microfluidic networks,” Science, 276(5313), pp. 779-781, (1997).
[27] A. Folch and M. Toner, “Cellular Micropatterns on Biocompatible Materials, Biotechnol. Prog., 14(3), pp. 388-392, (1998).
[28] A. Folch, A. Ayon, O. Hurtado, M. Schmidt and M. Toner, “Molding of deep poly(dimethylsiloxane) microstructures for microfluidics and biological applications,” J. Biomech. Eng., 121, pp. 28-34, (1999).
[29] S. Takayama, E. Ostuni, P. LeDuc, K. Naruse, D.E. Ingber and G.M. Whitesides, “Laminar flow: Subcellular positioning of small molecules,” Nature, 4, 11, (2001).
[30] M. Julia, Polak et al., ”Advances in Tissue Engineering,” , 2008-books.google.com
[31] M. Ozkan, T. Pisanic, J. Scheel, C. Barlow et al., “Electro-Optical Platform for the Manipulation of Live Cells,” Langmuir, 19(5), pp. 1532-1538, (2003).
[32] P. Y. Chiou, A. T. Ohta, and M. C. Wu, “Massively parallel manipulation of single cells and microparticles using optical images,” Nature, 436(7049), pp. 370-2, (2005).
[33] A. Ashkin, J. Dziedzic, M. and T. Yamane, “Optical trapping and manipulation of single cells using infrared laser beams,” Nature, 330(6150), pp. 769-71, (1987).
[34] D. G. Grier et al., “A revolution in optical manipulation,” Nature, 424(6950), pp. 810-6, (2003).
[35] Y. Nahmias, R. E. Schwartz, C. M. Verfaillie, and D. J. Odde, “Laser-guided direct writing for three-dimensional tissue engineering,” Biotechnology and Bioengineering, 92(2), pp. 129-136, (2005).
[36] H. Lee, A. M. Purdon, and R. M. Westervelta, “Manipulation of biological cells using a microelectromagnet matrix,” Applied Physics Letters, 85(6), pp. 1063-1065, (2004).
[37] T. Deng, and G. M. Whitesides, “Manipulation of mgnetic microbeads in
suspension using micromagnetic systems fabricated with soft lithography,”
Applied Physics Letters, 78(12), pp. 1775-1777, (2001).
[38] T. Matsue, N. Matsumoto and I. Uchida, “Rapid micropatterning of liver cells by repulsive dielectrophoretic force,” Electrochimica Acta, 42, pp. 3251-3256, (1997).
[39] M. Frenea, S. P. Faure, B. L. Pioufle, P. Coquet and H. Fujita, “Positoining living cells on a high-density electrode array by negtive dielectrophoresis,” Materials Science and Engineering Ｃ, 23, pp. 597-603, (2003).
[40] Z. Yu, G. Xiang, L. Pan, L. Huang, Z. Yu, W. Xing and J. Cheng, “Negative dielectrophoretic force assisted construction of ordered neuronal networks on cell positioning bioelectronic chips,” Biomedical Microdevices, 6(4), pp. 311-324, (2004).
[41] D. R. Albrecht, V. L. Tsang, R. L. Sah and S. N. Bhatia, “Photo- and electropatterning of hydrogel-encapsulated living cell array,” Lab on a chip, 5, pp. 111-118., (2005).
[42] P. Y. Chiou and M. C. Wu et al.,”Phototransistor-based optoelectronic tweezers for dynamic cell manipulation in cell culture media,” Lab on a chip, 10, Sep. (2010).
[43] R. Taub, “Liver regeneration: from myth to mechanism,” Nature Reviews Molecular Cell Biology 5, pp. 836-847, (2004).
[44] R. Langer, & J. P. Vacanti, “Tissue engineering,” Science, 260, pp. 920–926, (1993).
[45] J. R. Fuchs, B. A. Nasseri & J. P. Vacanti, “Tissue engineering: a 21st century solution to surgical reconstruction,” Ann. Thorac. Surg. 72, pp. 577-591, (2001).
[46] J. Tsiaoussis, P.N. Newsome, L.J. Nelson, P.C. Hayes & J.N. Plevris, “Which Hepatocyte Will it be ? Hepatocyte choice for bioartifical liver support systems,” Liver Tansplantation 7, pp. 2-10, (2001).
[47] D. Falconnet, G. Csucs, H. Michelle & M. Textor, “Surface engineering approaches to micropattern surfaces for cell-based assays,” Biomaterials, 27, pp. 3044-3063, (2006).
[48] K. Bhadriraju & C. S. Chen, “Engineering cellular microenvironments to improve cell-based drug testing,” Drug Discovery Today, 7, 11, pp. 612-620, (2002).
[49] K. Ali, R. Langer, Borenstein & J. P. Vacanti, “Microscale technologies for tissue engineering and biology,” Proc. Natl. Acad. Sci, 103, pp. 2480-2487, (2006).
[50] B. D. Foy, A. Rotem, M. Toner, R. G. Tompkins & M. L. Yarmush, “A device to measure the oxygen uptake rate of attached cells: importance in bioartificial organ design,” Cell Transplant. 3, pp. 515-527, (1994).
[51] G. I. Nedredal et al., “Liver sinusoidal endothelial cells represents an important blood clearance system in pigs,” Comp Hepatol. 2, 1, (2003).
[52] S.C. Strom, “Isolation of fetal hepatocytes for clinical transplantation,” Liver Transplant, 7: abstract C-16, (2001).
[53] N. Kobayashi, T. Okitsu and N. Tanaka, “Cell choice for bioartificial livers,” Keio J Med, 52(3), pp. 151-V157, September (2003).
[54] T. Hui, J. Rozga, A.A. Demetriou, “Bioartificial liver support,” J Hepatobiliary
Pancreat Surg, 8, pp. 1-15, (2001).
[55] S.M. Cascio, “Novel strategies for immortalization of human hepatocytes,” Artif. Organs 25, pp. 529-538, (2001).
[56] S. Wilkening, F. Stahl and A. Bader, “Comparison of primary human hepatocytes and hepatoma cell line HepG2 with regard to their biotransformation properties,” Drug Metab. Dispos. 31, pp. 1035-1042, (2003).
[57] N.B. RENA & BHANDARI et al.,”Liver Tissue Engineering: A role for co-culture systems in modifying hepatocyte function and viability,” Tissue Enguneering, 7, (2001).
[58] T. B. Jone, “Electromechanics of particles,” Cambridge University Press, 1995- books.google.com.
[59] M. P. Hughes, “Nanoelectromechanics in Engineering and Biology,” CRC Press, 2003-books.google.com.
[60] P. R. Gascoyne, and J. Vykoukal, “Particle separation by dielectrophoresis,” Electrophoresis, 23(13), pp. 1973-83, (2002).
[61] J. Voldman, M. L. Gray, M. Toner, and M. A. Schmidt, “A microfabrication-based dynamic array cytometer,” Anal Chem, 74(16), pp. 3984-90, (2002).
[62] R. S. Thomas et al., “Negative DEP traps for single cell immobilization,” Lab on c chip, 9, pp. 1534-1540, Mar. (2009).
[63] M. S. Jaeger et al.,”Contact-free single-cell cultivation by negative dielectrophoresis, “ Journal of physics D: Applied Physisc, 41, Aug. (2008).
[64] J. Voldman et al., “Electrical forces for microscale cell manipulation,” Annu. Rev. Biomed. Eng. 2006. 8:pp. 425-454, (2006).
[65] T. Heida, J.B. Wagenaar, W.L. Rutten, E. Marani, “Investigating membrane breakdown of neuronal cells exposed to nonuniform electric fields by finite-element modeling and experiments,” IEEE Trans. Biomed. Eng,
49(10), pp. 1195-1203, (2002).
[66] 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, pp. 53-68, (1999).
[67] C. Holzapfel, J. Vienken, and U. Zimmermann, “Rotation of cells in an alternating electric field: Theory and experimental proof,” J. Membr. Biol. 67, 13-V26, (1982).
[68] U. Zimmermann, U. Friedrich, H. Mussauer, P. Gessner, K. Hamel, and V. Sukhorukov, “Elecromanipulation of Mammalian Cells: Fundamentals and Application,” IEEE Transactions on Plasma Science, 28, NO. 1, (2000).
[69] P. Tabeling and S. Cheng, “Introduction to Microfluidics,” Oxford University Press, USA, 2006-books.google.com.
[70] F. M. White, “Viscous Fluid Flow,” McGraw-Hill Companies, Inc, Boston, 2006-books.google.com.
[71] P. Liesbesny, Arch. Phys. Ther., 19, pp. 736-740., (1939).
[72] A.A. Texeira-Pinto, L.L. Nejelski, J.L. Curtler and J. H. Heller, “The behavior of unicellular organisms in an electromagnetic field,” Exp. Cell. Res. 20, pp. 548-564, (1960).
[73] N. Kaplowitz, “Idiosyncratic drug hepatotoxicity,” Nat. Rev. Drug Discov. 4, pp. 489-499, (2005).
[74] Walter M.A. Westerink , Willem G.E.J. Schoonen, “Cytochrome P450 enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells,” Toxicology in vitro, 21, (2007).
[75] S.R. Khetani & S.N. Bhatia, “Microscale culture of human liver cells for drug development,” Nature Biotechnology, 26, 1, (2007).
[76] A.B. Okey, E.A. Roberts, P.A. Harper and M.S. Denison, “Induction of drug-metabolizing enzyme: mechanisms and consequences,” Clin. Biochem, 19, pp. 132-141, (1986).
[77] T. Matsushita, K. Nakano, Y. Nishikura, K. Higuchi, A. Kiyota and R. Ueoka, “Spheroid formation and functional restoration of human fetal hepatocytes on poly-L-amino acid-coated dishes after serial proliferation,” Cytotechnology, 42, pp. 57-66, (2003).
[78] J. J. Chen, G. S. Chen, N. 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, pp. 115-119, (2003).
[79] R.A. Lubet, R.W. Nims, R.T. Mayer, J.W. Cameron and L.M. Schechtman, “Measurement of cytochrome P-450 dependent dealkylation of alkoxyphenoxazones in hepatic S9s and hepatocyte homogenates: effects of dicumarol,” Mutat. Res, 142, pp. 127-131, (1985).
[80] A. Lizette Granberg et al.,”Cytochrome P450-dependent binding of 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P) in murine heart, lung, and liver endothelial cells,” Arch Toxicol, 74, (2000).
[81] L. Granberg, A. Ostergren, I. Brandt, and E. Brittebo, “CYP1A1 and CYP1B1 in blood-brain interfaces:CYP1A1-dependent bioactivation of 7,12-dimethylbenz(a) anthracene in endothelial cells,” Drug Metabolism and Disposition, 31, (2002).
[82] C.T. Ho, R.Z. Lin, H.Y. Chang and C.H. Liu, Micromachined T-switches for cell sorting applications, Lan on c a chip, 5, pp. 1248-1258, (2005).
[83] P. Roy & J. Washizu et al., “Effect of Flow on the Detoxification Function of Rat Hepatocytes in a Bioartificial Liver Reactor,” Cell Transplantation, 10, (2001).
[84] T. Torii et al., “Effect of Continuous Application of Shear Stress on Liver Tissue: Continuous Application of Appropriate Shear Stress Has Advantage in Protection of Liver Tissue,” Transplantation Proceedings, 37, (2005).
[85] Edmond W. K. Young et al., “Matrix-dependent adhesion of vascular and valvular endothelial cells in microfluidic channels,” Lab on a chip, 7, Sep. (2007).