Abstract

In this thesis, an extracorporeal liver tissue vitalizing system is reported. To investigate and test metabolism and accumulation of toxic chemicals in liver tissues, long-term in-vitro culture is very much needed. Presently tissue slices can only receive nutrients on the surface, and tissue degradation is resulted because the core is deficient of nutrients. Liver tissues cannot survive over 3 to 5 days by using conventional static culture method. Conventional static culture method without fresh culture medium supplement does not provide an appropriate environment for culturing liver tissues in-vitro. Therefore, a brand new system must be developed. The extracorporeal liver tissue vitalizing system with a bioreactor is demonstrated. Well-design nozzle-shaped wells in bioreactor are employed. These nozzle-shaped structures bring out extremely high efficiency of nutrient supplements. Liver tissues express better architecture, morphology, and viability by using this on-chip hydrodynamic culture system. A 9-day culture experiment is demonstrated in this research, and there is a great result of longer culture period which can provide more time for doctors to develop cure strategies for each individual patient. For preliminary research, liver tissues of transgenic mice are used in this research. Livers of these transgenic mice secrete HBsAg antigen. By examining the expression of HBsAg antigen, basic liver function remains during culture period. In addition, a Polydimethylsiloxane (PDMS) layer which is cultured with mesothelial cells is employed. Mesothelial cells have been presumed that can secrete some special proteins which are good for liver tissues. To study pathology of liver tissues, we culture liver tissues by using the extracorporeal liver tissue vitalizing system. This hydrodynamic system contains a syringe pump, silicone tubes, and the bioreactor. The syringe pump is set at the optimized flow velocity which can provide enough perfusion pressure without generate destructive shear stress on liver tissue and mesothelial cells. In this research, we develop a system which can prolong lifetime of liver tissues in-vitro. Therefore, patients who are suffered from liver diseases could have an opportunity to find treatments in a short time.

Key words: liver tissue; in-vitro culture; transgenic; nozzle; hydrodynamic
摘要
近年來，生醫工程技術在臨床醫學中扮演重要的角色，目的為突破既有醫學診斷、檢驗上的限制，因此，許許多多如生醫晶片(Biochip)、生物反應器(Bioreactor)等微型化技術相繼誕生，大幅提升了醫療服務的效率與準確性。然而，在肝臟醫學領域，傳統組織體外培養法已達極限，諸如肝臟病理、新陳代謝、B型肝炎、C型肝炎等研究，須仰賴更先進的技術以延長肝臟組織在體外的生存時間。因此，本文提出一體外肝臟組織維生系統，目的為提高肝臟組織於體外長時間培養的生存率，進而達到延長肝臟組織在體外的生存時間，醫生們得以獲得更多寶貴的時間針對不同病人來研擬治療的策略與方針。此系統包含一具有特殊噴嘴型幾何結構的生物反應器，具有高效率將養分帶入肝臟組織，並將代謝廢物攜出的特色。另外，將人類間皮細胞(Mesothelial Cell)培養於一特殊高分子材料聚二甲基矽氧烷(PDMS)，肝臟組織與間皮細胞直接接觸，兩者的交互作用將有利於肝臟組織於體外的培養。本實驗使用基因轉殖鼠(Transgenic Mice)之活體肝臟組織，該組織能生產B型肝炎表面抗原，文中描述肝臟組織於九天期體外培養實驗的過程與檢驗結果，檢驗其B型肝炎表面抗原的表現量、組織化學染色切片(H&E Stain)以及細胞凋亡檢測(TUNEL Assay)。結果顯示，透過此生物反應器與流體動力學搭配之體外肝臟組織維生系統，肝臟組織表現出較高生存率。然而，使用傳統靜態培養法的組織，在組織結構、細胞形態以及最重要之生存率方面，表現均不如使用體外肝臟組織維生系統。全世界每年有極多的B型肝炎、C型肝炎病患，每一位病患的病徵也不盡相同，如何針對單一病患提供適切的治療方式，是未來重要的目標之一。因此，體外肝臟組織維生系統提供一獨立的操作平台，肝臟組織在此平台下的存活時間大幅超越當前傳統的組織培養方法，為未來藥物測試及病理相關研究創造出更多的可能性。

關鍵字: 肝臟組織; 體外培養; 基因轉殖; 噴嘴;流體動力學

Reference
[1] Linda G. Griffith, Gail Naughton, “Tissue Engineering—Current Challenges and Expanding Opportunities,” Science, vol. 295, 8 February, 2002
[2] 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,” Transducers & Eurosensors, June, 2007
[3] Chen-Ta Ho, Ruei-Zeng Lin, Hwan-You Chang, and Cheng-Hsien Liu, “Rapid On-Chip Heterogeneous Bone-Cell Patterning via the Enhanced Dielectrophoresis Trap for Tissue Engineering Application,” μTAS, November, 2006
[4] Lin, R.Z., Ho, C.T., Liu, C.H. and Chang, H.Y., “Dielectrophoresis based-cell patterning for tissue engineering,” Biotechnol. J, 1, 949–957, 2006.
[5] Ho, C.T. and Lin, R.Z., Chang, H.Y. and Liu, C.H., “Micromachined Electrochemical T-switches for cell sorting applications,” Lab Chip, 5(11), pp. 1248–1258, 2005.
[6] Chen-Ta Ho, Ruei-Zeng Lin, Wen-Yu Chang, Hwan-You Chang and Cheng-Hsien Liu, “Rapid heterogeneous liver-cell on-chip patterning via the enhanced
field-induced dielectrophoresis trap,” Lab Chip, 6, 724–734, 2006
[7] Mark J. Powers, Karel Domansky, Mohammad R. Kaazempur-Mofrad, Artemis Kalezi, Adam Capitano, Arpita Upadhyaya, Petra Kurzawski, Kathryn E. Wack, Donna Beer Stolz, Roger Kamm, Linda G. Griffith, “A Microfabricated Array Bioreactor for Perfused 3D Liver Culture,” Biotechnology and Bioengineering, vol 78, NO. 3, MAY, 2002
[8] Ivan Martin, R. Suetterlin, W. Baschong, M. Heberer, G. Vunjak-Novakovic, and L. E. Freed, “Enhanced Cartilage Tissue Engineering by Sequential Exposure of Chondrocytes to FGF-2 During 2D Expansion and BMP-2 During 3D Cultivation,” Journal of Cellular Biochemistry, 83, 121-128, 2001
[9] KARL SCHUMACHER, M.D., YUET-MEI KHONG, M.S., SHI CHANG, M.D., JUN NI, M.S., WANXIN SUN, Ph.D., and HANRY YU, Ph.D., “Perfusion Culture Improves the Maintenance of Cultured Liver Tissue Slices,” Tissue Engineering, Volume 13, Number 1, 2007
[10] KATRIN ZEILINGER, Dr.Vet.Med., GUDRUN HOLLAND, IGOR M. SAUER, Dr.Med., EKATERINA EFIMOVA, Dr.Med., DIMITRIOS KARDASSIS, NICOLE OBERMAYER, MARCUS LIU,1 PETER NEUHAUS, Prof.Dr.Med., and J.RG C. GERLACH, Prof.Dr.Med., Ph.D., “Time Course of Primary Liver Cell Reorganization in Three-Dimensional High-Density Bioreactors for Extracorporeal Liver Support: An Immunohistochemical and Ultrastructural Study,” Tissue Engineering, Volume 10, Number 7/8, 2004
[11] Song-En Gong, Rong-Jhe Chen, Chung-Kuang Chin, Wen-Chen Chu, Chen-Ta Ho,
Hwan-You Chang, and Cheng-Hsien Liu, “On-Chip Lobule-Mimetic Construction of Heterogeneous Cells and Co-Culture via a Logarthmical-Concentration Varying Bioreactor,” Transducers, Denver, CO, USA, June 21-25, 2009
[12] Daniel A. J. Bow, Jennifer L. Perry, David S. Miller, John B. Pritchard, and Kim L. R. Brouwer, “Localization of P-gp (Abcb1) and Mrp2 (Abcc2) in Freshly Isolated Rat Hepatocytes,” Drug Metabolism and Disposition, Vol. 36, No. 1, 2008
[13] http://www.redboxdirect.com/
[14] Peter Olinga, Kees Groen, Ingrid H. Hof, Ruben De Kanter, Henk J. Koster, Winfried R. Leeman, Alphons A.J.J.L. Rutten, Klaas Van Twillert, and Geny M.M. Groothuis, “Comparison of Five Incubation Systems for Rat Liver Slices Using Functional and Viability Parameters, ” Journal of Pharmacological and Toxicological Methods, 38, 59-69, 1997
[15] De Kanter R., Koster H. J., “Cryopreservation of Rat and Monkey Liver Slices,” Alternatives to laboratory animals Y., vol. 23, No. 5, pages 653-665, 1995
[16] Olinga P., Merema M. T., Meijer D. K. F., Slooff M. J. H., Groothuis G. M. M., “Human Liver Slices Express The Same Lidocaine Biotransformation Rate as Isolated Human Hepatocytes,” Netherlands Alternatives to Animal Experiments Platform., Bilthoven vol. 21, pp. 466-469, 1993,
[17] W. R. Leeman, I. A. van de Gevel, A. A. J. J. L. Rutten , “Cytotoxicity of Retinoic Acid, Menadione and Aflatoxin B, in Rat Liver Slices Using Netwell Inserts as a New Culture System, ” Toxic. in Vitro, Vol. 9, No. 3, p.2 91-298, 1995
[18] Azri S., Mata H. P., Reid L. L., Gandolfi A. J. and Brendel K., “Further Examination of The Selective Toxicity of CCI in Rat Liver Slices, ” Toxicology and Applied Pharmacology, 112, 81-86, 1992
[19] Azri-Meehan S., Mata H. P., Gandolfi A. J. and Brendel K., “The Hepatotoxicity of Chloroform in Precision-cut Rat Liver Slices,” Toxicology, 73, 239-250, 1992
[20] Beamand J. A., Price R. J., Cunninghame M. E. and Lake B. G., “Culture of Precision-cut Liver Slices: Effect of Some Peroxisome Proliferators,” Food and Chemical Toxicology, 31, 137-147, 1993
[21] Chan, H. M., Tabarrok R., Tamura Y. and Cherian M. G., “The Relative Importance of Glutathione and Metallothionein on Protection of Hepatotoxicity of Menadione in Rats,” Chemico-Biological Interactions, 84, 113-124, 1992
[22] Dogterom P., “Development of a Simple Incubation System for Metabolism Studies with Precision-cut Liver Slices,” Drug Metabolism and Disposition, 21, 699-704, 1993
[23] Lake B. G., Beamand J. A., Japenga A. C., Renwick A., Davies S. and Price R. J.
, “Induction of Cytochrome P-450-dependent Enzyme Activities in Cultured Rat Liver Slices,” Food and Chemical Toxicology, 31, 377-386, 1993
[24] P.F. Smith, A.J. Gandolfi, C.L. Krumdieck, C.W. Putnam, C.F. Zukoski III, W.M. Davis, K. Brendel, “Dynamic Organ Culture of Precision Liver Slices for in vitro Toxicology,” Life Sciences, Volume 36, Issue 14, Pages 1367-1375, 1985
[25] Yoshida, T., Arisaka, Y., Nakagawa, S., and Takahashi, H., “Rotation Culture with a Newly Developed Holder Enables Longterm Liver Slice Culture for Study of Lliver Fibrosis,” Hepatology Research, 28, 198, 2004
[26] Vickers, A.E.M., Saulnier, M., Cruz, E., Merema, M.T., Rose, K., Bentley, P., and Olinga, P., “Organ Slice Viability Extended for Pathway Characterization: an in vitro Model to Investigate Fibrosis,” Toxicol. Sci, 82, 534, 2004
[27] Dogterom, P.,“Development of a Simple Incubation System for Metabolism Studies with Precision-cut Liver Slices,” Drug Metab. Dispos, 21, 699, 1993
[28] Smith, P., Krack, G., McKee, R., Johnson, D., Gandolfi, A., Hruby, V., Krumdieck, C., Brendel, K.,“Maintenance of Adult Rat Liver Slices in Dynamic Organ Culture,” In Vitro Cell. Dev. Biol, 22, 706, 1986
[29] Yuet Mei Khong, Jing Zhang, Sibo Zhou, Christine Cheung, Kai Doberstein, Victor Samper, Hanry Yu, “Novel Intra-Tissue Perfusion System for Culturing Thick Liver Tissue,” Tissue Engineering, Volume 13, Number 9, 2007
[30] Bancroft, G.N., Sikavitsas, V.I., Mikos, A.G., “Design of a Flow Perfusion Bioreactor System for Bone Tissue-engineering Applications,” Tissue Engineering, 9, 549, 2003
[31] Singh, H., Teoh, S.H., Low, H.T., and Hutmacher, D.W., “Flow Modelling within a Scaffold under The Influence of Uniaxial and Bi-axial Bioreactor Rotation,” J. Biotechno, 119, 181, 2005
[32] Eiselt, P., Kim, B.S., Chacko, B., Isenberg, B., Peters, M.C., Greene, K.G., Roland, W.D., Loebsack, A.B., Burg, K.J.L., Culberson, C., Halberstadt, C.R., Holder, W.D., and Mooney, D.J., “Development of Technologies Aiding Large-tissue Engineering,” Biotechnol. Prog, 14, 134, 1998
[33] Yi-Chin Toh, Teck Chuan Lim, Dean Tai, Guangfa Xiao, Danny van Noort and Hanry Yu, “A microfluidic 3D hepatocyte chip for drug toxicity testing,” Lab Chip, 2026–2035, 9, 2009
[34] Lily Kim, Yi-Chin Toh, Joel Voldman and Hanry Yu, “AA practical guide to microfluidic perfusion culture of adherent mammalian cells,” Lab Chip, 7, 681-694, 2007
[35] Medicine & NewSpace (http://62mileclub.com/62mileblog/?p=510)
[36] Chang-Gung Memorial Hospital, Linkou, Taiwan
[37] Steven E. Mutsaers, “Cells in Focus The Mesothelial Cell,” The International Journal of Biochemistry & Cell Biology, 36, 9–16, 2004
[38] Hausmann, M. J., Rogachev, B., Weiler, M., Chaimovitz, C., Douvdevani, A., “Accessory Role of Human Peritoneal Mesothelial Cells in Antigen Presentation and T-cell Growth,” Kidney International, 57, 476–486, 2000
[39] Liberek, T., Topley, N., Luttmann, W., & Williams, J. D., “Adherence of Neutrophils to Human Peritoneal Mesothelial Cells: Role of Intercellular Adhesion Molecule-1,” Journal of American Society of Nephrology, 7, 208–217, 1996
[40] Bellingan, G. J., Xu, P., Cooksley, H., Cauldwell, H., Shock, A., Haslett, C., Bottoms, S., Mutsaers, S. E., & Laurent, G. J., “Adhesion Molecule-dependent Mechanisms Regulate the Rate of Macrophage Clearance During The Resolution of Peritoneal Inflammation,” Journal of Experimental Medicine, 196, 1515–1521, 2002
[41] Rennard, S. I., Jaurand, M. -C., Bignon, J., Kawanami, O., Ferrans, V. J., Davidson, J., & Crystal, R. G., “Role of Pleural Mesothelial Cells in The Production of The Submesothelial Connective Tissue Matrix of Lung,” American Reviews of Respiratory Diseases, 130, 267–274, 1984
[42] Scott L. Friedman, “Molecular Regulation of Hepatic Fibrosis, an Integrated Cellular Response to Tissue Injury,” The Journal of Biological Chemistry, Vol. 275, No. 4, Issue of January 28, 2247–2250, 2000
[43] Florian Winau, Christian Quack, Alexandre Darmoise and Stefan HE Kaufmann, “Starring stellate cells in liver immunology,” Current Opinion in Immunology, 20, 68–74, 2008
[44] Luigi Atzori, Giuseppe Poli, Andrea Perra, “Hepatic stellate cell: A star cell in the liver,” The International Journal of Biochemistry & Cell Biology, 41, 1639–1642, 2009
[45] http://www.twinsoft.com.tw/index.htm
[46] http://en.wikipedia.org/wiki/Hepatitis_B
[47] Statistics from http://www.asianliver.com/en/