Hepatocytes: Methods and Protocols (Methods in Molecular Biology, 640)

✍ Scribed by Patrick Maurel (editor)

• Publisher: Humana
• Year: 2010
• Tongue: English
• Leaves: 558
• Category: Library

✦ Synopsis

Hepatocytes account for approximately 80% of the liver mass and play a significant role in various aspects of liver physiopathology, exhibiting unrivaled complexity and diversity of functions. In Hepatocytes: Methods and Protocols, expert researchers provide the reader with methods, technical protocols, and review chapters focusing on selected areas of hepatocyte biology including isolation, culture, differentiation and stem cells, and hepatocyte use in clinical, basic, and applied research. With a specific emphasis on human hepatocytes, the volume presents chapters covering subjects including hepatocyte culture models, cryopreservation methods, differentiation assessment, liver ontogenesis, production of hepatocytes from stem cells, drug/xenobiotic metabolism, toxicity and transport, bile acid and blood coagulation factor production, infection by HBV and HCV, humanized animals, biortificial liver devices, hepatocyte transplantation. As a volume in the highly successful Methods in Molecular Biology™ series, protocol chapters include brief introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls.

Comprehensive and cutting-edge, Hepatocytes: Methods and Protocols will be useful to all those who are currently using or planning to use human, or animal, hepatocytes to investigate any aspect of liver physiopathology or who are interested in liver development or liver stem cells and liver biotherapy.

✦ Table of Contents

Foreword
Preface
1 References
Contents
Contributors
1 General Review on In Vitro Hepatocyte Models and Their Applications
1 Introduction
2 Culture Conditions of Hepatocytes
2.1 Primary Adult Hepatocytes
2.2 Other Liver Cell Models
2.2.1 Precision-Cut Tissue Slices
2.2.2 Hepatocyte Cell Lines
2.2.3 Hepatocyte-Like Cells Derived from Stem Cells
3 Applications to the Biology of the Hepatocyte
3.1 Hepatocyte Differentiation
3.1.1 Commitment and Stability of the Hepatocyte Phenotype
3.1.2 Expression and Regulation of Liver Functions
3.2 Hepatocyte Proliferation
3.3 Bile Formation and Secretion
4 Applications to Xenobiotic Metabolism and Toxicity
4.1 Xenobiotic Metabolism
4.2 Xenobiotic Toxicity
4.2.1 Cellular Toxicity
4.2.2 Genetic Toxicology
4.2.3 New Strategies for Hepatotoxicity Testing
4.3 Regulation of Detoxifying Pathways
5 Applications to Hepatocyte Therapies
5.1 Extracorporeal Bioartificial Liver Devices
5.2 Hepatocyte Transplantation
5.2.1 Chronic Liver Failure
5.2.2 Inherited Metabolic Disorders
5.2.3 Acute Liver Failure
5.3 Perspectives of Hepatocyte-Based Therapies
6 Applications to Virology and Parasitology
6.1 Virology
6.1.1 HBV
6.1.2 HCV
6.2 Parasitology
7 Conclusions
References
2 Human Foetal Hepatocytes: Isolation, Characterization, and Transplantation
1 Introduction
2 Materials
2.1 Human Foetal Hepatic Cell Isolation
2.2 Foetal Hepatic Cell Culture
2.3 Reverse Transcription-Polymerase Chain Reaction Analysis
2.4 Double Immunostaining
2.5 Immunocytochemistry for Green Fluorescent Protein Expression
2.6 Western Blot Analysis for ERK Expression
2.6.1 Cell Lysis
2.6.2 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
2.6.3 Western Blotting
2.7 Lentiviral Vectors
2.8 Transplantation
2.9 Immunohistochemistry for Albumin Expression
3 Methods
3.1 Cell Isolation and Culture
3.2 Characterization
3.2.1 RT-PCR Analysis
3.2.2 Fluorescence Double Immunostaining
3.2.3 Western Blot Analysis
3.3 Cell Labelling with the Hoechst Fluorescent Dye
3.4 Retroviral Transduction
3.4.1 Cell Transduction
3.4.2 Detection of Transduced Cells
3.5 Cell Transplantation
3.6 Identification of Engrafted Hepatocytes by Albumin Histochemical Staining
4 Notes
References
3 Isolation and Culture of Primary Hepatocytes from Resected Human Liver Tissue
1 Introduction
2 Materials
2.1 Human Liver Tissue
2.2 Collection of Liver Samples
2.3 Supplies and Equipment
2.4 Reagents
3 Methods
3.1 Preparation for Liver Perfusion
3.2 Perfusion of Resected Human Liver Tissue
3.2.1 Preparation and Cannulation of Tissue
3.2.2 Perfusion of Resected Liver Tissue
3.3 Isolation of Hepatocytes from Digested Liver Tissue
3.3.1 Disaggregation of Liver Tissue
3.4 Cell Count and Viability Assessment
3.5 Monolayer Culture of Primary Human Hepatocytes
3.5.1 Plating Hepatocytes
3.5.2 Maintenance and Dosing of Hepatocyte Cultures
3.5.3 Harvest of Hepatocyte Monolayers
3.6 Overlay with Extracellular Matrix (Optional)
3.6.1 Overlay with Collagen
3.6.2 Overlay with Matrigel or Geltrex
4 Notes
Acknowledgments
References
4 Optimisation of the Cryopreservation of Primary Hepatocytes
1 Introduction
2 Programmable Freezing and Storage
3 Cell Treatment Prior to Freezing
3.1 Percoll Purification
3.2 Pre-culture
3.3 Pre-incubation with Medium Supplements
4 Selection of Human Donors and Hepatocyte Quality
5 Cryoprotectants
5.1 Cryoprotectant Properties
5.2 DMSO
5.3 Other Co-cryoprotectants
5.4 Membrane Stabilisers
6 Basal Freezing Medium
7 Freezing Cell Density
8 Thawing and Handling of Cryopreserved Hepatocytes
9 Percoll Purification After Freezing
10 Species Differences
11 Conclusions
12 Notes
References
5 Cryopreservation of Human Hepatocytes for Clinical Use
1 Introduction
2 Materials
2.1 Human Hepatocytes
2.2 Chemicals and Solutions
2.3 Main Equipment and Other Materials
3 Methods
3.1 Preparation of Cryopreservation and Thawing Solutions
3.2 Preparation of Human Hepatocyte Suspension in Cryopreservation Solution
3.3 Freezing Hepatocytes Using CRF
3.3.1 Starting the CRF
3.3.2 Starting the Cell Freezing
3.4 Thawing Cryopreserved Hepatocytes
4 Notes
References
6 Hepatocyte Differentiation
1 Introduction
2 Cell Culture
2.1 Three-Dimensional Bioreactors
2.2 Two-Dimensional Sandwich Culture
2.3 Defined Media Conditions
3 Markers of a Differentiated Hepatocyte
3.1 Morphology
3.2 Immunofluorescence
3.3 Plasma Proteins
3.4 Cytochromes P450 and Hepatic-Enriched Nuclear Factors
4 Stress Pathways and Hepatocyte Integrity
5 Functional Assessment of Hepatic Phenotype
5.1 Xenobiotic/Drug Induction Responses
6 Species-Specific Considerations
7 Conclusion
References
7 Reversible Manipulation of Apoptosis Sensitivity in Cultured Hepatocytes by Matrix-Mediated Manipulation of Signaling Activities
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Preparation of Rat Tail Collagen
2.3 Preparation of Collagen Gel in ''Sandwich'' Cultures
2.4 Antibodies, Chemicals, and Cytokines
2.5 Cell Lysis and Protein Quantification
2.6 SDS Polyacrylamide Electrophoresis
2.6.1 Sample Denaturation Buffer
2.6.2 SDS/PAGE (Protein Electrophoresis on Denaturating Conditions)
2.7 Western Blot
2.8 Phase-Contrast and Fluorescent Microscopy
2.8.1 Cell Fixation and Permeabilization
2.8.2 Microscopy
3 Methods
3.1 Cell Culture and Treatment
3.2 Western Blot Analysis of Apoptosis (PARP Degradation and Cleaved Caspase-3)
3.2.1 Cell Lysis and Protein Quantification
3.2.2 Sample Denaturation
3.2.3 SDS/PAGE (Protein Electrophoresis on Denaturating Conditions)
3.2.4 Western Transfer and Immunoblot
3.3 Apoptosis Detection by Chromatin Condensation
3.3.1 Cell Fixation, Permeabilization, and Chromatin Staining
3.4 Enhancement of Apoptosis Sensitivity in Stiff Collagen-Coated Cultures by Inhibiting MAPK and Akt Signaling
3.5 Reversion of Hepatocyte Apoptosis Resistance by Trypsination and Re-plating in Collagen Gel Sandwich
4 Notes
Acknowledgments
References
8 Markers and Signaling Factors for Stem Cell Differentiation to Hepatocytes: Lessons from Developmental Studies
1 Introduction
2 Development of a Regionalized Endoderm
3 Differentiation of Hepatoblasts from Foregut Endoderm Cells
4 Differentiation of Bipotent Hepatoblasts Toward the Hepatocyte Lineage
5 Hepatocyte Heterogeneity
6 Conclusion
References
9 Hepatic Stem Cells
1 Introduction
2 Basic Biology of Stem Cells
3 Adult Liver as a Source of Hepatic Progenitors
4 Plasticity of BM, HSC, and Their Differentiation into Hepatocytes
References
10 Hepatic Stem Cells and Liver Development
1 Introduction
2 Defining a Stem Cell
2.1 Stem Cell Niche
3 Liver Development
4 Liver Renewal
5 Liver Stem Cells
5.1 Definition
5.2 Fetal Liver
5.3 Adult Liver
5.3.1 Intrahepatic Stem Cells
5.3.2 Extrahepatic Stem Cells
6 In vitro Hepatic Differentiation Potential of Stem Cell
6.1 Adult Stem cells
6.1.1 Bone Marrow and Hematopoietic Stem Cells
6.1.2 Peripheral Blood
6.1.3 Mesenchymal Cells
6.1.4 Multipotent Adult Progenitor Cells (MAPCs)
6.2 Embryonic Stem Cells
6.2.1 Endoderm Induction from ES cells
6.2.2 Hepatic Induction
6.2.3 Hepatic Specification
6.2.4 Hepatic Maturation
6.2.5 Current Status of Human ES Cell Differentiation to Hepatocyte-like Cells
6.2.6 Conclusions and Prospects
7 Identifying a Hepatocyte
7.1 Hepatocyte Drug Metabolism
7.2 Hepatic Transporters
7.3 Hepatic Transcription Factors, Homeostasis, and Clinically Relevant Hepatic Enzymes
7.4 Conclusions
8 Therapeutic Potential of Stem Cell-derived Hepatocytes
9 Conclusions
References
11 Generation of Hepatocytes from Human Embryonic Stem Cells
1 Introduction
2 Materials
2.1 HESCs Medium
2.2 EBs Medium
2.3 Murine Embryonic Fibroblasts (MEFs) Medium
2.4 Antibiotic-Resistant MEF
2.5 Transfection Medium
3 Methods
3.1 Establishment of HESC Lines Stably Transfected by Reporter Gene Under the Control of Hepatic Promoter (e.g., Albumin-eGFP)
3.1.1 Preparation of Plasmid DNA for Stable Transfection
3.1.2 Transfection of HESCs by Albumin-eGFP Using ExGen 500
3.2 Differentiation of HESCs into Hepatic Cells by EBs Formation
3.3 Sorting of eGFP-Labeled Hepatic Cells Using FACS
4 Notes
References
12 Isolation and Culture of Adult Human Liver Progenitor Cells: In Vitro Differentiation to Hepatocyte-Like Cells
1 Introduction
2 Materials
2.1 Human Liver Samples
2.2 Materials
2.3 Reagents for Cell Isolation
2.4 Buffers and Solutions for Cell Isolation
2.5 Collagenase Solution
2.6 Cell Culture Media
2.7 Buffers Solutions and Materials for Cell Differentiation Analysis
2.8 Antibodies
3 Methods
3.1 Preparation of Non-parenchymal Epithelial Cells
3.1.1 Safety Conditions
3.1.2 Liver Perfusion
3.1.3 Non-parenchymal Epithelial Cell Isolation
3.2 Culture of Non-parenchymal Epithelial Cells
3.3 Differentiation of Non-parenchymal Epithelial Cells to Hepatocyte-Like Cells
3.3.1 Indirect Immunofluorescence
3.3.2 Immunoblotting
3.3.3 RT-PCR
4 Notes
Acknowledgments
References
13 The HepaRG Cell Line: Biological Properties and Relevance as a Tool for Cell Biology, Drug Metabolism, and Virology Studies
1 Introduction
2 Establishment of the HepaRG Cell Line
3 Characterization of HepaRG Cells
3.1 Morphologic Aspects
3.2 Hepato-specific Markers
3.3 Genotype and Phenotype of HepaRG Cells
4 Progenitor Features
5 In Vitro and In Vivo Differentiation of HepaRG Cell
5.1 Transcriptional Control and Hepatocyte Differentiation
5.2 Translational Control and Hepatocyte Differentiation
5.3 In Vivo Differentiation
6 HepaRG Cell Line: An Interesting Tool for Studying Liver Cell Biology and Hepatitis Viruses
6.1 Liver Metabolism and Iron Storage
6.2 Drug Metabolism and Toxicity Studies
6.3 HepaRG and Hepatitis B Virus Infection
6.4 HepaRG, Viral Infection, and Antiviral Innate Response
7 Conclusions
References
14 Transdifferentiation of Pancreatic Cells to Hepatocytes
1 Introduction
2 Materials
2.1 Culture and Transdifferentiation of AR42J Pancreatic Acinar Cell Lines
2.2 Culture and Transdifferentiation of Primary Mouse Pancreatic Cells
3 Methods
3.1 Transdifferentiation of Pancreatic AR42J or AR42J-B13 Cells to Hepatocytes
3.2 Isolation and Transdifferentiation of Pancreatic Exocrine Cells to Hepatocytes
3.3 Immunofluorescence Analysis of Transdifferentiated Hepatocytes
3.4 RT-PCR Analysis of Transdifferentiated Hepatocytes
4 Notes
Acknowledgments
References
15 Evaluation of Drug Metabolism, Drug_Drug Interactions, and In Vitro Hepatotoxicity with Cryopreserved Human Hepatocytes
1 Introduction
2 Materials
3 Methods
3.1 Cryopreserved Human Hepatocyte Thawing and Viability Determination
3.1.1 Thawing and Recovery of Cryopreserved Hepatocytes
3.1.2 Viability Determination
3.2 Applications of Human Hepatocytes in Drug Development
3.2.1 Metabolic Stability Screening
3.2.2 Metabolite Profiling and Species Comparison
3.2.3 Drug--Drug Interaction Evaluation
3.3 Hepatotoxicity Screening
3.4 Conclusion
4 Notes
References
16 The Use of Human Hepatocytes to Investigate Drug Metabolism and CYP Enzyme Induction
1 Introduction
2 Materials
2.1 Human Tissue
2.2 Cell Culture
2.3 Chemicals
2.4 Analytical Materials and Equipments
3 Methods
3.1 Evaluation of Drug-Metabolizing Capacities
3.2 Evaluation of CYP Gene Inducibility
4 Notes
References
17 The Use of Hepatocytes to Investigate UDP-Glucuronosyltransferases and Sulfotransferases
1 Introduction
2 Materials
2.1 Assessment of Drug Conjugation by Human Hepatocytes
2.1.1 Hepatocyte Culture and Ex Vivo Drug Glucuronidation and Sulfation
2.1.2 Preparation of Microsomes and Cytosol from Hepatocytes
2.1.3 In Vitro Measurement of UGT Activity Toward Drugs
2.1.4 In Vitro Measurement of SULT Activity Toward Drugs
2.2 Induction Studies
2.3 Evaluation of UGT and SULT Expression in Hepatocytes
2.3.1 Total mRNA Preparation
2.3.2 cDNA Synthesis and Real-Time PCR Quantification
3 Methods
3.1 Assessment of Drug Conjugation by Human Hepatocytes Ex Vivo
3.1.1 Glucuronide and Sulfate Formation from Various Drugs
3.1.2 Separation, Structure Identification, and Quantification of Drug Conjugates
3.2 Assessment of Drug Glucuronidation by Human Hepatocytes In Vitro
3.2.1 Preparation of Microsomes and Cytosol from Hepatocytes
3.2.2 Measurement of the In Vitro UGT Activity Toward Acceptor Substrates
3.2.3 Measurement of the In Vitro SULT Activity Toward Acceptor Substrates
3.3 Use of Hepatocytes to Assess Induction
3.3.1 Hepatocyte Culture, Inducer Treatment, and UGT/SULT Activity
3.3.2 Analysis of Expression of UGT and SULT in Hepatocytes by Quantitative PCR
4 Notes
References
18 The Use of Hepatocytes to Investigate Drug Uptake Transporters
1 Introduction
2 Materials
2.1 Isolation of Rat Hepatocytes by Collagenase Perfusion
2.2 Thawing Procedure for Human Cryopreserved Hepatocytes
2.3 Uptake Experiments Using Isolated Hepatocytes
2.4 Construction of Cell Lines Stably Transfected with cDNA for OATPs
2.5 Uptake Experiments Using Transporter-Expressing Cell Lines
2.6 SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis of OATPs in Expression Systems and Hepatocytes
3 Methods
3.1 Isolation of Rat Hepatocytes by Collagenase Perfusion
3.2 Thawing Procedure for Human Cryopreserved Hepatocytes
3.3 Uptake Experiments Using Isolated Hepatocytes
3.4 Construction of Cell Lines Stably Transfected with cDNA for OATPs
3.5 Uptake Experiments Using Transporter-Expressing Cell Lines
3.6 SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis of OATPs in Expression Systems and Hepatocytes
3.7 Calculation of Uptake Clearance of Compounds in Hepatocytes and Expression Systems
3.8 Calculation of the Quantitative Contribution of OATPs to the Overall Hepatic Uptake of Compounds
3.8.1 RAF Method for Estimating the Uptake Clearance of Reference Compounds for Each Transporter
3.8.2 Comparison of the Expression Level of Each Transporter Using Western Blot Analysis
3.8.3 Inhibitable Portion of Uptake in Human Hepatocytes in the Presence of a Specific Inhibitor for Each Transporter
3.9 Prediction of In Vivo Hepatic Clearance of Transporter Substrates
4 Notes
References
19 Metabonomic Studies on Human Hepatocyte in Primary Culture
1 Introduction
2 Materials
2.1 Cell Origin and Culture
2.2 Samples Analysis
2.3 Data Analysis
3 Methods
3.1 Primary Culture of Human Hepatocytes (HHPCs) and Sample Preparation
3.2 Sample Analysis by UPLC--TOF-MS
3.3 Data Analysis (Data Treatment and Chemometric Analysis)
3.4 Metabolic Profile Interpretation
3.5 Conclusion
4 Notes
References
20 The Application of HepRG Cells in Evaluation of Cytochrome P450 Induction Properties of Drug Compounds
1 Introduction
2 Materials
2.1 Cell Culture
2.2 mRNA Measurements
2.3 CYP Enzyme Activities
2.4 Analytical Equipment
3 Methods
3.1 Exposure of HepaRG Cells to Inducers
3.2 Real-Time PCR
3.2.1 RNA Extraction, 96-Well Plates
3.2.2 cDNA Preparation and Real-Time PCR
3.3 Activity Assay
3.4 Chromatographic Method
3.5 Calculation of Enzyme Activities
4 Notes
Acknowledgements
References
21 The Use of Hepatocytes to Investigate Drug Toxicity
1 Introduction
2 Hepatotoxins
3 Mechanisms of Hepatotoxicity
4 Hepatocytes: A Tool for Drug Toxicity Evaluation
5 Conventional In Vitro Hepatotoxicity Protocols
6 Omic Technologies to Investigate DILI
6.1 Toxicogenomics
6.2 Proteomics
6.3 Metabonomics
6.4 Cytomics
References
22 The Use of Human Hepatocytes to Investigate Bile Acid Synthesis
1 Introduction
2 Materials
2.1 Collagen Extraction
2.2 Matrigel Extraction
2.3 Cell Cultures
2.4 Bile Acid Analysis
2.5 Gene Expression Analysis
3 Methods
3.1 Collagen Extraction
3.2 Matrigel Extraction
3.2.1 Thawing of Tumors for Injection
3.2.2 Injection
3.2.3 Harvest of Tumors for Passage
3.2.4 Harvest of Tumors for Extraction of Matrigel
3.2.5 Harvest of Tumors for Storage
3.3 Cell Plating
3.4 Bile Acid Analysis
3.5 Gene Expression Analysis
3.6 Statistics
4 Notes
References
23 Use of Human Hepatocytes to Investigate Blood Coagulation Factor
1 Introduction
2 Materials
2.1 Human Liver Samples
2.2 Materials and Reagents
2.3 Hepatocyte Preparation and Culture
2.4 Hepatocyte Culture Media
2.4.1 Short-Term Culture Medium
2.4.2 Submixes of Additives for the Short-Term Culture Medium
2.4.3 Long-Term Culture Medium
2.4.4 Additives for Preparing the Mix for the Long-Term Culture Medium
2.5 Collagenase Solution
2.6 Enzyme-Linked ImmunoSorbent Assay (ELISA) for Quantification of Factor II Antigen and Antithrombin Antigen
2.7 Enzyme-Linked ImmunoSorbent Assay (ELISA) for Quantification of Factor V Antigen
2.8 Enzyme-Linked ImmunoSorbent Assay (ELISA) for Quantification of Factor VII Antigen, Factor VIII Antigen, Protein Induced by vitK 1 Absence or Antagonist II Antigen (PIVKA-II:Ag) and Free Protein S Antigen
2.9 Coagulant Activity
2.10 Immunofluorescence Staining of the Fibrillar Material
3 Methods
3.1 Preparation of Hepatocytes
3.2 Hepatocyte Plating and Culture
3.3 Preparation of Samples for Assays for Quantification of Haemostasis Proteins by Enzyme-Linked ImmunoSorbent Assay or for Functional Assays
3.4 Enzyme-Linked ImmunoSorbent Assay (ELISA)
3.5 Coagulant Activity Measurement
3.6 Immunofluorescence Staining of the Fibrillar Material
4 Notes
References
24 Use of Human Hepatocytes to Investigate HCV Infection
1 Introduction
2 Materials
2.1 Human Hepatocyte Culture
2.2 Bank of HCV-Positive Serum Samples
2.3 Production of Huh7.5/JFH1-Derived HCVcc Particles
2.4 Materials and Reagents
3 Methods
3.1 Human Hepatocyte Infection
3.2 RNA Extraction
3.3 Strand-Specific rTth RT-PCR
3.4 Real-Time PCR Quantification of Positive- and Negative-Strand HCV RNA
3.5 Standard for HCV RNA Quantitation as Extracted from Pool of Sera or Serum of Various Genotypes
3.6 Standard for HCV RNA Quantitation
3.7 Treatment of Primary Hepatocytes with IFN
3.8 Evaluation of Hepatocyte Innate Response Gene Expression After Infection with HCVser or HCVcc Particles
4 Notes
Acknowledgments
References
25 The Use of Hepatocytes to Investigate HDV Infection: The HDV/HepaRG Model
1 Introduction
2 Materials
2.1 Plasmids Required for Production of HDV Particles
2.2 Huh-7 Cell Culture
2.3 HepaRG Cell Culture
2.4 Northern Blot Analysis for Detection of HDV RNA
3 Methods
3.1 Production of HDV Particles in Huh-7 Cells
3.2 HDV Infection Assays in HepaRG Cells
3.3 Detection of HDV RNA in Inocula and Infected Cells
3.3.1 Detection of HDV RNA in Culture Medium of Transfected Huh-7 Cells
3.3.2 Detection of HDV RNA in HepaRG Infected Cells
4 Notes
Acknowledgments
References
26 Rodent Models of Liver Repopulation
1 Introduction
2 A Review of Existing Models
2.1 Two Historical Models of Mouse Liver Repopulation
2.2 A Second Generation of Liver Repopulation Models
3 Creating Mouse Models of Liver Repopulation: What for? What Future Challenges?
3.1 Demonstrating Therapeutic Efficacy
3.2 Developing Rodent Models with a Humanized Liver
3.3 Entering the ''Stem Cells Field Forever''
Acknowledgements
References
27 Chimeric Mice with Humanized Liver: Tools for the Study of Drug Metabolism, Excretion, and Toxicity
1 Introduction
2 Mouse Models
3 Properties of a Model Useful for Generating Chimeric Mice
4 Methods for Generating Chimeric Humanized Mice in the FRG Model
5 Expression and Induction of CYP450 Genes, Proteins, and Metabolic Activities in Humanized Mice
6 Expression of Phase II Enzymes in Chimeric Mice
7 Toxicology Studies with Chimeric Mice with Humanized Liver
8 Summary and Future Directions
References
28 Bioartificial Liver Support Systems
1 Introduction: Therapies of Liver Failure
2 Cell Source
3 Bioreactor Design
4 Bioreactor Systems for Clinical Application
4.1 Extracorporeal Liver Assist Device (ELAD)
4.2 HepatAssist
4.3 The Bioartificial Liver of the Amsterdam Medish Centrum (AMC-BAL) and the Radial Flow Bioreactor (RFB)
4.4 Bioartificial Liver Support System (BLSS), TECA Hybrid Artificial Liver Support System (TECA-HALSS), and Hybrid Bioartificial Liver (HBAL)
4.5 Extracorporeal Bioartificial Liver Support System (EBLSS)
4.6 Modular Extracorporeal Liver Support System (MELS)
4.7 Limitations of Bioartificial Liver Support Systems
References
29 Human Hepatocyte Transplantation
1 Introduction
2 Hepatocyte Transplantation
3 Methods for Isolation of Human Hepatocytes
3.1 Source of Liver Tissues
3.2 Hepatocyte Isolation
4 Pre-clinical Studies
5 Clinical Hepatocyte Transplantation
6 Cell Administration and Safety Concerns
7 Immuno-suppression
8 The Future
8.1 Limitations of Hepatocyte Transplantation
8.2 Alternative Cell Sources
References
SUBJECT INDEX