Microfluidic Bioreactors for Long-Term Culture and Histopathological Studies of Transgenic Mice Liver Tissue

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研究生：	李聰 SHILPA SIVASHANKAR
論文名稱：	Microfluidic Bioreactors for Long-Term Culture and Histopathological Studies of Transgenic Mice Liver Tissue 肝組織長期培養之微流控生物反應器研發
指導教授：	劉承賢 Liu, Cheng-Hsien
口試委員:	盧向成 Lu, Shiang-Cheng 葉昭廷 Yeh, Chau-Ting 彭慧玲 Peng, Hwei-Lin 張晃猷 Chang, Hwan-You 劉承賢 Liu, Cheng-Hsien
學位類別：	博士 Doctor
系所名稱：	工學院 - 動力機械工程學系 Department of Power Mechanical Engineering
論文出版年：	2012
畢業學年度：	101
語文別：	英文
論文頁數：	154
中文關鍵詞：	肝組織、生物反應器、基因轉殖老鼠、蘇木精&曙紅
外文關鍵詞：	liver tissue, bioreactor, transgenic mice, hematoxylin &eosin
相關次數：	點閱：2 下載：0
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Nurturing thick liver tissue for long-term allowing regeneration within 3D environment with perfusion system to improve clinical predictivity of preclinical testing is described in this thesis. At first, we have tried to prolong the culture period of primary tissue sample >2mm that were well supported on 3D microfluidic architecture or scaffold thereby aiding tissue samples to meet high oxygen demands of liver tissue mimicking 3D microenvironment present in vivo. Secondly to promote regeneration in tissue section we have added growth factors like hepatocyte growth factor (HGF) and insulin-like growth factor (IGF) and were successful in culturing tissue that could survive for at least 16 days allowing one round of ex vivo regeneration.. In order to supply the tissues with in vivo microenvironment there is a need for pumping system, reservoirs; microfluidic structures/hydrogels with appropriate porosity to meet the oxygen demand. The bioreactor comprised of microstructures or hydrogels that were bio-compatible to support the tissue and minimize the shear stress that was caused due to medium perfusion at high flow rates to meet the oxygen demand. The bioreactor comprises of a pumping device, supporting device, breathers, and reservoirs to meet the essential requirements of the tissue sample. Initiation of hepatocyte proliferation
ii
was observed in the ex vivo liver tissues between the eighth and twelfth days,
which could be demonstrated by proliferating cell nuclear antigen (PCNA)
staining. Furthermore we tried to observe the decrease in viable cells after 16days
and found that cells were adopting extrinsic apoptotic mechanism for cell death
when stained with different antibodies for intrinsic and extrinsic apoptosis. Once
we found that there could be possibility of regeneration, we wanted to optimize the
growth factor concentration and test with a drug Sorafenib which would be
effective when combined with another drug or the growth factor. Therefore we
have utilized a multi layered device enabling two different functions
(encapsulation and gradient generation) on the same chip. Here we determined the
efficacy of combined Sorafenib and HGF treatment against hepatoma
tumorogenesis and progression using the transgenic mice model as a pilot study.
The long-term culture and re-growth of cells within the primary liver tissue
samples provided a promising model to study liver pathophysiology or to develop
an efficient, safer and effective drug filling the gap between in vivo and cell culture
models. The development of these in vitro cell and tissue culture models can
contribute substantially to the reduction, refinement, and possibly to the
replacement of animal experiments. The efficacy and tolerability of combined
Sorafenib and HGF may provide rationale to use in treatment of HCC models.

Abstract………………………………………………………...…………………..i
Acknowledgements................................................................................................. iii
Abbreviations ........................................................................................................... v
Contents .................................................................................................................. vi
List of Figures ......................................................................................................... xi
List of tables ............................................................................................................xv
Chapter 1: Introduction .......................................................................................... 1
1.1 Background ...................................................................................................... 1
1.2 Tissue engineering ........................................................................................... 1
1.3 Problems faced in liver tissue engineering ..................................................... 5
1.4 Tissue engineering techniques ..................................................................... 5
1.4.1 Static/dynamic liver tissue culture technique ......................................... 6
1.4.2 Perfusion liver tissue culture technique .................................................. 9
1.4.3 2D liver tissue constructs technique .....................................................11
1.4.4 3D liver tissue constructs technique .....................................................13
1.4.5 Scaffold for liver tissue engineering .....................................................16
1.4.6 Microscale technologies for liver tissue engineering ...........................19
1.5 Comprehensive analysis of bio-artificial livers (BAL’S) ..........................22
1.5.1 Tissue engineered constructs ................................................................23
1.5.2 Cell Seeded BAL Systems ....................................................................24
vii
1.5.3 Membrane incorporated micro-bioreactors ..........................................25
1.6 How Liver Biopsies are Evaluated? .................................................26
1.7 Introduction to bioreactor cultures ............................................................27
1.7.1 Cell seeded bioreactors .......................................................................28
1.7.2 Tissue inserted bioreactors ...................................................................29
1.7.3 Scaffold bioreactor ...............................................................................30
1.7.4 Engineered constructs ..........................................................................31
1.8 Current therapies for liver diseases ...........................................................32
1.9 Aims and scope of this thesis .....................................................................34
Chapter 2: Theory and Design ............................................................................36
2. 1 Liver structure ..........................................................................................36
2.1.2 The Hepatic Vascular System ..............................................................38
2.1.3 The Biliary System ................................................................................38
2.2 Mechanism involved: perfusion flow and microfluidic structures ..........42
2.3 Design of the microfluidic bioreactor ........................................................42
2.4 Working principle of the bioreactor ..........................................................43
2.5 Repair and regeneration ............................................................................45
2.6 Tissue proliferative activity ........................................................................47
2.7 Liver regeneration and apoptosis mechanism...........................................50
2.8 Study of histopathology with growth factors .............................................53
2.9 Sorafenib functions ....................................................................................58
viii
Chapter 3: Experimental Methods ..............................................................60
3.1 Preparation of PEG-DA structures ...........................................................60
3.2 Maskless lithography for fabrication of PEG-DA structures ...................61
3.3 Cell culture and medium ............................................................................62
3.4 Tissue preparation for perfusion culture ..................................................63
3.5 Surface modification for attachment of PEG-DA structures ...................64
3.6 Experimental setup for perfusion of liver tissues .....................................65
3.7 Histopathological investigation and apoptosis assessment of tissue ........65
3.7.1 HBsAg expression .................................................................................65
3.7.2 H&E assay .............................................................................................66
3.7.3 TUNEL assay ........................................................................................66
3.8 Construction of flow perfusion bioreactor ................................................66
3.9 Microfabrication of PDMS structures .......................................................67
3.10 Surface modification for enhancing the cell-to-substrate adhesion and establishing extracellular matrix .........................................................................68
3.11 Fabrication of the microfluidic chip .........................................................69
3.12 Medium preparation ...................................................................................70
3.13 Tissue Degradation Procedure ..................................................................71
Chapter 4: Experimental Results and Discussion...............................................73
ix
4.1 The parameter set for tissue culture ..........................................................73
4.2 Characterization of PEG-DA structures ...................................................74
4.2.1 Stability retention of PEG-DA microstructures ....................................74
4.2.2 Characterization of swelling behaviors ................................................75
4.3 Assessment of antigen expression in tissue ...............................................77
4.4 Histo-architecture assessment of cultured liver tissue .............................79
4.5 Viability assessment via quantifying apoptotic cells in liver tissue ..........82
4.6 Suitability of the bioreactor ........................................................................85
4.6.1 Liver tissue structure characterization by histology .......................87
4.6.2 Viability quantification assay using TUNEL ........................................89
4.6.3 Urea quantification assay ......................................................................92
4.6.4 PCNA staining assay .............................................................................93
4.7 Tissue culture allowing one round of ex vivo regeneration…………….94
4.8 Cell death mechanism involved .................................................................95
4.8.1. Intrinsic- mitochondrial mediated apoptosis ...........................................96
4.8.2. Extrinsic- activator mediated apoptosis ...................................................97
4.8.1.1. Staining for BCL complexes ..................................................... 99
4.8.1.2. Staining for P53: ................................................................... 100
4.8.2.1. Staining for FAS protein ........................................................ 102
4.8.2.2. Staining for FADD protein ..................................................... 103
4.9 Viability via trypan blue assay .................................................................106
4.9.1 Cytochrome p450 1A1 subfamily enzyme activity ............................107
x
4.9.2 LDH cytotoxicity assay .......................................................................108
4.9.3 Urea secreted by liver tissue ...............................................................110
4.10 Histoachitecture of liver tissues ...............................................................111
4.10.1 Hematoxylin & Eosin ...........................................................................111
4.10.2 TUNEL assay .....................................................................................113
4.11 Gradient generation..................................................................................114
4.12 Adjuvant Therapy Drug-Sorafenib .......................................................118
4.12.1 H&E .........................................................................................................120
4.12.2 TUNEL .....................................................................................................120
Chapter 5: Conclusions ..........................................................................................129
5.1 Trends in liver tissue engineering............................................................130
5.1.1 Human Ectopic Artificial Livers (HEALS) ........................................130
5.1.2 Scientists grow human liver in a laboratory .......................................133
5.1.3 Simulation of regenerative phenomena in liver ..................................134
5.2 Future work ..............................................................................................136
Towards BrDU incorporation ...........................................................................136
References ............................................................................................ 140
Appendix A: VITA ........................................................................... 149
Appendix B: List of Publications ........................................................ 150
xi
List of Figures
Chapter 1
Figure 1.1: Population affected with liver disease and its remedy……………….3
Figure 1.2: Major liver tissue engineering techniques ....................................... 6
Figure 1.3: (a) Static culture, (b) Dynamic culture: rocker platform allows the movement of the culture media ............................................................................. 7
Figure 1.4: Microfluidic device used for maintaining and probing tissue samples. …………………………………………………………………………….10
Figure 1.5: Schematic diagram showing sequence of steps in Pattern I and Pattern II co-culture. ...........................................................................................12
Figure 1.6: Development of 3D tissue engineering. ...........................................15
Figure 1.7: Fabrication of HEALs. ....................................................................19
Figure 1.8: Soft lithographic process to fabricate microscale liver hepatocyte cultures in a multiwell format. ............................................................................21
Chapter 2
Figure 2.1: Typical liver lobule structure. ..........................................................37
Figure 2.2: Structure of liver. .............................................................................41
Figure 2.3: Fabrication of our bioreactor chip and the PEG-DA microstructures.. ..................................................................................................43
Figure 2.4: Illustration, working principle and the photo picture of our bioreactor chip.. ...................................................................................................44
Figure 2.5: Static culture method. ......................................................................45
xii
Figure. 2.6. Overview of healing responses after injury. ...................................46
Figure. 2.7. Role of extra cellular matrix in regeneration and repair. .............48
Figure 2.8 Repair, regeneration and fibrosis after injury and inflammation. …………………………………………………………………………….49
Figure 2.9: Illustration of the microfluidic device. ............................................55
Figure 2.10: Cartoon of gradient generation chip. ............................................56
Figure 2.11: Simulation results of the gradient generator.. ..............................57
Chapter3
Figure 3.1: Preparation for fabricating PEGDA microstructures……………….60
Figure 3.2: Fabrication of 3D PEG-DA microstructures.. ................................62
Figure 3.3: HBsAg genome organization.. .........................................................64
Figure 3.4: Fabrication of microstructures.. ......................................................67
Figure 3.5: Fabrication of PDMS microstructures.. .........................................69
Chapter 4
Figure 4.1: Stability of PEG-DA based microstructures. ..................................75
Figure 4.2: Influence of concentration of PEG-DA microstructures on EWC. …………………………………………………………………………….76
Figure 4.3: Influence of the feed ratio of (PEG-DA+PETA)/PBS on EWC of the PEG-DA hydrogels.. ......................................................................................77
Figure 4.4: HBsAg antigen expression. ..............................................................78
Figure 4.5: H&E stain representing structural morphology of liver tissue. .....82
xiii
Figure 4.6: Cell viability of liver slices measured by TUNEL assay.. ...............85
Figure 4.7: Histopathological examination for the structural morphology of cultured liver tissues.. ..........................................................................................89
Figure 4.8: The changes of the percentages of apoptosis cells in liver slices measured by TUNEL assay.. ...............................................................................92
Figure 4.9 Amount of urea secreted during the culture period. ........................93
Figure 4.10: Liver tissue sections from rats were analyzed for PCNA distributions.. ........................................................................................................94
Figure 4.11: Difference between apoptosis and necrosis cell death...... ...........98
Figure 4.12: Bcl-2 staining results......................................................................99
Figure 4.13: p53 staining results.................................................................... ...102
Figure 4.14: FAS staining results. ....................................................................103
Figure 4.15 (a): FADD signaling pathway and its presence in tissue.......... ..105
Figure 4.15(b): synopsis of cell death mechanism............................................105
Figure 4.16: Viability measured using trypan blue stain.................................106
Figure 4.17: Cytochrome P4501A1 subfamily enzyme activity of cells obtained from medium treated with gradient generator ..................................................108
Figure 4.18: Metabolic activity of cells treated with medium obtained gradient generator .............................................................................................................109
Figure 4.19: Urea secreted by the tissue slices during the culture period. .....110
Figure 4.20: Histoarchitecture assessment of the tissue slice.. .......................112
Figure 4.21: TUNEL assay results....................................................................114
xiv
Figure 4.22: Concentration profile of the gradient generated in our gradient generator. ............................................................................................................115
Figure 4.23: pH of the solutions measured at each outlet of the gradient generator. ............................................................................................................116
Figure 4.24: Structural integrity of tissues assessed by H&E stain. ...............121
Figure 4.25: Distribution of live and apoptotic cells. .......................................122
Figure 4.26: Growth curve to optimize the concentration of Sorafenib. ........123
Figure 4.27: Results of AKT staining ...............................................................124
Figure 4.28: Results of ERK staining ...............................................................125
Figure 4.29: Results of EGFR staining ............................................................126
Figure 4.30: Results of VEGFR staining .........................................................127
Chapter5
Figure 5.1:. Regeneration in the simulation model, starting with a representative liver lobule.. ................................................................................134
Figure 5.2: Assessment of regeneration via BrDU incorporation….. ...........137
Figure 5.3: BrDU optimization using a gradient generator…………………138
List of tables
Table 1.1 Comparison with other static methods ................................................. 8
Table 1.2 Comparison with other perfusion bioreactors ....................................11
Table 1.3 Comparisons between 2D liver constructs ..........................................13
Table 1.4 Alternative approaches to 3D constructs ............................................15
Table 1.5 Comparisons between scaffold utilized constructs .............................18
Table 1.6 Compares new microscale technology approaches ............................22
Table 4.1: Difference between apoptosis and necrosis.......................................97
Table 4.2. The concentration of HGF and IGF growth factors at the outlet of the gradient generator. .......................................................................................117
Table 4.3 Concentration of sorafenib at different outlets. ...............................122
Table 4.4: Tissue stained for different antibodies ............................................127
                                

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